key: cord-317632-2bjzn6p7 authors: jones, robert t; guest, claire; lindsay, steve w; kleinschmidt, immo; bradley, john; dewhirst, sarah; last, anna; logan, james g title: could bio-detection dogs be used to limit the spread of covid-19 by travellers? date: 2020-08-12 journal: j travel med doi: 10.1093/jtm/taaa131 sha: doc_id: 317632 cord_uid: 2bjzn6p7 nan the covid-19 crisis has not only deeply affected those with sars-cov-2 infections, and their families, but it has also had an unprecedented global economic impact. the travel and tourism sectors have been particularly hard hit, with aircraft grounded, hotels closed, and travel restrictions and quarantines introduced around the world. the united nations' world tourism organization predicts there will be declines of 58% to 78% in international tourist arrivals this year. 1 there are increasing numbers of covid-19 cases in many countries, including australia, brazil, france, spain, and japan. whilst some of the apparent surge may be a result of increased testing, there is a concern of a second wave. in the spanish flu pandemic of 1918, which killed 50-100 million people, it was the second wave that produced the greatest number of deaths. 2 as a defence against a second wave of covid-19, it is critically important that measures are put in place to prevent the importation of new cases from countries with a high incidence of screening travellers for covid-19 at airports is an attractive option that could prevent imported infections, and may reduce the need to enforce quarantine on travellers. infrared thermal image scanners have been used previously during epidemics, but modelling studies indicate that, even under best-case assumptions, thermal screening will miss more than half of infected people. 3 many cases are fundamentally undetectable through these scanners because infected individuals may be presymptomatic or do not have a fever at the time of scanning. there remains a need for a sensitive, rapid, non-invasive means of screening large numbers of asymptomatic people, both at departure and on arrival into ports. previous work with bio-detection dogs suggests that highly-trained dogs may offer a viable solution to covid-19 detection. there is evidence that respiratory infections cause the release of specific odours. the analysis of volatiles liberated in breath, urine, faeces and sweat has shown that microbial infections are associated with the production of specific volatile organic compounds (vocs), and that the ratio of these vocs differs depending on the infectious microorganisms. 4 this area of research has led to interest in using volatiles as biomarkers of infectious diseases, such as cholera. other, more recent work by the authors, demonstrated that asymptomatic malaria infection also causes changes in vocs, which vary throughout the infection. 5 further work by the same team demonstrated the use of trained dogs in identifying malaria parasites in asymptomatic children. 6 the dogs were trained using odour samples collected on socks worn by confirmed positive asymptomatic or negative children in the gambia. the dogs were then trained to distinguish between the odours of socks worn by infected and uninfected children. once training was completed, a second set of specimens was used in a double blinded study to determine the specificity and sensitivity of detection. the study demonstrated that dogs could identify malaria with a degree of sensitivity and specificity broadly in line with the world health organization's criteria for the procurement of rapid diagnostic tests. 6 in march this year, we commenced a prospective three-phase study in the uk involving collection of body and breath odour samples from more than three hundred asymptomatic and mildly symptomatic participants. phase 1 is a proof-of-principle study to demonstrate that medical detection dogs can be trained to identify asymptomatic or mild symptomatic infections with sars-cov-2 with high sensitivity and specificity. phase 2 is an assessment of the capability of the trained dogs to detect people with asymptomatic or mildly symptomatic sars-cov-2 infection. this important step will move the research team closer to deployment of dogs, and will require the sensitivity and specificity of the dogs to be assessed at covid-19 test centres where they can sniff individuals waiting to donate swab samples for formal diagnosis. if bio-detection dogs prove to be effective in the detection of asymptomatic or mildly symptomatic individuals, there is enormous potential for them to be used at key entry points, such as airports and sea ports, to support efforts to screen departing or incoming passengers, which is crucial for ensuring safer, and fully operational, global travel in the future, and for any elimination strategies. each trained dog can screen up to 250 people in an hour and they could be stationed before entering the terminal building, at the security checkpoint for outbound passengers and staff, and at border control for inbound passengers. positive individuals and their contacts could then isolate to prevent the spread of infection to others. this will be of significant benefit to the economy and the control of the pandemic, as it could eliminate the need to keep impact assessment of the covid-19 outbreak on international tourism the spanish flu of 1918 and how it changed the world estimated effectiveness of symptom and risk screening to prevent the spread of covid-19 the scent of disease: volatile organic compounds of the human body related to disease and disorder plasmodium-associated changes in human odor attract mosquitoes trained dogs identify people with malaria parasites by their odour cellular scent of influenza virus infection real-time detection of a virus using detection dogs scent dog identification of samples from key: cord-022664-jw0jvpc6 authors: jackson, ae title: in this issue – october 2010 date: 2010-09-20 journal: aust vet j doi: 10.1111/j.1751-0813.2010.00498_1.x sha: doc_id: 22664 cord_uid: jw0jvpc6 computed tomography diagnosis of brain infarction in dogs · tibial tuberosity advancement in canine stifles · holter monitoring in dogs with mitral valve disease · staggers in horses grazing paspalum infected with claviceps paspali · distal luxation of the patella in a horse · escherichia coli and salmonella serotypes in sheep at slaughter · alzheimer type ii astrocytes in the brains of pigs with salt poisoning · classification of infectious bronchitis viruses in poultry field specimens · mortality events in sea‐caged yellowtail kingfish seizures were the most common signs in five of the dogs. ischaemic, non-haemorrhagic infarction in the territory of the rostral cerebellar artery was identified in three dogs and telencephalic infarcts in five dogs, in the territories of the middle or rostral cerebral arteries. one of these was ischaemic, the others appeared haemorrhagic. all dogs were at least 8 years old, and six had concurrent medical conditions. one dog was euthanased after diagnosis because of the severity of its neurological signs and one dog was euthanased as a result of associated renal disease 2 months after diagnosis. six dogs were alive at least 1 year after diagnosis. the authors conclude that many of these dogs can recover with supportive care, but that an underlying disease may lead to a poorer prognosis. this retrospective study of short term clinical outcome and owner evaluations of dogs suggests that favourable results can be expected when cranial cruciate ligament (ccl)-deficient stifles are treated with tibial tuberosity advancement (tta) and that they have faster postoperative improvement compared with other stifle procedures. 2 tta is a recent procedure for treatment of ccl rupture in canine stifles, based on model analysis of the human knee. this retrospective study describes the first 92 procedures of tta repair in stifles of 72 dogs with partial or complete ccl rupture. labrador retrievers and rottweilers were the most the most common breeds, and the period of lameness ranged from 3 days to 24 months. the median pre-operative lameness score was 3/4 and meniscal injury was present in 51 stifles. minor complications occurred in 29% of cases with major complications in 6.5% of cases; these consisted of meniscal injury and two tibial tuberosity fractures. in the owner evaluation, 96% reported moderate to great improvement postoperatively, with no lameness at rest and mild to no lameness after vigorous exercise. results of a study of dogs with myxomatous mitral valve disease (mmvd) with or without clinical signs, shows that arrhythmias are a common finding. 3 holter monitoring is reliable and better than standard electrocardiogram (ecg) for both heart rate monitoring and diagnosis. mmvd is the most common acquired valvular disease in dogs, and the presence of arrhythmias has been thought to be a complication of the condition. thirty six small dogs with mmvd were divided into a preclinical group with no clinical signs and a clinical group that were showing clinical signs. each dog underwent standard echocardiogram, ecg and 24-h holter recording. minimum and mean holter heart rates were higher and there were more ventricular arrhythmias in the clinical group than in the preclinical group. an enlarged left atrium was associated with the presence of more supraventricular arrhythmias. the authors conclude that more studies are needed to establish if the presence of arythmias alters progression of disease or survival. this case report describes two occurrences of paspalum staggers in foals and mature horses. 4 claviceps spp. are an ergot fungus that invade the flowering heads of paspalum (paspalum dilatatum) grass and produce sclerotia (or ergots) that contain toxins. ingestion of these can induce a range of clinical symptoms including staggers, and cattle are the most commonly affected species. although it has been mentioned since the early part of the 20 th century, there are no published descriptions of paspalum staggers in horses. the foals presented with ataxia in all limbs after consuming infected paspalum. one foal died from misadventure and the other two recovered within 1 week of removal from the infected paddock. in the second case, two of eight mares and geldings grazing in an irrigation channel developed hindquarter paresis. after removal of all horses from the area, one of the affected horses continued to deteriorate and had to be euthanased. the authors report that diagnosis is based on clinical signs, history and the presence of the sclerota in the parspalum seed heads. they suggest that animals should be moved quietly to an uncontaminated pasture to recover, as other treatments do not appear to be warranted. the authors report a rare manifestation of patellar luxation in a horse that has only been reported once previously in the equine literature. 5 a 19-year-old thoroughbred gelding presented with sudden onset, non-weight bearing lameness in the right hindlimb. radiography confirmed distal luxation of the patella, which was replaced into its normal anatomical location under general anaesthesia. there were no pathological sequelae noted on follow-up examination 9 months after the initial injury. results of a study to determine the presence and concentration of escherichia coli o157 and salmonella spp. on fleece, faeces and carcases of sheep during slaughter, show a low risk of human infection from meat products from these animals. 6 food borne pathogens present on the intestinal contents and fleece of sheep presented for slaughter are reported to be significant sources of carcase contamination. faeces, fleece and pre-chill carcase samples were collected from 164 sheep slaughtered at two australian abattoirs, and the presence of e. coli o157 and salmonella spp. were determined. results showed that the prevalence and concentration of pathogens were low in the sheep tested; e. coli o157 was isolated from 5% of faeces, 3% of fleeces and 0.6% of pre-chill carcases. salmonella spp. were isolated from 20% of faeces, 13% of fleeces and 1.3% of pre-chill carcases. this paper reports the novel finding of alzheimer type ii astrocytes, in addition to the pathognomonic combination of laminar cerebrocortical necrosis and eosinophil infiltration, in the brains of pigs with salt poisoning. 7 this neurological disturbance is frequently fatal in pigs, and follows restoration of water after a period of water deprivation. pigs seem to be vulnerable because of their relatively high salt intake, and although the mechanism is unclear, appears to result from indirect salt poisoning that causes acute cerebral oedema. a type 3 coronavirus causes infectious bronchitis (ib) is a significant cause of production losses in poultry. a real-time polymerase chain reaction (pcr)/high-resolution melt (hrm) curve analysis protocol was developed to differentiate ib virus reference strains, and the method was used to detect and classify ib viruses in field submissions. 8 the study investigated samples from 40 cases of suspected ib virus of which 17 were positive for ib virus by pcr. hrm curve analysis classified each strain as subgroup 1, 2 or 3 in 12 submissions or a strain that was unable to be classified in the other 5 submissions. of the 12 ib field viruses that were classified as subgroup 1, 2, or 3 using hrm curve analysis, the gene nucleotide sequences studied were virtually identical to the respective subgroup reference strain. however, analysis of the gene nucleotide sequences for the five ib virus strains that could not be classified suggested that four belonged to one of the subgroups, but that one was a potential recombinant strain (between strains from subgroups 2 and 3). the authors conclude that hrm curve analysis can rapidly assign the majority of ib viruses present in field submissions to known subgroups. hrm curve analysis also identified variant genotypes that require further investigation, to determine its virulence for chickens and to establish if the current vaccines are protective. two cases of unexplained deaths in yellowtail kingfish, seriola lalandi, that occurred during the first attempt to grow them in sea cages in western australia, highlights the difficulties that can occur in investigating disease outbreaks in relatively new species in new areas. 9 a commercial sea-cage operator reported more than 70% mortality of the sea-caged fish. several parasites and potentially pathogenic bacteria were isolated from the fish, but despite a detailed laboratory investigation the cause of the mortality event could not be determined. a second smaller episode of deaths occurred later, with fish dying in a sea-cage that had been stocked several weeks earlier. similar pathogens, including parasitic infections, were again isolated but again a single causative agent could not be identified as the cause. the authors suggest that multiple stress factors resulting in immunosuppression may have precipitated the mortality events. computed tomography diagnosis of 8 dogs with brain infarction tibial tuberosity advancement in 92 canine stifles: initial results, clinical outcome and owner evaluation holter monitoring in 36 dogs with myxomatous mitral valve disease staggers in horses grazing paspalum infected with claviceps paspali distal luxation of the patella in a horse concentration and prevalence of escherichia coli o157 and salmonella serotypes in sheep during slaughter at two australian abattoirs alzheimer type ii astrocytes in the brains of pigs with salt poisoning (water deprivation/intoxication) application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens two mortality events in sea-caged yellowtail kingfish seriola lalandi valenciennes, 1833 (nannopercidae) from western australia t-shaped malformation of the ventral colon in a thoroughbred filly with colic the article in the august issue of the journal of the surgical management of a case of colic in a filly makes for interesting reading. 1 it would appear that it took three surgeries over a period of 2 days to manage this condition. three surgeries to manage a case of equine colic? this is not an isolated instance. today, when reading journal articles of equine laparotomies it is common to encounter re-laparotomies. the authors of the above article noted that "repeat laparotomy" is a risk factor for the development of ventral abdominal wound dehiscence.i began performing laparotomies for surgical colic cases in the 1970s. in those days colic surgery was performed as a last resort and conducted without aggressive iv fluid therapy. my success ratethe number of cases discharged from the hospital after surgeryranged from 20% to 25%. this was the result achieved from one laparotomy; if the condition could not be resolved after one surgery there was no question of performing a second operation. this meant that surgery was seldom recommended because of the poor prognosis and high cost.in 1980 i travelled to the usa and spoke with a number of equine surgeons there and the 20-25% success for this type of surgery was consistently described. a number of surgeons were of the opinion that the poor success rate mitigated against the procedure and recommended humane euthanasia as the better option. some older surgeons had stopped performing such surgery. today, with improved techniques, including going to surgery much earlier and the use of fluid therapy, new equipment and suture material, the success rate has improved -but not by that much! even with repeat laparotomies a success rate of 50% still leaves a lot to be desired and makes the ethics of such surgery questionable.if this is the case why is such surgery so readily undertaken? is it performed because of client demand or because of the willingness of the surgeon to go to surgery, once, twice or more times or is it for the welfare of the patient?is it because horse owners are now prepared to pay for the high cost of such surgeries? comprehensive insurance policies may allow this type of surgery to be undertaken more frequently.is it because the surgery can be done and is available? it would be a brave surgeon -or a foolish one -who did not advise the client that the prognosis for such surgery is guarded.but what about the welfare of the horse? is this surgery in the best interest of the patient? if the veterinary profession is to take ethics seriously, shouldn't this be the first consideration?in the same issue of the journal there was an opinion piece entitled "ethical dilemma -sheba. " perhaps a debate, as opposed to an opinion piece, regarding the ethics of the performance of laparotomies and re-laparotomies in the horse for the management of colic should be considered. key: cord-329150-9g5nu5ok authors: schredl, michael; bailer, christian; weigel, muriel sophie; welt, melina sandra title: dreaming about dogs: an online survey date: 2020-10-19 journal: animals (basel) doi: 10.3390/ani10101915 sha: doc_id: 329150 cord_uid: 9g5nu5ok simple summary: the findings of the survey indicate that waking-life experiences with dogs (owning a dog or negative experiences with dogs in the past) affect dreaming in a significant way. on the one hand, dog owners dream about dogs more often and had overall positively toned dreams, whereas persons with negative experiences with dogs in their waking life reported a higher percentage of dreams with threatening dogs. abstract: dogs have been close human companions for millennia and one would expect—according to the continuity hypothesis of dreaming—that dogs are also quite common in dreams. previous studies showed that the percentages of dreams that include dogs range from about 1.5% to 5%, but studies relating waking-life experiences with dogs with dreams about dogs have not been carried out. in total, 1695 persons (960 women, 735 men) completed an online survey that included questions about dreams and waking-life experiences that included dogs. the findings indicate that dogs show up, on average, in about 5% of remembered dreams, but this percentage is much higher in the dreams of dog owners and persons with close contacts with dogs. moreover, the active time spent with a dog and the proximity during sleep is also related to a higher percentage of dreams that include dogs. although dreams including dogs are on average more positively toned than dreams in general, about 11% of the dog dreams included threatening dogs. persons who had negative experiences with dogs in their waking lives reported more threatening dog dreams. the results support the continuity hypothesis and it would be very interesting to conduct content analytic studies with dream samples obtained from dog owners to learn more about the variety of interactions between dreamers and dogs. dogs have been human companions for millennia [1] and are widespread all over the world. in germany, for example, dogs are present in 19% of all households, with a total number of about 9.4 million dogs [2] . given the closeness of human-dog relationships, e.g., dogs as part of the family, dogs as best friends, treating dogs like persons, and daily interactions such as walking the dog, feeding, etc. [3] , one would expect-according to the continuity hypothesis of dreaming [4] that dogs would also be quite common in dreams. indeed, dogs are often the most frequent animals within dreams; the percentage of dreams including dogs ranges from about 1.5% to 5% [5] [6] [7] [8] [9] [10] [11] . there is a long history of speculating about the meaning of dog dreams that dates back to the second century ad [12] , including the ideas that animals in general, but especially dogs, represent the animal nature of humans [13, 14] , the impulsive self of the child [15] , the parents [16] , or sexual impulses in adolescence [17] . artemidorus (second century ace) differentiated between hunting dogs (representing the deeds of the dreamer), house dogs (representing family and belongings), and malteser dogs in dreams that represent the most delightful and pleasant things in life [12] . despite the large amount of literature on the symbolic interpretations of dogs in dreams, there is a lack of empirical research looking, for example, into the simple question as to whether dog owners dream about dogs more often than persons who do not own dogs. the finding that animal rights activists dream more often about animals in general, and also report more dog dreams (9.51%), would be in line with the continuity hypothesis, since one might assume that individuals investing considerable effort in protecting animals also have a great love for them. in a single case study [10] , it was shown that the newly bought dog of the romantic partner of the dreamer increased the number of dog dreams significantly from 0.53% in the year 2006 (overall, 189 dreams were remembered in that year) to 3.93% in the year 2007 (280 dreams). this is the first hint that living close to a dog affects the number of dog dreams. several studies [7, [18] [19] [20] found that most animal dreams are negatively toned, e.g., being bitten or being chased. van de castle [7] reported that animals were attacking the dreamer in about 30% of the dreams, but were companions or playmates in only about 15% of the animal dreams; however, if the dream animal interacted with the dreamer positively, it was most likely a dog. in the single case study based on 108 dog dreams (out of a series of 8400), 9 (8.33%) included negative interactions with dogs (being threatened, being bitten, fighting with the dog) whereas 30 (27.78%) included positive interactions between the dreamer and a dog (the dreamer cares for the dog, plays with the dog, or the dog helps the dreamer). this research indicates that dreams including dogs could be either positive or negative (in addition to neutral dreams). according to the continuity hypothesis of dreaming [4] , one would expect that individuals who have had negative experiences with dogs in their waking life would also have more negatively toned dog dreams. the aim of this study was to investigate whether or not waking-life experiences with dogs affect dreams. we hypothesized that dog owners dream about dogs more often when compared to persons who do not own a dog. moreover, it was expected that persons with negative dog experiences in their past would also report a higher percentage of negatively toned dog dreams. overall, 1695 persons (960 women, 735 men) completed an online survey between 13 april 2020 and 20 april 2020. the mean age of the sample was 53.84 ± 13.99 years (range: 20 to 96 years). concerning educational level, 0.53% had no degree, 12.80% had 9 years of schooling, 39.62% had o-levels (middle degree, "realschule", about 10 years), 23.13% a-levels ("abitur"), 30.91% obtained a university degree, and 3.01% had doctoral degrees. for eliciting dream recall frequency, a 7-point scale (coded as 0 = never, 1 = less than once a month, 2 = about once a month, 3 = about two to three times a month, 4 = about once a week, 5 = several times a week, 6 = almost every morning) was presented. the exact wording was [21] "how often have you recalled your dreams recently (in the past several months)?" the retest reliability of this scale is high-r = 0.85 for an average interval of about 55 days [22] . the overall emotional tone of the dreams was measured via five categories (−2 = very negative, −1 = somewhat negative, 0 = neutral, +1 = somewhat positive, and +2 = very positive). next, the participants were asked to estimate the percentage of dreams that included dogs; the following explanation was included: "the dog does not have to be the focus of the dream. it can be your own dog but also unfamiliar dogs. if you have never dreamed of dogs, please enter a zero." the emotional tone of the dreams that included dogs was-like the general emotional tone of dreams-measured on a five-point scale ranging from −2 to +2. the next question was: "what is the percentage of remembered dog dreams where the dog is threatening?" the next section included items about dogs in waking life, starting with the question: "are you or a household member currently the owner of one or more dogs?" there were four answering options: "yes, i myself own a dog/dogs", "yes, there is a dog/dogs in my household", "no, but i have owned a dog or lived with a dog in my household", and "no, i have never lived with a dog." the time spent with the dog was measured as follows: "how much time during a week do you actively spend with your own and/or another dog? note: active time includes, for example, playing, cuddling, petting, going for a walk, feeding, etc." the typical sleep locations of the dog(s) were also elicited-within the bed, within the bedroom but outside the bed, within the household but outside the bedroom, and outside the household. lastly, the participants were asked whether they had had negative/threatening experiences with dogs in the past. if so, the situation should be described briefly. within the wisopanel online panel, persons with an interest in online studies and with heterogenic demographic backgrounds are registered. at the time of the study, 14,277 individuals were in the database. almost all the participants live in germany with less than 5% living in austria and switzerland. the survey was conducted in german. all registered persons received an email with the link to the study entitled "everyday life and dreams". the participation was voluntary and unpaid. the study was carried out following the rules of the declaration of helsinki of 1975, revised in 2013. in germany, this type of study asking healthy human volunteers to participate in an online survey does not require ethical approval. this was confirmed by the ethics committee of the university of mannheim. statistical procedures were carried out with the sas 9.4 software package for windows. as the percentage variables (percentage of dog dreams, percentage of dog dreams with threatening dogs) were not normally distributed, they were categorized (see the results section below). the categorized variables were treated as ordinal variables. the reports about negative experiences with dogs in the past were classified into several groups: "being bitten", "feeling threatening", "being attacked without being bitten", "loud barking", "another person or the own dog was bitten", and "others". ordinal regressions (cumulative logit model) were used for analyzing the effects of waking-life variables, e.g., owning a dog, having owned a dog, time spent with the dog, or negative experiences in the past with dogs, on dog dream variables (dog dream percentage, emotional tone of dog dreams) controlled for age, sex, education, and dream recall frequency. all variables were entered simultaneously. effect sizes for each predictor were computed based on the wald chi-square values obtained by the ordinal regression analyses and sample size according to the formula given by cohen [23] . this option for computing effect sizes was chosen because (1) the dependent variable is ordinal and (2) the ordinal regression coefficient that is tested with the wald chi-square is controlled for possible confounding effects, as the effects of all other variables entered simultaneously into the regression are particalled out. the distribution of the estimated dream recall frequency was as follows: never (8.69%), less than once a month (18.79%), about once a month (8.87%), about two to three times a month (13.36%), about once a week (18.62%), several times a week (22.99%), almost every morning (8.69%), and there were three missing values. the mean of the percentage of dreams including dogs was 5.52 ± 14.04% (n = 1695). the distribution of the categorized variables is depicted in table 1 . overall, 525 participants (about 31%) reported that they had dreams that included dogs. the emotional tones of dreams that include dogs are shown in table 2 ; the positive dreams clearly outweigh the negative ones (mean: 0.64 ± 1.06). compared to the general emotional tone of the dreams (mean: 0.07 ± 0.81; see table 2 ), the emotional tone of dreams including dogs was significantly more positive (wilcoxon signed ranks test: z = 9.4, p < 0.0001, n = 497, effect size = 0.940). 227 participants owned a dog, whereas 49 participants stated that there is a dog within their household that they do not own. about 50% never owned a dog or lived with a dog, whereas 29% had owned or lived with a dog in the past (25 participants did not complete this item). the percentages of dreams that included dogs for the three groups (dog owners and persons with dog[s] in the household were collapsed into one group) differed considerably; see table 3 . the difference between the "dog owner/dog(s) within the household" group and the "never had a dog or lived with a dog" group was large (effect size = 1.079) and significant (for significance levels, see table 4 ). similarly, participants who had a dog or lived with a dog reported a higher dog dream percentage (effect size = 0.537) than participants that had never lived with a dog. in addition, high dream recall was associated with higher percentages of dreams including dogs, and females reported slightly higher percentages of dog dreams then males (see table 4 ). dog owners and/or persons with a dog living in the household also reported that their dog dreams are more positive than those of persons who had never lived with a dog (effect size = 0.689; see table 4 ). the same was true for persons who had owned a dog or lived with a dog (effect size = 0.482; see table 4 ). the average active time spent with the dog was, for the dog owners group, 20.28 ± 12.82 h per week (n = 198). several answers (above 60 h per week) had to be excluded as these participants most likely confused overall time spent with the dog and active time. regarding the dog's sleeping place, 66 participants stated that the dog sleeps predominantly within the bed, within the bedroom but outside the bed (n = 71), within the household but outside the bedroom (n = 78), or outside the household (n = 11; one missing value). within the dog owners group, the regression analysis indicated that the amount of active time spent with the dog is significantly associated with the percentage of dreams that include dogs (effect size = 0.430; see table 5 ). moreover, the closer the proximity of the dog's sleeping place to the dog owner, the more likely (marginally significant, effect size = 0.260) it is that the owner dreams about the dog (see table 5 ). about one third of the participants who reported dreams including dogs stated that at least in some of these dreams the dog(s) were threatening (see table 6 ). only a small percentage of individuals reported that their dog dreams are predominantly threatening. the mean percentage of dog dreams that included threatening dogs was 11.14 ± 25.67% (n = 522). about 25% of the 1583 participants who responded to the question reported negative experiences in the past. overall, 356 brief descriptions were provided in the study; 48.60% referred to being bitten by a dog, 37.64% included a threat such as being attacked or chased but not bitten, 6.46% included incidents in which another person or the dog of the participant was bitten, and 4.49% referred to experiences in which loud barking startled the participant. in total, 10 reports (2.01%) dealt with other topics like not being able to control a dog, being startled by a dog causing an injury, or dog feces (neighbor's dog) on the porch. as expected, the individuals reporting negative experiences with dogs in the past reported a higher percentage of threatening dog dreams (effect size = 0.371; see table 7 ). interestingly, younger persons also reported a higher percentage of threatening dog dreams, as did males and persons with lower education levels (see table 7 ). similar to the percentage of dog dreams when compared to all dreams, dream recall frequency was associated with a higher percentage of threatening dogs within their dreams that included dogs. the findings indicate that, on average, dogs show up in about 5% of remembered dreams, but the percentage is much higher with dog owners or persons with close contacts with dogs. moreover, active time spent with the dog and proximity during sleep are also related to a higher percentage of dog-including dreams. although dreams including dogs are on average more positively toned than dreams in general, about 11% of the dog dreams included threatening dogs. persons who had had negative experiences with dogs in their waking life reported more threatening dog dreams. from a methodological viewpoint, it has to be noted that the present sample was self-selected and not representative, despite the large age range and varying educational levels. comparing the dream recall frequency distribution to representative samples [21] clearly indicated that high recallers are overrepresented. this is of importance, as dream recall frequency was related to the percentage of dreams that included dogs, and thus it was necessary to include this possible confounder into the regression analyses. the positive relationship between dream recall frequency and the percentage of dog-including dreams in relation to all remembered dreams could reflect a methodological issue in the retrospective approach, since schredl [24] was able to demonstrate that low dream recallers are less accurate in estimating different dream characteristics retrospectively compared to high recallers. on the other hand, one might speculate that other variables play a role; dream recall frequency, for example, is related to openness to experience [25] , and it could be hypothesized that persons with high openness might also be more curious about dogs and dream more often about them. thus, it would be interesting to study the relationship between dream recall frequency and the percentage of dreams including dogs in more detail in the future. on the other hand, there was no selection regarding dog owners, as the study was entitled "everyday life and dreams", and included other topics like work-related dreams and family-related dreams. the figure of 16.5% of participants owning a dog or living with a dog in the household is close to the representative percentage of 19% of german households with a dog [2] . in contrast to previous studies [7, 9, 20] , the percentages of dog dreams were elicited retrospectively, but previous research [26, 27] comparing content analytic data derived from diary dreams with this retrospective format of eliciting percentages of specific dream topics (in that case, sport dreams) demonstrated that the obtained figures are comparable. moreover, the study was conducted during the covid−19 lockdown in germany, i.e., sleep behavior and dream recall might have changed due to this conditions. however, previous studies, e.g., schredl et al. (2014) [21] , carried out within the same panel (wisopanel), yielded a similar distribution of dream recall frequency, that is, markedly higher dream recall on average compared to representative samples. the percentage of dreams including dogs of about 5% is in line with previous results [5] [6] [7] [8] [9] [10] [11] , especially if one considers that the 5% figure is a slight overestimation due to many high dream recallers in the sample. overall, the emotional tone of dog-including dreams was more positive when compared to the overall general tone of dreams, especially in dog owners. this shift towards positive dream emotions was also found for music dreams [28, 29] ; since music is a pleasant leisure time activity, this is in line with the continuity hypothesis. the positive emotional tone of dreams including dogs can be interpreted in a similar way. it would be very interesting to elicit explicit dream content, especially in samples of dog owners, about the variety of interactions between the dog and the dreamer. the very high percentage of dog-including dreams (about 19%) in dog owners-comparable to figures of the romantic partner showing up in dreams [30] [31] [32] [33] -clearly indicate that dogs are typically very close companions [3] . the closeness to the dog (amount of active time spent with the dog, dog sleeping in the bed) is also related to the percentage of dreams including dogs. all this is in line with the continuity hypothesis of dreaming [4] . furthermore, persons who owned dogs or lived with dogs dream more often about dogs than persons who never owned a dog, indicating that not only do current experiences with dogs affect dreams, but past experiences do too (cf. [34] ). despite the finding that dreams including dogs are on average more positively toned than dreams in general, in about 11% of the dog dreams the dog threatens the dreamer, and a similar figure has been reported by schredl [10] . the higher percentage of aggression related to animals [7, [18] [19] [20] might be related to the type of animal (i.e., dogs are not as threatening as other animals), but also may be related to age, as the previous samples were much younger (children, students) compared to the present sample and, in the present sample, younger persons reported threatening dog dreams more often. interestingly, men also reported a higher percentage of threatening dog dreams-possibly reflecting the higher percentage of physical aggression found in men's dreams when compared to women's dreams [8, 35, 36] . about 25% reported negative experiences with dogs in waking life, clearly indicating that human-directed aggression in pet dogs is an important issue [37] . these persons reported higher percentages of threatening dog dreams, indicating that a negative experience in waking life, e.g., being bitten by a dog during childhood, can have lasting effects on dream life. to summarize, the findings of the present study clearly indicate that waking life experiences with dogs (positive and negative) affect dreaming and, thus, focusing exclusively on symbolic interpretations of dogs in dreams might not be appropriate. it would be very interesting to conduct content analytic studies with dream samples obtained from dog owners to learn more about the variety of interactions between dreamers and dogs. the dog: a natural history understanding dog-human companionship continuity between waking and dreaming: a proposal for a mathematical model the scientific study of dreams: neural networks gender differences in the dream content of children and adolescents: the uk library study animal figures in fantasies and dreams the content analysis of dreams animal dreams in a long dream series your child's dreams artemidorus' oneirocritica: text, translation, and commentary die tiere im traum des menschen the enceclopedia of dreams: symbols and interpretations children's dreams: longitudinal studies nightmares, conflict and egodevelopment in childhood a study of subject matter and motivation of children dreams dreams in children of preschool age finding meaning in dreams: a quantitative approach the mannheim dream questionnaire (madre): retest reliability, age and gender effects reliability and stability of a dream recall frequency scale statistical power analysis for the behavioral sciences questionnaire and diaries as research instruments in dream research dream recall frequency, attitude toward dreams, and the big five personality factors dreams reflecting waking sport activities: a comparison of sport and psychology students relationship between waking sport activities, reading and dream content in sport and psychology students music in dreams and music in waking: an online study music in dreams: a diary study dreams of singles: effects of waking-life social contacts on dream content attachment styles and emotional content, stress, and conflict in dreams of romantic partners script-like attachment representations in dreams containing current romantic partners reminiscences of love: former romantic partners in dreams researching dreams: the fundamentals gender differences in dreams: do they reflect gender differences in waking life? gender differences in dream content: related to biological sex or sex role orientation? human-directed aggression by pet dogs: a preliminary study the authors declare no conflict of interest. key: cord-258696-01wj76es authors: decaro, nicola; campolo, marco; lorusso, alessio; desario, costantina; mari, viviana; colaianni, maria loredana; elia, gabriella; martella, vito; buonavoglia, canio title: experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia date: 2008-04-30 journal: vet microbiol doi: 10.1016/j.vetmic.2007.10.008 sha: doc_id: 258696 cord_uid: 01wj76es a pantropic canine coronavirus (ccov) strain (cb/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. we report the clinical, virological and serological findings in dogs experimentally infected with strain cb/05. the dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral rna with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. considering the severity of the cb/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8–9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. at postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for ccov by real-time rt-pcr and virus isolation on cell cultures. all pups seroconverted for ccov, as shown by the high optical density values and antibody titres detected by elisa and virusneutralisation tests, respectively. the present study confirms that strain cb/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions. coronaviruses (covs) are enveloped, singlestranded rna viruses which included in three different antigenic groups. covs infecting dogs comprise canine enteric coronavirus (ccov) (enjuanes et al., 2000) and www.elsevier.com/locate/vetmic available online at www.sciencedirect.com veterinary microbiology 128 (2008) [253] [254] [255] [256] [257] [258] [259] [260] the newly recognised canine respiratory coronavirus (crcov) (erles et al., 2003; decaro et al., 2007a) , belonging to group 1 and group 2 covs, respectively. two ccov genotypes have been identified so far, namely ccov type i and ccov type ii, which are responsible for the occurrence enteritis in dogs and are frequently associated in mixed infections decaro et al., 2005c) . although its tropism is restricted to the gastroenteric tract, ccov has been recently associated to systemic disease followed by fatal outcome in pups . severe clinical signs were observed in the affected pups, whereas necropsy examination revealed remarkable gross lesions in lungs, liver, spleen and kidneys. virological and bacteriological investigations failed to detect common canine pathogens. unexpectedly, ccov type ii rna was detected at very high titres in the internal organs of the dead pups and the virus (strain cb/05) was isolated on canine cell cultures. the association of strain cb/05 to a severe, sometimes fatal disease of dogs, together with the isolation of the virus from organs with severe lesions, strongly suggests that ccov has changed its tropism, acquiring the ability to spread from the enteric tract to the internal organs (decaro et al., 2007b) . in this study, we report the results of the experimental infection with isolate cb/05 in pups with different age, showing that in contrast with classical ccovs, this virus is able to cause systemic disease followed by fatal outcome in younger pups. canine fibroma a-72 cells were grown in dulbecco's minimum essential medium supplemented with 10% foetal calf serum. strain cb/05 was isolated from the lungs of a dead pup (117/05-c) and adapted to growth on a-72 cells. at the 3rd passage, the virus was titrated on cell cultures and inocula containing 10 6.25 tcid 50 /ml of viral suspension were stored at à70 8c. contaminations by other canine pathogens, such as canine parvovirus type 2 (cpv-2), canine distemper virus (cdv) and canine adenoviruses (cadvs), were ruled out by specific molecular assays (hu et al., 2001; decaro et al., 2005b; elia et al., 2006) . the experimental study was performed according to the animal health and well-being regulations and was authorised by the ministry of health of italy (authorization no. 53/2005-c) . six mixed-bred female dogs including four 2.5-month-old (n = 1-4) and two 6-month-old (n = 5, 6) pups were housed at the ''infectious disease unit'' of the animal hospital, faculty of veterinary medicine of bari. the dogs had tested negative for ccov rna by a real-time rt-pcr assay carried out on the faeces and for ccov antibodies by an elisa test (pratelli et al., 2002) carried out on serum samples. all dogs were housed individually in separate boxes, fed twice daily with a commercial dry dog food and provided water ad libitum. after an acclimatization period of 5 days, 5 animals (n = 1, 2, 3, 5, 6) were administered oronasally 3 ml of a viral suspension of strain cb/05, with a titre of 10 6.25 tcid 50 and 7.85 â 10 7 rna copies per ml, whereas one dog (n = 4), 2.5-monthold, was maintained uninfected by oronasal administration of 3 ml of sterile saline solution. the clinical condition of each dog was monitored daily for 21 days. a scoring system was devised taking into account rectal temperatures, total white blood cell (wbc) counts, appearance of clinical signs (vomiting, diarrhoea, depression, loss of appetite, dehydration), following the scheme adopted in a previous study (decaro et al., 2005a) and derived by nakamura et al. (2001) , with some modifications (table 1 ). due to ethical reasons, dogs whose total clinical score reached a value !15 were euthanized by intravenous administration of 10 mg/ kg of body weight of zoletil 100 (virbac s.r.l., italy) followed by 0.5 ml/kg body weight of tanax (intervet italia, italy). edta-treated blood samples were collected daily for total and differential wbc counting and for testing for ccov rnaemia by real-time rt-pcr . the presence of the viral rna in the blood was also evaluated at hours 3, 6, 9, 12 and 18 after inoculation. plasma samples were prepared daily to evaluate free viral rnaemia and weekly to determine ccovantibody titres by virus neutralisation (vn) and elisa tests (pratelli et al., 2002) . to evaluate the viral shedding in the faeces, the rectal swabs collected daily from the control dog and from the dogs inoculated with strain cb/05 were subjected to rna extraction using qiaamp 1 viral rna mini kit (qiagen s.p.a.). in addition, tissue samples from parenchymatous organs were withdrawn from the two dead pups (table 2) . rna was extracted from the wbc pellets using qiaamp 1 rna blood mini kit (qiagen s.p.a.), from the plasma samples using qiaamp 1 viral rna mini kit and from the tissue samples using qiaamp 1 rneasy mini kit. attempts to isolate the virus in a-72 cells were carried out on rectal swabs of all infected pups and on organs of the sacrificed animals as described previously (decaro et al., 2007b) . real-time rt-pcr targeting the m gene of ccov type ii (genbank accession number d13096) was carried out on the rna extracts as described elsewhere (decaro et al., 2005c) . the genotypespecific rt-pcr assays were undertaken in an i-cycler iq tm real-time detection system (bio-rad laboratories srl, milan, italy) and the data were analyzed with the appropriate sequence detector software (version 3.0). after reverse transcription, triplicates of the ccov type ii standard dilutions and rna templates were simultaneously subjected to realtime analysis. the 50 ml reaction mixture contained 25 ml of iq tm supermix (bio-rad laboratories srl), 600 nm of each primers ccovii-f (tagtgcat-taggaagaagct) and ccovii-r (agcaatttt-gaacccttc), 200 nm of probe ccovii-pb (fam-cctcttgaaggtgtgcc-tamra) and 20 ml of c-dna. the thermal profile consisted of activation of itaq dna polymerase at 95 8c for 10 min, followed by 45 cycles of denaturation at 95 8c for 15 s, annealing at 48 8c (type ii-specific assay) for 30 s and extension at 60 8c for 1 min. plasma samples from inoculated dogs were tested in parallel by virus neutralisation (vn) and elisa tests (pratelli et al., 2002) . for vn test, duplicates of serial twofold dilutions of heat-inactivated plasmas (starting from dilution 1:2) were mixed with 100 tcid 50 of the isolated strain cb/05 in 96-well microtitre plates. after preincubation at room temperature for 90 min, 20,000 a-72 cells were added to each well. the plates were read after 4 days of incubation at 37 8c. vn titres were calculated with the karber method and expressed as the highest plasma dilution that was able to neutralise the virus. for elisa, microtitre plates were coated with ccov antigen (enteric strain s-378) and, after treatment with blocking solution (0.2% gelatin in carbonate buffer [15 mm na 2 co 3 , 35 mm nahco 3 , ph 9.6]) and repeated washing, the 1:50 dilutions of the plasma samples were added to each well. then the plates were incubated for 90 min at 37 8c, washed positive and negative controls were used as described previously (pratelli et al., 2002) . neither clinical signs or viral shedding were observed in the control dog, whose leukocyte counts did not draw away from the baseline values. all the inoculated animals displayed severe clinical signs similar to those observed in dogs infected naturally, although the outcome of the disease was different on the basis of the age (fig. 1) . in fact, two out of the three youngest pups (dogs 1 and 3) had to be euthanized for ethical reasons, at days 8 and 9 postinfection (p.i.), respectively, while both the 6-month-old pups recovered from the disease, albeit very slowly. irrespective of the final outcome, i.e, euthanasia or recovery, clinical signs were remarkably similar in all inoculated animals. the 2.5-month-old dogs that underwent a fatal outcome (fig. 1a) showed fever at days 1-2 p.i., with a peak of 39.9 8c at day 2 p.i. (dog 1), and from days 1 to 6 p.i., with a peak of 40.0 8c at day 3 p.i. (dog 3). depression (days 3-8 p.i.), anorexia/dysorexia (days 3-9 p.i.), haemorrhagic diarrhoea (days 2-7 p.i.) and vomiting (days 4-5 p.i.) also occurred. leukopenia appeared at day 3 (dog 1) or 2 p.i. (dog 3), with total wbc counts remaining below 60% of the baseline values until euthanasia. acute lymphopenia was observed in both dogs, with lymphocyte numbers dropping below 60% of the initial counts from day 3 p.i. until death (mean, 16.3%; 0.9 â 10 3 lymphocytes/ ml at day 8 p.i.). postmortem examination revealed severe changes in the intestines and major organs, which were very similar to those observed in dogs infected naturally (data not shown). the 2.5-month-old dog that survived (fig. 1b ) displayed less severe symptoms, consisting of fever (up to 39.2 8c) from days 1 to 5 p.i. and at days 11-12 p.i., and mucoid diarrhoea (days 2-6 p.i.). total wbc counts dropped below the 60% of the initial counts from days 3 to 6 p.i., whereas severe lymphopenia (below 60% of baseline values) was registered from days 1 to 8 p.i., with a peak at day 5 (19%; 1.6 â 10 3 lymphocytes/ml). subsequently, the total number of peripheral blood lymphocytes started to rise again and the clinical signs subsided. a mild loss of appetite occurred from days 1 to 6 p.i. in the two 6-month-old pups (fig. 1c) , fever showed a biphasic course, with a first peak at day 2 p.i. of 39.8 and 40.1 8c in dogs 5 and 6, respectively. a transient remission was observed from days 5 to 8 p.i. (dog 5) and at day 5 p.i. (dog 6), and a second episode of pyrexia occurred from days 9 to 14 p.i. with a peak of 39.5 8c at day 10 p.i. (dog 5), and from days 6 to 8 . dogs inoculated oronasally with cb-05 strain were monitored for up to 21 days for total wbc, lymphocyte and polymorphocyte counts (top graphs). in addition, fever, viral rna shed in faeces and clinical score where determined (bottom graphs). total wbc, lymphocyte and polymorphocyte counts are presented as percentages of the cell counts determined at day 0. viral rna titres as determined by real-time rt-pcr are expressed as log copy numbers (log 10 ) per ml of template. clinical scores were calculated as shown in table 1 and are reported for each day in correspondence of the temperature curves. p.i. with a peak of 39.4 8c at day 7 p.i. (dog 6). depression was observed between days 3 and 8 p.i., whereas vomiting appeared only sporadically in the same period (days 3, 4, 8 p.i. in dog 5; days 1, 3, 8 p.i. in dog 6), with 1-4 episodes per day. both dogs displayed anorexia (days 3-7 p.i.), mucoid or fluid diarrhoea (days 3-10), and leucopenia, with wbc values dropping below 60% of the baseline from days 3 to 6 (dog 5) or 3 to 8 p.i. (dog 6). lymphocytes dropped below 60% of the initial cell counts from days 3 to 6 p.i. in dog 5 and from days 3 to 8 p.i. in dog 6 (mean, 32.2%; 1.6 â 10 3 lympholymphocytes/ml at day 3 p.i.). starting from day 10 (dog 5) or 9 p.i. (dog 6), the clinical conditions of the dogs improved progressively, with a complete recovery at days 15-16 p.i. the control (uninfected) pup (fig. 1d ) remained in a good clinical status during the entire observation period and no variations in total wbc and lymphocyte counts were observed. the uninfected dog tested constantly negative for ccov rna. all the ccov-infected dogs tested negative for other common pathogens of dogs, including ccov type i (decaro et al., 2005c) , cdv , cadvs (hu et al., 2001) and cpv-2 (decaro et al., 2005b) . the faecal shedding of the infected dogs followed the similar pattern, although higher viral rna titres were obtained from the two sacrificed animals in comparison to survivors (fig. 1) . the pups that succumbed shed virus starting between days 1 and 3 p.i. and lasting until the day of death, with a peak at day 6 (titre of 4.97 â 10 5 rna copy numbers/ml of template) or 9 (titre of 8.72 â 10 5 rna copy numbers/ ml of template). after their death, ccov type ii rna was detected in the organs at titres slightly lower than those observed in the dogs of the natural outbreak ( table 2 ). the 2.5-month-old pup that recovered shed ccov rna starting from day 1 p.i. (titre of 2.97 â 10 1 rna copy numbers/ml of template) and lasting for the entire observation period (21 days), with a peak at day 6 p.i. (3.24 â 10 4 rna copy numbers/ml of template). shedding of ccov in the faeces of the two 6-month-old dogs was observed from day 2 p.i. (mean titre, 1.40 â 10 3 rna copy numbers/ ml of template) to day 21 p.i. (last day of observation) reaching the maximal mean value of 6.79 â 10 5 rna copy numbers/ml of template at day 10 p.i. surprisingly, ccov rna was never detected in the blood of the 6-month-old pups, as well as in the euthanized animals, in whose organs remarkable viral rna titres were found. traces of ccov rna were detected only in the blood of the survived 2.5-monthold pup between days 7 and 10 p.i., with plasma viral titres ranging from 5.54 â 10 0 to 9.30 â 10 1 rna copies/ml of template. the virus was successfully isolated on cell cultures from the rectal swabs of all inoculated dogs (data not shown) and from some organs of the euthanized pups ( table 2) . all the infected animals seroconverted for ccov, whereas antibodies were not detected in the control dog (fig. 2d ). in the dogs that were euthanized the antibody titres were determined only at day 7 p.i., with vn titres of 1:8 and od values of 0.089 (geometric means, fig. 2a ). in survivors, the maximal antibody titres were reached at days 14 and 21 p.i. by vn and elisa test, respectively ( fig. 2b and c) . in a previous study, a pantropic variant of ccov was associated to a fatal disease of dogs, characterised by leucopenia, gastroenteritis and severe changes in the internal organs . the disease induced by strain cb/05 in pups of the natural outbreak was reproduced in dogs infected experimentally. all inoculated dogs were successfully infected, as shown by the occurrence of faecal shedding, seroconversion and severe clinical signs. the viral excretion was similar to those observed during enteric ccov infection (decaro et al., , 2005c , but the course of disease was more severe, as clinical signs characteristic of systemic infection were observed in the infected dogs. the pantropism of the virus was confirmed by the presence of gross lesions in the internal organs of the dead dogs, as well as by the detection of viral rna in those tissues. interestingly, ccov rna was detected also in the brain of the dead dogs. in contrast, enteric ccov has been never associated to systemic infection, although the virus has been isolated previously from some tissues (tonsils, lungs and liver) of experimentally infected pups (tennant et al., 1991) . two of the three inoculated 2.5-month-old pups had to be euthanized after few days of postinfection, whereas the other dogs (the remaining dog of the same age and the two dogs 6-month of age) recovered after a severe disease. considering that dogs infected naturally were all between 45 and 56 days of age, it could be hypothesised that the age of the infected animals plays a role in determining the fate of cb/05 infection, with a very severe clinical course in the youngest pups. moreover, in the experimental infection, the organs of the dead dogs contained lower ccov rna titres with respect to dogs infected naturally, so that virus isolation was not obtained from all pcr-positive tissues. despite the drop of the wbc counts registered in all infected dogs and the detection of the viral rna in the internal organs of the sacrificed dogs, free or cell-associated ccov rnaemia was not found at any time either in euthanized or survived dogs, with the exception of the recovered 2.5-month-old pup which showed very low rna viral titres in the plasma between days 7 and 10 p.i. thus, at this moment, the mechanisms of lymphopenia and viral spread to internal organs remained unknown. albeit strange, our findings are compatible with those obtained from cats experimentally infected with feline infectious peritonitis virus (fipv). cats succumbed to fipv display very high-viral titres in the haemolymphatic tissues (kipar et al., 2006) , in contrast with the low loads detected in the blood (de groot-mijnes et al., 2005) . in our study, cb/05-infected dogs were found to contain lower viral loads in the lymphoid tissues in comparison to fipv-infected cats, thus likely accounting for the undetected viral rnaemia in euthanized dogs. in conclusion, we have confirmed the pantropism of strain cb/05, reproducing the natural disease even in experimental conditions. further studies will contribute to better understand the epidemiological distribution and the pathogenetic mechanisms of the virus, including the possible involvement of the different lymphocyte classes. canine coronavirus highly pathogenic for dogs natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr maternally-derived antibodies in pups and protection from canine parvovirus infection a real-time pcr assay for rapid detection and quantitation of canine parvovirus type 2 dna in the feces of dogs genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs serological and molecular evidence that canine respiratory coronavirus is circulating in italy molecular characterisation of the virulent canine coronavirus cb/05 strain detection of canine distemper virus in dogs by real-time rt-pcr family coronaviridae detection of a group 2 coronavirus in dogs with canine infectious respiratory disease detection and differentiation of cav-1 and cav-2 by polymerase chain reaction natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats pathogenic potential of canine parvovirus types 2a and 2c in domestic cats prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of italy two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea canine coronavirus infection in the dog following oronasal inoculation key: cord-337977-vzue2p6p authors: ellwanger, joel henrique; chies, josé artur bogo title: the triad “dogs, conservation and zoonotic diseases” – an old and still neglected problem in brazil date: 2019-09-30 journal: perspectives in ecology and conservation doi: 10.1016/j.pecon.2019.06.003 sha: doc_id: 337977 cord_uid: vzue2p6p abstract the presence of domestic/free-ranging dogs in brazilian protected areas and native vegetation fragments is an important problem, mainly because these animals pose a threat to wild species that live in such areas. in addition, dogs constantly circulate between wildlife environments and urban regions, acting as “bridges” in spillover events. dogs are traditionally recognized as vectors of zoonoses, which are correct, but their roles as facilitating agents for the “jump” of pathogens from wild animals to humans (and vice versa) are sparsely debated. in this context, this work briefly describes the different roles of dogs in the dynamics and ecology of infectious diseases, using the brazilian scenario as a study model. the presence of domestic/free-ranging dogs in brazilian protected areas is an important problem that affects wildlife, including various endangered species (lessa et al., 2016) . also, dogs can be considered the most abundant carnivore in some areas of the brazilian atlantic forest (srbek-araujo and chiarello, 2008; paschoal et al., 2016; ribeiro et al., 2019) . dogs threaten wild animals due to predation, competition for territory, and disturbance (chasing or harassment) (young et al., 2011; silva-rodríguez and sieving, 2012; lessa et al., 2016; doherty et al., 2017) . in addition, the risk of pathogen transmission from dogs to other animals, mainly mammals, is also an important problem. dogs can act as pathogen reservoirs and can transmit leishmania spp. (leishmaniasis), leptospira interrogans (leptospirosis), toxoplasma gondii (toxoplasmosis), neospora caninum (neosporosis), dirofilaria immitis (dirofilariasis/heartworm disease), brucella canis (brucellosis), sarcoptes scabiei (scabies), echinococcus spp. (echinococcosis), rickettsia rickettsii (brazilian spotted fever), different canine viruses (e.g., distemper virus, adenovirus, coronavirus, herpesvirus, parvovirus) , rabies virus, among other pathogens, to both humans and wildlife (craig et al., 1992; fiorello et al., 2006 fiorello et al., , 2017 dantas-torres, 2007; labruna et al., 2007; pinter et al., 2008; piranda et al., 2008; yabsley et al., 2008; moraes-filho et al., 2009; brunetti et al., 2011; ogrzewalska et al., 2012; furtado et al., 2013; millán et al., 2013; parrish et al., 2015; basano et al., 2016; campos et al., 2016; curi et al., 2016; doherty et al., 2017; lessa et al., 2016; faccini-martínez et al., 2017) . importantly, dogs are amplifier hosts of different pathogens (dantas-torres, 2007; piranda et al., 2011; szabó et al., 2013) , which means that they can develop infection at sufficient levels to infect other species, such as vectors, increasing the disease transmission (kilpatrick and altizer, 2010; labruna et al., 2011; piranda et al., 2011) . recently, kossel et al. (2018) evaluated the occurrence of domestic dogs in the tijuca national park (city of rio de janeiro, southeastern brazil). this study demonstrated that dogs were widely present throughout the entire park. also, the absence of dog pups in the park area indicated that dogs were probably derived from regions around the park, suggesting a steady flow of these animals between the park and urban areas. these results, together with other fundamental studies (galetti and sazima, 2006; srbek-araujo and chiarello, 2008; lacerda et al., 2009; torres and prado, 2010; curi et al., 2016; lessa et al., 2016; paschoal et al., 2016; fernandez et al., 2017; rosa and souza, 2017; vieira et al., 2017; paschoal et al., 2018) , clearly confirm that brazilian forest areas (mainly those close to urban centers) are strongly influenced by the activity of domestic/free-ranging dogs. in a study addressing the factors involved in the invasion of dogs in forest remnants, ribeiro et al. (2019) evaluated 12 landscapes (2830 ha each) of the brazilian atlantic forest. the number of detected dogs ranged from 5 to 27 per landscape, with an estimated number of up to 46 dogs per landscape. overall, the authors found a high abundance of dogs in the studied forest. according to the same study, high human population density, density of dogs, and landscape disturbances are important factors involved in the ecological problems caused by dogs in brazil, which invade even forest remnants far from their homes (ribeiro et al., 2019) . dogs circulate in forest fragments of protected and nonprotected areas, being registered in the most different environments (doherty et al., 2017) , such as agroforests (frigeri et al., 2014; santos et al., 2017) , caatinga (bezerra et al., 2014; lessa et al., 2016) , and the amazon region (basano et al., 2016) . in brazil, 53% of native vegetation is located on private properties (soares-filho et al., 2014) . therefore, it is important to consider that the ecological problems caused by dogs are more critical in protected regions, but they also occur in wildlife environments outside these areas. furthermore, such problems are probably more frequent in non-protected areas due to the extension of these territories and greater interaction with human agglomerates. the consequences of dogs-associated disturbances are more complex and worrisome than just the direct impact of dogs on wild species living in the invaded areas. from an ecological perspective, the circulation of dogs between wildlife environments (considering protected and non-protected areas) and urban regions make the dogs facilitators of the movement of zoonotic diseases between wild animals and humans. the interaction of domestic dogs with wildlife is a driver for disease emergence/re-emergence in dogs, humans, and in native animals (lessa et al., 2016; fiorello et al., 2017) . the transmission of a particular pathogen from domestic dogs to wild animals will depend on various factors, including the susceptibility of the wild species, the pathogen circulation among dogs, and the contact of dogs with the wildlife (fiorello et al., 2006) . predation of dog pups or debilitated/sick dogs by wild animals also facilitates the transmission of pathogens from dogs to wild animals (millán et al., 2013) . importantly, the movement of pathogens can also occur in the opposite direction, from wild animals to dogs (otranto et al., 2015a,b) . as a consequence, dogs can potentially act as drivers for spillover events, the "jump of pathogens" from wild animals to other animal species, including humans. this event is also called "host jump", and describes the process by which a particular pathogen infects a new previously unaffected (and usually taxonomically distant) host species (stukenbrock and mcdonald, 2008) . humans and our most classical companion animals (cats and dogs) share more than 60 parasitic zoonoses (macpherson, 2005) , which shows that many microorganisms capable to infect dogs can also infect humans. indeed, different studies have shown that dogs are pathogen reservoirs or amplifiers hosts with an important role in the transmission risk of some zoonotic diseases to humans, evidencing that dogs can act as "bridges" for the transmission of pathogens between wild animals and human (craig et al., 1992; dantas-torres, 2007; pinter et al., 2008; salb et al., 2008; brunetti et al., 2011; piranda et al., 2011; ramírez et al., 2013; szabó et al., 2013; campos et al., 2016; fiorello et al., 2017) . as mentioned above, some pathogens detected in dogs may have the ability to infect humans as well, even considering they were originated from wild animals. however, spillover is a very complex event, with pathogens having to overcome a number of ecological, physical and molecular barriers, including reservoir distribution and density, pathogen prevalence, infection intensity, pathogen release from reservoir host, pathogen survival and spread, human exposure, structural barriers, host immune response, among others (plowright et al., 2017) . genetic factors from host species and pathogens also influence the spillover events . in the case of viruses, to consider dogs as drivers/bridges for the movement of pathogens between wild animals and humans, the pathogen in question needs to infect the hosts using generalist receptors, present in the wild species source of the pathogen, in dogs, and in humans. the cellular machinery used for viral replication also needs to be present in all three hosts (woolhouse et al., 2001; woolhouse and gowtage-sequeria, 2005; plowright et al., 2017; barclay, 2019) . although these demands require complex and seemingly unlikely events, rabies virus and bat influenza viruses can infect different animal species, being good examples of generalist pathogens (woolhouse et al., 2001; barclay, 2019; karakus et al., 2019) . in brief, host plasticity (pathogens with a diverse host range: generalist pathogens) is a fundamental aspect for the occurrence of a zoonotic spillover (cleaveland et al., 2001; woolhouse et al., 2001; woolhouse and gowtage-sequeria, 2005; elena et al., 2009; johnson et al., 2015) . although most spillover events do not result in outbreaks, they can cause important isolated cases of human infections. moreover, when the spillover happens, it creates a chance for the emergence of a new disease in humans and should be considered a concern of the public health agents and translated into prevention actions. dogs can also transfer pathogen vectors from wild animals to humans, such as ticks and fleas, carriers of various bacterial and viral pathogens that can infect both wild animals and humans (parola and raoult, 2001; labruna et al., 2007; bitam et al., 2010; chomel, 2011; szabó et al., 2013; campos et al., 2016; mansfield et al., 2017; vogel et al., 2018) . we speculate that the role of dogs as "vectors of vectors" can be a problem even harder to handle than the direct participation of dogs in spillover events. it is also important to remember that humans can transmit pathogens to other animal species, a phenomenon known fig. 1 . potential roles of dogs in the transmission of pathogens. dogs transit between wildlife environments and urban areas, interacting with wild animals and humans. dogs can transmit pathogens to wild animals (a) and to humans (b, zoonotic diseases). transmission of pathogens from wild animals to dogs (c) and the subsequent transmission of the pathogen to humans (d) is less likely because the pathogen would need to cross a number of barriers. however, considering the existence of multi-host pathogens, the role of dogs as "spillover bridge" is possible and should not be overlooked. moreover, dogs can transport pathogen vectors (e.g., ticks) from wild animals to humans, facilitating human infection with pathogens derived from wild animals. finally, the role of dogs as mediators of reverse-zoonosis (e, pathogen transmission from humans to wild animals) is little studied and should be evaluated in greater detail. see text for references. this figure was created using mind the graph illustrations (available at www.mindthegraph.com). as reverse-zoonosis or zooanthroponosis (messenger et al., 2014; nelson and vincent, 2015) . although this process is poorly studied, it can have a major impact on wildlife and the role of dogs as mediators of reverse-zoonosis should be evaluated in greater detail (messenger et al., 2014) . the various roles of dogs in the transmission of pathogens between different species were summarized in fig. 1 . looking at this scenario from a different perspective, the participation of dogs in the transmission of infectious diseases creates a valuable opportunity when it is not possible, in practice, to control the circulation of dogs in protected or vulnerable areas. in such cases, dogs can be used as sentinels for infectious disease surveillance and even the discovery of new pathogens with potential of infecting humans (labruna et al., 2007; pinter et al., 2008; campos et al., 2016; bowser and anderson, 2018; ellwanger et al., 2019) . currently, there are a number of genetic-based methods which could be employed to achieve these goals (ellwanger et al., 2017; gardy and loman, 2018) . we acknowledge that the use of sentinel dogs to quantify the risk of zoonotic diseases in humans can be difficult, although the monitoring of dog populations already in contact with wildlife environments (such as the previously mentioned animals found in the tijuca national park) could, alternatively, be used to assess trends and patterns involved in microbial epidemiology of zoonotic pathogens, and ultimately to detect emerging infectious diseases (bowser and anderson, 2018) . however, we emphasize that the use of dogs as sentinels must be considered only when it is not possible to remove these animals from protected areas. when this can be done, the use of native animal species as sentinels to study the dynamics of infectious diseases (or for surveillance purposes) is the best option. an eventual low potential for transmission of a particular pathogen can be compensated by the high number of infecteddogs generally present in urban areas (mackenstedt et al., 2015) . based on data collected in são paulo state, we can assume that the dog/human ratio in brazil is very high (alves et al., 2005) . moreover, considering all the ecological and health problems associated with the large number of dogs in brazil, some actions need to be put into practice more intensively. firstly, understanding the extent of dogs' impacts on wildlife will help to structure public policies and action plans to mitigate such impacts (young et al., 2011) . next, effective policies of dog population control need to be implemented in brazil. dog control plans must involve residents and users of areas surrounding and in the vicinity of parks, protected or vulnerable areas, park managers, professionals responsible for public health promotion and control of zoonoses, politicians, and representatives of animal protection organizations. these plans have to consider prevention, control, and eradication strategies (galetti and sazima, 2006; lessa et al., 2016) . more specifically, the following actions are required: evaluate the impacts of dogs-wildlife interactions; promote campaigns explaining to the public and dogs' owners the negative impacts of dogs on wildlife, indicating how to avoid these impacts; exclude domestic dogs from critical wildlife habitats and protected/vulnerable areas; monitor and control the circulation of dogs in the vicinity of critical and protected areas (buffer zones and transition areas); vaccination of domestic animals; sterilization of dogs; when hunting is necessary for subsistence, hunters need to be advised not to include ill dogs in hunting activities; keep dogs healthy and in hygienic conditions; remove garbage from vulnerable areas; promote actions focused on responsible dogownership (macpherson, 2005; fiorello et al., 2006; lacerda et al., 2009; young et al., 2011; furtado et al., 2013; soto and palomares, 2015; . in other words, the one health perspective must be applied, in which animal, human, and environmental factors are considered together aiming the control of infectious diseases (cunningham et al., 2017; destoumieux-garzón et al., 2018) . in conclusion, it is clear that the circulation of dogs between wildlife environments and urban areas facilitate the transmission of infectious diseases, besides damaging native species. however, the set of problems caused by the poor management of the dog populations in brazil is still a neglected issue. the contact between humans and dogs is historical (macpherson, 2005) , but needs to be carefully controlled in order to balance the issues and the benefits arising from this relationship. jhe received a doctoral scholarship from coordenaç ão de aperfeiç oamento de 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free-roaming dogs on wildlife populations key: cord-273573-a9inlk96 authors: jaeger, gry; skogmo, hege kippenes; kolbjørnsen, øyvor; larsen, hans jørgen søiland; bergsjø, bjarne; sørum, henning title: haemorrhagic pneumonia in sled dogs caused by streptococcus equi subsp. zooepidemicus one fatality and two full recoveries: a case report date: 2013-09-11 journal: acta vet scand doi: 10.1186/1751-0147-55-67 sha: doc_id: 273573 cord_uid: a9inlk96 in spite of yearly vaccination, outbreaks of canine infectious respiratory disease are periodically seen amongst domestic dogs. these infections compromise host defense mechanisms, and, when combined with other stressful events, allow opportunistic pathogens like streptococcus equi subsp. zooepidemicus to create serious disease. early recognition and treatment are tremendously important for a successful outcome in these cases. a polyvalent vaccine was given to 22 racing dogs three days after a competition, followed by two days of rest, and then the dogs were returned to regular training. coughing was noticed among the dogs four days after immunisation. three days after this outbreak one of the dogs was unusually silent and was found dead the next morning. simultaneously two other dogs developed haemorrhagic expectorate, depression and dyspnea and were brought in to the veterinary hospital. streptococcus equi subsp. zooepidemicus was isolated in pure culture from all three cases. they were treated and rehabilitated successfully, and won a sledge race three months later. this paper discusses the necropsy results, treatment regime, rehabilitation and the chronology of vaccination, stressful events and disease. contagious upper airway infections in dogs occur regularly and are most commonly caused by canine parainfluenza virus (cpiv) or bordetella bronchiseptica, amongst other agents [1] . this clinical syndrome has also been named infectious tracheobronchitis (itb), canine infectious respiratory disease (cird), "kennel cough" or "kennel croup", so named due to its occurrence in environments where many dogs live or stay close together for shorter periods of time. characteristic clinical signs include a self-limiting paroxysmal cough lasting for up to two weeks, which usually resolves without treatment. in norway, immunisation against cird is performed using live attenuated viruses, annually with cpiv and every third year against canine adenovirus type 2 (cav-2) [1] . however, in spite of vaccination, outbreaks of cird remain common. some dogs with cird will develop serious pneumonia due to an immature immune system or other causes of immunodeficiency. occasionally, bacteria such as streptococcus equi subsp. zooepidemicus can cause fatal pneumonia [2] [3] [4] [5] [6] [7] [8] . this case-report describes the first canine outbreak of haemorrhagic pneumonia in the nordic countries caused by s. equi subsp. zooepidemicus. most of the animals in the pack of athletic sled dogs showed symptoms of cird with three dogs demonstrating symptoms of severe peracute infection. one sled dog died while two were successfully treated, rehabilitated and returned to competition. to the author's knowledge, this is the first report documenting the chronology from onset of clinical signs, through convalescence to complete recovery for peracute haemorrhagic pneumonia in dogs. the vaccination regimen related to season and extreme training will also be discussed. the affected animals belonged to a pack of 26 racing sled dogs with one owner. twenty-two dogs where in active competition/training at an international level, competing annually in the norwegian, european and world championships. the dogs were a mix of pure bred german shorthaired pointer (gsp), mixed-bred alaskan husky/gsp or mixed-bred gsp/greyhounds. the dogs were mainly kept as outdoor dogs in standard heated kennels, but at times allowed indoor as family pets. in october 2008 they participated in the norwegian national championship in 4-dogs and 8-dogs classes. prior to the race all dogs were healthy with no signs of infection or disease. three days after the race the 22 competing dogs were vaccinated with duramune dappi + lc vet. fort dodge containing attenuated distemper virus (cdv), strain onderstepoort, attenuated adenovirus type 2 (cav-2), attenuated parvovirus (cpv), attenuated parainfluenza virus (cpiv), inactivated enteric coronavirus (ccov) as well as inactivated leptospira interrogans serovar canicola and serovar icterohaemorrhagiae. after vaccination the dogs rested for two days with no exercise or training. on the third day the dogs continued regular training. following training on the 4th day post-vaccine, several dogs in the kennel started to cough, and 24 hours later there were more animals in the pack with an intensive cough. all animals had normal body temperature and showed no other clinical signs. six days after the vaccine two of the dogs became anorectic and depressed. one of these dogs stayed overnight in the shelter and was found dead the next morning (case 1). the second dog was taken into the owner's family house for the night due to anorexia, intensive coughing and salivation (case 2). according to the owner this dog had been vomiting blood during the night and demonstrated a rectal body temperature of 39.7°c. after a telephone consultation with the local veterinarian the owner administered 500 mg (19 mg/kg) of oxytetracycline (oxytetral; alpharma) orally and thereafter brought the dog to the small animal hospital at norwegian school of veterinary science (nsvs) 140 km away, together with case 1 for post-mortem examination. the next day a third dog was found anorectic and depressed (case 3) and was brought immediately to the nsvs. the rest of the kennelled dogs continued to cough, though no other animals developed further clinical signs. cases 2 and 3 were more intensively trained than the other dogs on day 3 and 4 postvaccination. post-mortem examination revealed epistaxis and haemorrhagic frothy fluids in the trachea and bronchial airways on cut sections. haemorrhages were present in the thymus, epicardium, intercostally, and in the pleural space 200 ml of uncoagulated blood were present. the lungs were congested, wet, consolidated and diffusely to cavernous haemorrhagic, these changes being more severe in the left lung lobes (figure 1) . histopathology of the lungs revealed a subacute necrotising suppurative pneumonia, with haemorrhagic, often cavernous areas in the lungs and intra-lesional gram-positive cocci. a large number of macrophages with phagocytosed erythrocytes were present (figures 2 and 3) . a subacute pleuritis was also seen. streptococcus equi subsp. zooepidemicus was isolated in pure culture from the lung tissue with identification based on morphology, microscopy, lancefield grouping (streptococcal grouping kit, oxoid, basingstoke, hampshire, england) and biochemical testing. properties included βhaemolytic colonies on bovine blood agar, gram-positive, katalase negative cocci belonging to lancefield group c. glucose, lactose, and sorbitol were fermented, trehalose was not. the isolate was also tested by using api20strep (biomérieux®, lyon, france) with the same conclusion. toxicological diagnostic screening of the liver tissue showed no evidence of anticoagulant poisons. clinical data for cases 2 and 3 are present in table 1 . on presentation, the dog was depressed, dehydrated, shivering, hypersalivating with blood stained saliva, and coughed spontaneously with haemorrhagic expectorate. the neck was slightly stretched and auscultation of thorax revealed increased vesicular sounds. thoracic radiographs showed moderately increased attenuation of the ventral part of the right middle lung lobe, moderately to severely increased attenuation of the ventrocaudal part of the right caudal lung lobe as well as air bronchograms (figures 4 and 5 ). these changes are consistent with acute pneumonia. a faint soft-tissue opacity was seen in the lung fissures, interpreted as a possible low amount of free pleural fluid. the dog was treated with intravenous (iv) ringer acetate 100 ml/kg/hr for eight hours, thereafter 50 ml/kg/hr, enrofloxacin (baytril, vet. bayer; 5 mg/kg bodyweight (bw) iv once daily), ampicillin (pentrexyl, bristol-myers squibb; 35 mg/kg bw iv three times daily) and buprenorfin (temgesic, schering-plough; 0.02 mg/kg bw iv three times daily). all medications were given for four days. the dog was hospitalised in an oxygen cage. simultaneously as the treatment was initiated an expectorate sample was sent for routine bacteriological cultivation. s. equi subsp. zooepidemicus was isolated in pure culture and directly demonstrated to belong to the lancefield group c of streptococci. complete blood count with serum biochemistry analysis was normal except for a mild leucopenia ( table 2 ). the coagulation profile was normal. on the second day of hospitalisation the dog showed substantial clinical improvement and was normothermic (38.1°c), less dyspneic, less tachypneic (respiration rate (rr) = 44/min), and had reduced salivation and coughing. there was no longer blood in the saliva nor epistaxis. the result from the bacteriological investigation of the expectorate demonstrated sensitivity against penicillin, tetracyclin, cefalexin, ampicillin, amoxicillin/ clavulanate, enrofloxacin and linkomycin. the antibiotic regimen was switched to phenoxymethylpenicillin (apocillin; actavis) 660 mg per os (po) three times daily for 14 days. no diagnostic tests for respiratory viruses were performed. clinical signs gradually resolved over the next few days and the dog was sent home seven days after hospitalisation. control radiographs before departure from the clinic revealed absence of air bronchograms, though a mild to moderate increased attenuation with an interstitial pattern was still present. this animal was presented to nsvs one day after case 1. the dog had been coughing for several days and gradually worsened with reduced appetite and depression developing on the day of presentation. on physical exam there was moderate dyspnoea with abdominal respiration and increased vesicular sounds, slight neck extension, blood stained saliva as well as fever (table 1) . haematology showed moderate leucocytosis due to neutrophilia and monocytosis ( table 2 ). radiography of the thorax showed the same changes as described for case 2. the dog was hospitalised and medically treated in the same way as for case 2. clinical progression was similar to case 2, with normalisation of temperature (38.8°c), respiration rate (28 breaths/min), heart rate (100 beats/ minute) and appetite on the second day of hospitalisation. no salivation or spontaneous coughing was observed unless whilst excited after visiting the exercise figure 4 left lateral radiograph of the lung of case 2, first day of admission to hospital. the radiograph reveals marked increased attenuation of the ventral part of the right caudal lung lobe and lobus accessorius, and moderate increased attenuation of the right middle lunglobe, with air bronchograms (white arrows). pen. repeat thoracic radiographs on the seventh day of hospitalisation revealed air bronchograms in the right middle lung lobe, but reduced consolidations. the dog was sent home on phenoxymethylpenicillin (apocillin; actavis) 660 mg po three times daily for another 14 days. follow-up hospital care of both dogs included radiographs of the thorax after one, three, five and eight weeks (figures 6 and 7) together with complete blood counts (table 2) . after thoroughly scrutinising the last taken radiographs together with assessing their clinical condition they started a step-wise training program. the radiographs taken during recovery revealed a very mild interstitial attenuation of the lung lobes that had been most severely affected and mild to faint visualisation of fissure lines which was interpreted as either mild amount of free fluid or mild fibrosis. these minor findings were gradually reduced, but faint fissure lines could still be seen after five weeks for case 3 and after eight weeks for case 2 (figures 6 and 7) . the dogs were kept confined for one week after they were released from the hospital, and did run free on a large (2-3 acres) fenced yard for two weeks. case 2 was in full training eight weeks post infection and case 3 was slightly behind. early in january the following year both dogs participated in a sled race resulting in a time track record, and medal placements in various championships were achieved the following season. the dogs in this highly trained sled-dog pack competed in a sledge dog race after prolonged transportation and were additionally moderately exercised before the race. three days after the sled race the dogs were given a commercial polyvalent canine vaccine containing cdv, cav-2, cpv, cpiv, ccov and leptospira. clinical signs of respiratory disease were observed four days after vaccination. immunosuppression in dogs immunised with a similar polyvalent vaccine was reported by phillips and others [9] . they demonstrated that the absolute lymphocyte count in blood was suppressed on day five and seven following immunisation and that one group of dogs immunised with cdv and cav-2 also had decreased lymphocyte count on day three post vaccination. all the dogs produced neutralising antibodies to canine distemper virus and some parts of the natural immunity were not influenced by the vaccination. however, the immunisation with polyvalent vaccines significantly suppressed the lymphocyte response to phytohaemagglutinin, a mitogen that induce proliferation in tlymphocytes in vitro on day 5, 7 and 11 post inoculation, but returned to normal response level by day 14 following immunisation. the combination of cdv/canine adenovirus type 1 (cav-1) or cdv/cav-2 was believed to be responsible for the suppression in lymphocyte responsiveness. the duration of the suppressed lymphocyte response was at least seven days and was most pronounced on day 7 post inoculation with up to 90% suppression of mitogen-induced lymphocyte response compared to control dogs. in the present study, the use of modified live polyvalent canine vaccine combined with stress associated with training, competition and prolonged transportation may have suppressed the innate immune response to s. equi subsp. zooepidemicus, which seldom causes pathology in dogs. the possibility of a co-infection with viral pathogens cannot be excluded and may have contributed to this outbreak. outbreaks of haemorrhagic pneumonia in dogs due to s. equi subsp. zooepidemicus have been described following transportation to a race track associated with sudden change in the weather [8] , in newly arrived densely housed research dogs [4, 6, 7] and in kennelled dogs [3, [10] [11] [12] . in most of these reports the cause of the outbreak of cird is believed to be primary viral agents alone or in combination with environmental factors that induce secondary peracute pneumonia. some of these papers report the use of cdv/cav-2 vaccine prior to infection with s. equi subsp. zooepidemicus [3] or a possible dual infection with cdv [4] , though do not postulate that immunosuppression due to vaccination with modified live polyvalent canine vaccine is significant in this pathogenesis. in a study of experimental parvovirus infection in dogs, potgieter and others [13] observed that dogs vaccinated with modified live cdv and cav-1 five days before figure 6 control radiograph of the lung of case 2 eight weeks after admission to hospital. left lateral radiograph reveals a faint line (white arrow) which is interpreted to represent either mild amount of pleural fluid or mild thickening pleura. challenge with virulent canine parvovirus resulted in disease caused by canine parvovirus whereas unvaccinated dogs remained healthy. this may indicate that these animals had reduced immunity associated with recent vaccination. histopathologically, the pneumonia in case 1 was severe with the presence of inflammatory cells, leakage of blood into the lung tissue, and intralesional gram positive cocci, which were likely to be s. equi subsp. zooepidemicus (figures 2 and 3) . similar histopathological findings were also described in the paper of priestnall and others [14] , who specified that s. equi subsp. zooepidemicus is associated with acute and often fatal clinical disease in dogs. a possible source of s. equi subsp. zooepidemicus in the outbreak described in this paper was neighboring horses. however, there may have been sub-clinical carrier animals within the pack as chalker and others [3] isolated this pathogen from 9.7% of clinically healthy kennel dogs, but where the kennel had a history of endemic cird. s. equi subsp. zooepidemicus is not normally carried by dogs, but it can probably cause disease in a situation where there is high level exposure from diseased dogs or other sources in the environment, an on-going viral infection, temperature stress, transport stress, intense exercise associated with training and competition and vaccine induced immunosuppression. in a retrospective study of 393 cases of streptococcal infections in dogs, four cases (1%) were found to be caused by s. equi subsp. zooepidemicus [15] . in humans, s. equi subsp. zooepidemicus represents a potential virulent zoonotic pathogen, with infection inducing a toxic shock syndrome following contact with horses [16] [17] [18] . one report used a study population from a re-homing shelter with an endemic respiratory disease, including 39 dogs positive for s. equi subsp. zooepidemicus culture on post-mortem lung wash and 16 negative control dogs. an objective scoring system based on histopathological lung examination demonstrated that 26 (67%) of dogs with a positive culture had evidence of pneumonia caused by s. equi subsp. zooepidemicus [14] . the lungs of the dogs affected with severe pneumonia caused by s. equi subsp. zooepidemicus had significantly elevated levels of expression of the genes of pro-inflammatory cytokines indicating a strong innate immune response caused by the invading pathogen. paillot and others [19] demonstrated that three novel genes (szef, n and p) for production of superantigens in s. equi subsp. zooepidemicus are occurring in half of the isolates from cases of disease. the authors indicate that the introduction of these genes to the population of s. equi subsp. zooepidemicus may be the cause of an emerging trend of severe pneumonia caused by this pathogen. however, in the study of priestnall and others [14] there was no indication that the carriage of these superantigen encoding genes is related to the severity of the histopathological changes. the authors emphasise that further knowledge and alertness of early clinical signs will contribute to enhance the treatment success and avoid transmission of the causative strain of s. equi subsp. zooepidemicus to other animals and humans. the rate of resolution of pneumonia in humans has been defined as the resolution of radiographic abnormalities associated with the disease [20] . patients with moderately severe pneumonia have a radiological clearance rate of 70% after 1 month, and more delayed if the pneumonia is severe [21] . a multicenter cohort study that included adult patients classified as at low risk of short-term mortality concluded that full resolution of symptoms from community-acquired pneumonia (cap) may take more than 28 days [22] . this study was not specific to athletes, as they are thought to have a better physical health status compared with the general population. when to recommence training for athletes following pulmonary infection depends upon the underlying microbial aetiology and should be individualised [23] . there is no reason to believe that dog athletes should be advised differently. the long-term rehabilitation of high performance sled dogs may be complicated because of the dual challenge of strenuous exercise and low temperature. as a general rule, caution should be taken to prevent athletes from returning to competition too fast. in human figure 7 control radiograph of the lung of case 2 eight weeks after admission to hospital. ventrodorsal view reveals a mild, mild increased attenuation in the right caudal lung lobe, which was interpreted as likely mild "scar tissue" from the previous severe pneumonia. athletes, potential complications such as pneumothorax, bronchiectasis and hemoptysis has been recorded, as well as acute respiratory failure. furthermore, intense exercise too early in the rehabilitation process can increase susceptibility to viral illness and weaken the muscle performance [24] . we may speculate that the environmental stress related to the transportation, high-end competition, time of vaccination and too early exercise all contributed to the immunosuppression and consequently the susceptibility to virus-and/or bacterial infection, and where potentially more aggressive and opportunistic bacteria like s. equi subsp. zooepidemicus can progress quickly. it may also be advisable to choose a later moment for vaccination than close up to a challenging physical competition, and to avoid any strenuous exercise for at least ten to 14 days after immunisation. return to play is a hot issue within the top-level sports, but without any specific guidelines post pneumonia. these dogs were in a very good physical shape at the start of the outbreak of haemorrhagic pneumonia, and the two surviving individuals recovered relatively fast and returned to their previous performance level by setting time track records in their first race after recovery. environmental stress associated with intense exercise, competition and prolonged transportation combined with canine vaccination may suppress the innate immune response to viral infections and subsequent s. equi subsp. zooepidemicus infection that only rarely cause pathology in dogs. knowledge and alertness of early clinical signs of acute haemorrhagic pneumonia will enhance treatment success and shorten the rehabilitation period. however, caution should be taken to prevent athletes from return to the competition arena too fast. these high performance sled dogs recovered relatively fast and could prove their fitness by competing and winning international sled race championships three months later. canine infectious tracheobronchitis. in infectious diseases of the dog and cat an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs the association of streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease hemorrhagic streptococcal pneumonia in newly procured research dogs sudden deaths in greyhounds due to canine haemorrhagic pneumonia outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus streptococcus zooepidemicus as the cause of septicemia in racing greyhounds effects of vaccines on the canine immune system peracute haemorrhagic pneumonia syndrome in dogs deaths in racing greyhounds haemorrhagic streptococcal pneumonia in a dog experimental parvovirus infection in dogs characterization of pneumonia due to streptococcus equi subsp. zooepidemicus in dogs streptococcal infection in dogs: a retrospective study of 393 cases isolation of streptococcus zooepidemicus from three dogs in close contact with horses fatal case of toxic shock-like syndrome due to group c streptococcus associated with superantigen exotoxin streptococcus zooepidemicus (group c) pneumonia in a human identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep the radiographic resolution of streptococcus pneumonia pneumonia recovery: discrepancies in perspectives of the radiologist, physician and patient measuring symptomatic and functional recovery in patients with community-acquired pneumonia pulmonary infections in the athlete lower respiratory infections and potential complications in athletes submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors wish to thank field veterinarian steinar dagestad for supplement information about the kenneled dogs. we would also like to thank the staff from the norwegian school of veterinary science including radiologists bernadette helmer & lena stenhaug and all the animal nurses for their intensive loving care. a warm thank to john debenham for language consulting and valuable comments. the authors declare that they have no competing interests.authors' contributions gj did the clinical examinations, drafting and revising the manuscript. . hks evaluated the radiographs initially and the controls. øk did the postmortem examination. bb and hs did the microbiology testing. hjsl and hs made substantial contribution to the discussion in the manuscript. all authors read, revised and approved the final version of the manuscript. key: cord-293274-ysr1l557 authors: perisé-barrios, ana judith; tomeo-martín, beatriz davinia; gómez-ochoa, pablo; delgado-bonet, pablo; plaza, pedro; palau-concejo, paula; gonzález, jorge; ortiz-diez, gustavo; meléndez-lazo, antonio; gentil, michaela; garcía-castro, javier; barbero-fernández, alicia title: humoral response to sars-cov-2 by healthy and sick dogs during covid-19 pandemic in spain date: 2020-09-22 journal: biorxiv doi: 10.1101/2020.09.22.308023 sha: doc_id: 293274 cord_uid: ysr1l557 covid-19 is a zoonotic disease originated by sars-cov-2. infection of animals with sars-cov-2 are being reported during last months, and also an increase of severe lung pathologies in domestic dogs has been detected by veterinarians in spain. therefore it is necessary to describe the pathological processes in those animals that show symptoms similar to those described in humans affected by covid-19. the potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. forty animals with pulmonary pathologies were studied by chest x-ray, ultrasound study, and computed tomography. nasopharyngeal and rectal swab were analyzed to detect canine pathogens, including sars-cov-2. twenty healthy dogs living in sars-cov-2 positive households were included. immunoglobulin detection by different immunoassays was performed. our findings show that sick dogs presented severe alveolar or interstitial pattern, with pulmonary opacity, parenchymal abnormalities, and bilateral lesions. forty dogs were negative for sars-cov-2 but mycoplasma spp. was detected in 26 of 33 dogs. five healthy and one pathological dog presented igg against sars-cov-2. here we report that despite detecting dogs with igg α-sars-cov-2, we never obtained a positive rt-qpcr, not even in dogs with severe pulmonary disease; suggesting that even in the case of a canine infection transmission would be unlikely. moreover, dogs living in covid-19 positive households could have been more exposed to be infected during outbreaks. covid-19 is a zoonotic disease originated by sars-cov-2. infection of animals with sars-cov-2 are being reported during last months, and also an increase of severe lung pathologies in domestic dogs has been detected by veterinarians in spain. therefore it is necessary to describe the pathological processes in those animals that show symptoms similar to those described in humans affected by covid-19. the potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. forty animals with pulmonary pathologies were studied by chest x-ray, ultrasound study, and computed tomography. nasopharyngeal and rectal swab were analyzed to detect canine pathogens, including sars-cov-2. twenty healthy dogs living in sars-cov-2 positive households were included. immunoglobulin detection by different immunoassays was performed. our findings show that sick dogs presented severe alveolar or interstitial pattern, with pulmonary opacity, parenchymal abnormalities, and bilateral lesions. forty dogs were negative for sars-cov-2 but mycoplasma spp. was detected in 26 of 33 dogs. five healthy and one pathological dog presented igg against sars-cov-2. here we report that despite detecting dogs with igg α-sars-cov-2, we never obtained a positive rt-qpcr, not even in dogs with severe pulmonary disease; suggesting that even in the case of a canine infection transmission would be unlikely. moreover, dogs living in covid-19 positive households could have been more exposed to be infected during outbreaks. we are currently in an international health emergency generated by the emerging zoonotic coronavirus sars-cov-2 that began its expansion by the end of the year 2019 in wuhan (china) and has caused a pandemic in a few months. 1 covid-19 is a pathology with various clinical manifestations caused by sars-cov-2, and the severity of its infection is mainly associated with lung injury, with findings similar to macrophage activation syndrome that causes hyperinflammation and lung damage by an uncontrolled activation and proliferation of t lymphocytes and macrophages. 2, 3 four genera of coronavirus have been described: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (α-cov, β-cov, γ-cov, and δ-cov) according to their genetic structure. with the detection of sars-cov-2 in humans, seven coronaviruses had been isolated from people, but only two of them (sars-cov and mers-cov) predominantly infect the lower airways and cause fatal pneumonia. severe pneumonia is associated with rapid viral replication, massive inflammatory cell infiltration, and proinflammatory cytokine responses. 4 in humans, the other coronaviruses cause mild upper respiratory tract infections in immunocompetent adults and serious symptoms in children and elderly people. α-cov and β-cov infect mammals and have been also described in dogs and cats; mostly they are responsible for respiratory infections in humans and gastroenteritis in animals. in dogs, canine enteric coronavirus (ccov), an α-cov, causes an enteritis of variable severity (rarely fatal) and develop immunity; however some of the recovered dogs become carriers with the ability to infect other dogs. however, the described fecal-oral transmission pattern for ccov includes only the canine species, and is not currently postulated as a possible zoonotic agent. however, canine respiratory coronavirus (crcov), that belong to the β-cov (like sars-cov-2), cause respiratory symptoms in dogs, in general with mild clinical signs 5 and occasionally as a coinfection with other respiratory pathogens. recently, the first cases of asymptomatic dogs infected with sars-cov-2 have been described. 6 due to the zoonotic origin of sars-cov-2 and the described transmission between species, the hypothesis of the spread between animals becomes more plausible. 7 cases of infected cats, dogs, tigers, lions, minks, and ferrets have been reported during sars-cov-2 outbreaks, and all of them had close contact with infected people. 6, 8 experimental infections using different animal species report an increased susceptibility to infection by ferrets and cats, and indicate a low susceptibility to sars-cov-2 infection by golden hamsters, macaques, fruit bats, and dogs. 9 furthermore, under natural conditions, infections in more than 20 mink farms have been reported. the transmission pattern postulated is that virus was transmitted to the animals by infected people and then the virus spread between minks. 10 later, in may 2020, two workers of the mink farms that could have acquired the infection from the minks have been reported. these would be the first described transmissions of sars cov-2 from animal to human (apart the first one that originated the pandemic). 11 some data suggests that also transmission from minks to cats and dogs occurred in the farms. the world organization for animal health-oie stated that some animals can become infected by being in permanent contact with infected people, although they note that there are no evidences defining the role of infected pets in the spread of sars-cov-2. to date, no cases of transmission from domestic or captive wild animals to humans have been described (excluding, if confirmed, mink farm workers). information regarding the possibility of companion animals becoming infected is confusing and controversial. some authors describe that dogs whose owners were positive for sars-cov-2, showed serological negative to sars-cov-2, postulating that pets are not virus carriers. 12 by contrast, some cases of companion dogs have been reported that were positive by rt-qpcr detection 6 and others that have developed neutralizing antibodies against sars-cov-2. 13 currently, around ten rt-qpcr-positive dogs have been detected worldwide (hong kong, denmark, and usa, but none in spain), all of them in close contact with covid-19 positive humans. 14 less than half of them were asymptomatic, one presented mild respiratory illness, and only one present also neutralizing antibodies coursing with hemolytic anemia. 15 on the other hand, two negative-pcr dogs have developed neutralizing antibodies, one was asymptomatic and the other had breathing problems, but it is not clear if those were related to the infection. 15 further, molecular testing of 3500 dogs, cat and horse companion animals were done by idexx company in usa and korea and no positive cases were found. no positive cases were reported in dogs exposed to sars-cov-2 in france. 16 another recent study in italy carried out with pets has shown that none of the 817 animals studied was positive to sars-cov-2 by rt-qpcr test but 13 dogs and 6 cats had neutralizing antibodies. 13 in this manuscript we report that, during the months of the pandemic, an increase of aggressive lung pathologies in dogs was detected by veterinarians in spain. therefore, it is important to determine the infectious agent and a potential role of a sars-cov-2 infection. considering the information that is currently available, it is therefore necessary to better describe the pathological processes that could occur in those animals that could be infected by sars-cov-2 and also showing symptoms similar to those described in humans affected by covid-19. it is also highly relevant to determine if dogs could become infected in a home environment where close humanpet relationships occur. therefore, the potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. here, we describe the study of sick and healthy dogs regarding a potential infection with sars-cov-2. a prospective study with forty dogs presenting pneumonia was performed between april and june 2020 in spain. a clinical follow-up of all patients was performed and mortality was also recorded. twenty healthy dogs living with people affected by covid-19 were included as animals exposed to the virus. inclusion/exclusion criteria and more information are available in suppl. methods. the study was approved by the ethical committee of the faculty of health sciences, alfonso x el sabio university and all dog owners gave written informed consent. chest x-ray (cxr), thoracic radiographic and a study ultrasound were performed in sick dogs. the pattern type, distribution and intensity were analyzed. the pathological lung was recognized when the ultrasound lung rockets also called b lines were observed ( figure 1 ) or by the presence of other pulmonary ultrasound findings for consolidation (crushing, tissue, nodule sign) ( figure 1 ). computed tomographic (ct) was perform to assess the lesions distribution, classified as generalized, focal, and uni or bilateral. more information is available in suppl. methods. blood samples from either sick or healthy-exposed dogs were analyzed to determine immunoglobulins (igg) against sars-cov-2. a high-sensitive sars-cov-2 spike s1 protein elisa kit was used. values >2·5od of the negative control were considered as positives. to determine antibodies (igm and igg) against ccov, dog plasma samples were analyzed by eia assays. to determine neutralizing antibodies (igg) against canine adenovirus (cav), canine parvovirus (cpv) and canine distemper virus (cdv), plasma samples were analyzed by an elisa in solid phase. more information is available in suppl. methods. nasopharyngeal and rectal swabs were collected from sick dogs and analyzed by laboklin gmbh & co.kg using conventional pcr or real-time pcr (qpcr and rt-qpcr). all samples were tested for canine adenovirus type 2 (cav-2), bordetella bronchiseptica, cdv, canine parainfluenza virus (cpiv), canine influenza a virus (civ), canine herpesvirus-1 (canid alphaherpesvirus-1: cahv-1), and sars-cov-2 by taqman real-time pcr, and for mycoplasma spp. by conventional pcr. more information is available in suppl. methods. lungs of two dogs (ser209 and ser222) were histologically evaluated after necropsy. the macroscopic exam evaluated congestion, oedema and the lung injury pattern. lung samples were fixed in formalin 4% for 24 hours, paraffin-embedded and 3µm thick sections stained with hematoxylin-eosin. more information is available in suppl. categorical variables were presented as percentages. for continuous variables, data distribution normality was evaluated with the kolmogorov-smirnov test. continuous data were presented as mean with standard deviation (sd) or median with interquartile range (iqr). forty pathologic dogs met the inclusion criteria with the mean age of 8 years (range: 2 months to 13 years). fifteen breeds were recorded being the most common cross-breed 22·5% (9/40), yorkshire terrier 12·5% (5/40), and german shepherd 10% (4/40). there were 22 females and 18 males. the most common clinical signs were crackles on lung auscultation, followed by cough, tachypnoea, fatigue, fever, tachycardia, vomiting, and diarrhoea ( table 1 ). the radiographic findings in all analyzed dogs were consistent with mild to severe alveolar or interstitial pattern, with pulmonary opacity accentuated in the caudodorsal lung field. in 32·5% (13/40) in order to assess the overall status of dogs, and considering that the number of white blood cells is frequently altered in an infection, we consider it relevant to perform a hematologic evaluation on twenty-four pathologic patients. the count of white blood cells was out of range in 58·3% of dogs being the number of neutrophils abnormal in 75%, lymphocytes in 37·5%, and monocytes in 45·8% (table 2) . considering the altered number of immune cells observed in peripheral blood, together with the clinical course, we proceeded to evaluate possible pulmonary pathogens. in order to determine whether the observed pathologies could be related to a sars-cov-2 infection or to other pathogens, pcr analysis was performed. all forty dogs were negative for sars-cov-2 (table 3) . furthermore, thirty-three dogs were analyzed for a complete profile including the most common canine infectious agents. all of them were negative for cpiv, civ, and cahv-1. mycoplasma spp. and cdv were detected as a single agent with 57·6% (19/33) and 3% (1/33), respectively ( table 3 ). the pathologies in our patients were very aggressive and 42·5% (17/40) of dogs died of pneumonia during follow-up ( table 1) scant and disperse inflammatory cells, mainly macrophages with intracytoplasmic brown granular pigment were observed in alveolar septa. interestingly, some of these findings, mainly the scattered syncytia, are usually present in some viral infections. 17 after analyzing the possible pathogens in the diagnosed dogs and the findings in the evaluated tissues, we set out to study the immune response against some of these infectious agents in 17 dogs. further, we also decided to study 20 dogs that lived with people diagnosed with covid-19, as a group of dogs exposed to the virus but which did not present symptoms at the time of sampling. first of all, information about vaccination was gathered to determine the immune status of dogs. ten sick dogs had been vaccinated routinely as it is recommended by veterinarians but eight sick dogs did not received any vaccine (suppl. table s1). we have not detected any association between the vaccination patterns of pathological animals compared to healthy dogs. immunoglobulins g (igg) against cav, cpv, and cdv were analyzed in peripheral blood samples from these sick and healthy dogs (table 4 ). further, antibodies (igm and igg isotypes) against canine coronavirus that affects enteric tract, and also igg against sars-cov-2 were studied in both groups ( table 4 ). the number of dogs that presented igg antibodies against sars-cov-2 was higher in the group of healthy dogs (25%; 5/20), compared to the pathological ones (5·88%; 1/17). interestingly, the sick dog that presented antibodies against sars-cov-2 was negative for the detection of the virus in swabs studied by rt-qpcr, however mycoplasma spp. and cdv were detected in this patient ( table 3 ). all of the five igg α-sars-cov-2 positives healthy dogs showed the same pattern of antibodies against the other studied pathogens, being positives for igg α-cav, igg α-cpv, and igg α-cdv (table 4 ). nevertheless, two of them presented igg α-ccov while the remaining three were not protected against canine coronavirus. twelve healthy dogs presented igg α-ccov and two of them were positive for igg α-sars-cov-2 (table 4 ). seven pathological dogs presented igg α-ccov but in this group all of them were negative to α-sars-cov-2 (table 4 ). professionals and pet owners demand more information about a possible infection of their animals with sars-cov-2. a survey of us veterinarians reported that 60% of them seeing owners concerned that their pets had covid-19, 18 so many owners are restless and demand a more in-depth clinical study from their animals. pets are often in close contact with humans, and thus, it is important to determine their susceptibility to sars-cov-2 as well as the risk that infected pets are as a source of infection for humans. dogs are currently not considered to be susceptible hosts for sars-cov-2, despite few positive rt-qpcr test results in dogs were reported. 6 surveillance data from idexx laboratories, a multinational veterinary diagnostics company, showing that there were no positive results for sars-cov-2 in any of more than 1500 dog´s specimens submitted for respiratory pcr panels, according with the idea of transmission from human to pet is very rare. however, veterinarians, in spain, have observed an increase in aggressive lung pathologies in dogs in the months of the human pandemic, which did not respond to conventional antibiotic treatments. moreover, in veterinary medicine the respiratory disease is rarely lethal in the pet dog population. a mortality rate of 1·2% due to respiratory disease (only 0·3% due to pneumonia) has been reported, 19, 20 nevertheless in our study we found a mortality rate of 42·5% during follow-up without a clarified etiology. these dogs, with very aggressive lung diseases, showed a very similar appearance to those described for covid-19 pneumonia in human medicine. 21 historically, the most common pathogens associated with canine infectious respiratory disease complex have been cpiv, cav-2, bordetella bronchiseptica, streptococcus equi subsp. zooepidemicus, mycoplasma cynos, chv-1, cdv, civ, and crcov. 20 in our study, we detect eight of 33 analyzed dogs presenting classical primary respiratory pathogens, and we also detect igm for ccov in four dogs (3/17 pathological dogs and 1/20 healthy dog). in this regard, the presence of crcov is detected more frequently in dogs with mild clinical signs than in dogs with moderate or severe clinical signs, 5 therefore, we would rule out that it was the agent responsible for the severe respiratory pathologies in these three dogs. moreover, mycoplasma cynos is the only mycoplasma spp. significantly associated with pneumonia in dogs but it is still also unclear if m. cynos is a primary or secondary pathogen in dogs, because it can be cultured from the lungs of dogs, both with and without other identifiable infectious agents. 22 in a european study of dogs with canine infectious respiratory disease seroprevalence of mycoplasma spp. levels ranging from 20·7% to 61·9%, 23 but in other study with healthy dogs mycoplasma were isolated from 78% to 93% of throat swabs. 20 moreover, mycoplasma infections are usually associated with other infections. it is interesting to note that mycoplasma coinfections are very common in covid-19 human patients 24 and it also has been suggested that a co-infection or activation of latent mycoplasma infections in covid-19 disease may be important in determining a fatal disease course. 25, 26 normally, the therapy response when treating respiratory tract diseases with drugs (antibiotics, bronchodilators, anti-inflammatories, antitussives, decongestants, mucolytics, mucokinetics or expectorants) is adequate or complete, nevertheless our patients did not respond adequately to the therapeutic protocol. a major pathogen has not been detected in our patients, so at the moment the causative agent of the pathologies is unknown. further, the number of deaths was more than 30 times higher than expected without clarified etiology and curiously during the peak of the covid-19 pandemic in spain. when analyzing deceased dogs, interstitial pneumonia that usually courses with nonspecific lesions was detected, that has been described also in canine pathologies, such as canine distemper, herbicide poisoning or systemic processes (septicemia or uremia principally). 17 however, it should be noted that showed lesions are similar to described for covid-19 affected humans. 1 specially, striking lesions observed in vessels, both lymphocytic vasculitis, and the hyalinosis of the arteriolar wall. 27 however, all of them were negative in rt-qpcr tests for sars-cov-2 using nasopharyngeal and rectal samples. these results agree with a large-scale study that recently has been shown to assess sars-cov-2 infection in 817 companion animals living in northern italy and neither animals tested positive using rt-qpcr were found. 13 likewise, it should be considered that viral particles have been detected in the skin endothelium of human patients despite they were negative when tested by rt-qpcr. therefore, it would be useful analyze sars-cov-2 by ihc in necropsy samples from our patients. 28 regarding the presence of immunoglobulins against sars-cov-2 in peripheral blood of pets, in a previous study 487 dogs were tested in china. they were serological negative for anti-sars-cov-2 iggs. among them, 15 pet dog and 99 street dog sera were collected from wuhan city but it should be noted that only one pet dog living with a confirmed covid-19 human patient presented antibodies against sars-cov-2. 12 however, in the italian study, 3·4% of 188 dogs (and 3·9% of 63 cats) had measurable sars-cov-2 neutralizing antibody titers. none of these animals with neutralizing antibodies displayed respiratory symptoms at the time of sampling. interestingly, dogs from covid-19 positive households seem to be significantly more likely to test igg positive than those from covid-19 negative households. 13 finally, it has been determined that only half of the dogs artificially inoculated with sars-cov-2 seroconverted. 8 here we detected specific anti-sars-cov-2 canine immunoglobulins in one sick dog (1/17) infection; nevertheless, they were not tested and confirmed. other owners had not exhibited symptoms during the pandemic. therefore, it was decided to exclude this data from the statistical study due to a lack of reliable information. most people infected with sars-cov-2 display an antibody response between day 10 and day 21 after infection, and several studies have suggested that previous antibodies and t cells against endemic human coronavirus may provide some degree of crossprotection to sars-cov-2 infection. 29 further, it has been reported pre-existing memory cd4+ t cells that are cross-reactive with sars-cov-2 and the common human cold coronaviruses hcov-oc43, hcov-229e, hcov-nl63, or hcov-hku1. 30 in our data we did not find any correlation between ccov and sars-cov-2 igg-positive dogs, although the low number of cases makes it difficult to reach a valid conclusion. in sum, we analyzed dogs affected by severe pulmonary disease, all of them being negative for sars-cov-2 by rt-qpcr, however some of them present igg α-sars-cov-2, as well as the healthy dogs; suggesting that even in the case of a canine infection it would be little transmissible. moreover, dogs with owners positive for sars-cov-2 could have been more exposed to be infected during outbreaks. a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex infection of dogs with sars-cov-2 the proximal origin of sars-cov-2 susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 transmission and response to re-exposure of sars-cov-2 in domestic cats sars-cov2 infection in farmed mink sars-cov-2 infection in farmed minks, the netherlands serological survey of sars-cov-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals evidence of exposure to sars-cov-2 in cats and dogs from households in italy in: associate administrator u-a, united states department of agriculture absence of sars-cov-2 infection in cats and dogs in close contact with a cluster of covid-19 patients in a veterinary campus s pathology of domestic animals owner concerns that pets have covid-19 methods and mortality results of a health survey of purebred dogs in the uk aetiology of canine infectious respiratory disease complex and prevalence of its pathogens in europe point-of-care lung ultrasound in patients with covid-19 -a narrative review canine infectious respiratory disease european surveillance of emerging pathogens associated with canine infectious respiratory disease precautions are needed for covid-19 patients with coinfection of common respiratory pathogens covid-19 coronavirus: is infection along with mycoplasma or other bacteria linked to progression to a lethal outcome? characterization of microbial co-infections in the respiratory tract of hospitalized covid-19 patients the pathological autopsy of coronavirus disease 2019 (covid-2019) in china: a review sars-cov-2 endothelial infection causes covid-19 chilblains: histopathological, immunohistochemical and ultrastructural study of seven paediatric cases targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals selective and cross-reactive sars-cov-2 t cell epitopes in unexposed humans the authors would like to thank the owners of the dogs for their participation, as well as the veterinary clinics and hospitals that have collaborated in this study (veterinary hospital vetcare, veterinary hospital madrid norte, among others). the authors would like to thank the "centro de transfusión veterinario" for their donation of samples of virus not-exposed dogs. key: cord-021655-ojfm5rt3 authors: langan, jennifer n.; jankowski, gwen title: overview of african wild dog medicine date: 2018-09-28 journal: fowler's zoo and wild animal medicine current therapy, volume 9 doi: 10.1016/b978-0-323-55228-8.00077-1 sha: doc_id: 21655 cord_uid: ojfm5rt3 nan the african wild dog (lycaon pictus)-also referred to as the african hunting dog, painted dog, and cape hunting dog-is one of africa's most endangered carnivores. 1 owing to its decreasing numbers, it holds an international union for conservation of nature (iucn) red list priority status for the conservation of canid species in africa. 2 african wild dogs were formerly distributed throughout sub-saharan africa but are now mostly confined to southern africa and the southern portion of east africa (fig. 77.1 ). 3, 4 they require large ranges and live at low population densities. the population is currently estimated at 6600 animals and continues to decline as a result of ongoing habitat fragmentation, conflict with humans, and infectious disease. 1, 5 predation by lions and competition with spotted hyenas also contribute to population suppression. 6 there are approximately 600 african wild dogs in zoos, which serve to educate the public and fulfill an important role as ambassadors aiding the recovery effort for this species. the african wild dog is a member of the family canidae in the order carnivora; it likely diverged from wolves in the pleistocene period. 1 genetic studies demonstrate that it is sufficiently different from other canid species to warrant being classified into a separate genus. adults weigh 18-35 kg, with males slightly larger than females. 2, 3, 7 the average age of survival in zoos is 10.3 years 8 ; a few animals live 12-16 years. 2, 7, 9 the african wild dog's most striking characteristic is the tricolored spotted coat, for which it received its latin name, lycaon pictus, meaning "painted wolf." it has large round ears, lacks a supracaudal (tail) gland, has four digits on each limb (lacks dew claws), and the pads of the middle digits are connected by dermal webbing. reproductive anatomy is similar to that of domestic dogs in both males and females, but females have 12-14 mammae. they have very sharp, large premolars relative to their body mass, which allow them to consume 77 overview of african wild dog medicine jennifer n. langan and gwen jankowski sizable quantities of meat and bone with impressive speed. the dental formula is i3/3, c1/1, pm3/4, m3/3 = 21, of which the last mandibular molar is vestigial and generally not visualized. 1 the typical wild pack is composed of an older dominant female paired with a young dominant male and subordinates of both sexes. dominant males may be displaced as they age or lose strength. juvenile males are most likely to stay with the pack, whereas females often emigrate. following the death of the dominant female, significant social changes occur within the group, which can result in pack dispersal in the wild. 1, 7 social management of african wild dogs under human care is challenging and can have significant health impacts. it involves working across institutions to create and maintain packs that thrive socially, support a healthy population, and sustain genetic diversity. the most stable social groups include a well-established dominant pair with male offspring of any age and young female offspring. the inability to disperse may result in conflict between female offspring above 18 months of age and the dominant female. the decreasing frequency of "hoo-calls" (long-distance communication calls) and distance between resting sites of same-sex groups suggest that unrelated individuals under human care are more likely to integrate into a pack successfully. 10 if animals need to be separated due to social incompatibility, it is recommended that individuals be split up as same-sex packs or with littermates less than 18 months of age. 7 contraception for reproductively mature individuals intended to reduce aggression has not been successful. 7 measurement of fecal corticosteroids may be a useful management tool and has demonstrated that dominant animals generally have the highest stress levels. [11] [12] [13] behavior is a key indicator of social and physical wellbeing in african wild dogs. "normal" behavior varies by an individual's status within the pack, and establishing pack hierarchy is essential for avoiding excessive aggression. have access to multiple heated areas if the temperature regularly drops below 4.4°c-7.2°c (40°f-45°f) and should have shelter from the elements. 7 additionally, a heated den should be provided if breeding is planned. facilities should have sufficient holding space to accommodate separating animals for long periods. with the exception of fish, housing african wild dogs with other species is not recommended due to their high predatory drive. african wild dogs should be moved only in sturdy metal or wood crates with good ventilation that meet us department of agriculture (usda) and international air transport association (iata) requirements for live animal transport. completing a written transport plan, health evaluation, and crate training facilitates transitions when animals are relocated. african wild dogs are generalist predators, occupying a range of habitats where they hunt medium-sized antelope. 6 in natural settings, reduced prey populations and competition from other predators inhibit population growth. 3, 17 african wild dogs in zoos are fed a nutritionally complete raw meat-based diet (1-1.36 kg/adult/day) and are supplemented with small whole prey, knuckle/rib/shank bones (one to two times week), and carcasses (pig, deer, calf, horse). lactating bitches require up to three times permanent or even brief temporary removal of an established pack member may have profound social impacts, including changes in social hierarchy with aggression so substantial that reintroduction may not be possible. 7, 15 detailed plans to reduce stress and promote normal behaviors should be implemented if a dog must be isolated, including maintaining olfactory and visual contact. if separation is required, it may be helpful to subdivide the pack and then reintroduce them all simultaneously. alternatively, introductions of subordinate dogs first, then dominant pairs, may be effective. successful implementation of enrichment has included environmental devices, sensory stimulants, and food, behavioral, and habitat variance. 16 piles of leaves, dirt, and mulch allow natural digging and rolling behaviors. rotating exhibits with other predators provides habitat diversity and promotes scent-marking behavior. offering carcass feeds hung from trees or on zip lines, feeding bones, providing several types of enrichment to the pack simultaneously, and permitting breeding when possible are recommended for this species. 15, 16 enclosures should be large and contain ample space for exercise to meet the animals' physical, social, behavioral, and psychological needs. 7 specific size and perimeter recommendations may be found in the association of zoos & aquariums (aza) large canid care manual. 7 facilities that allow the public to observe the animals should prevent close contact or inadvertent access to the enclosure. dogs should ah, inc., fort worth, texas). current estimates show 23% of females have some degree of reproductive pathology, 28 the most frequently reported of which is cystic endometrial hyperplasia (ceh) with or without pyometra and adenomyosis ( fig. 77.2 ). [27] [28] [29] adenocarcinoma, uterine rupture, and pyometra without other pathology have also been reported (kinsel, personal communication, april 10, 2017). 23, 27, 28 the species survival plan program (ssp) currently recommends that all postreproductive females (>10 years) be spayed. 8 deslorelin implants have been used for contraception and behavioral alteration in males with variable results (see table 77 .1 for a summary of reproductive information) (see also chapter 22) . 26, 30, 31 healthy adult african wild dogs are not physically restrained due to safety concerns. noninvasive procedures including visual examination, hand injections, wound treatment, venipuncture, and crate training may be accomplished with operant conditioning. restraint cages are useful and provide a safe, controlled environment to facilitate intramuscular injections of anesthetics. a quiet area away from the pack promotes quick inductions and smooth recoveries. remote injection systems are recommended for immobilizing free-ranging african wild dogs or in situations when a chute is not available. anesthetic regimens selected should take into account health status, age, and environmental conditions. in cases where cardiovascular disease has been confirmed or cardiac status is unknown, alpha-2 agonists should be avoided and alternatives such as ketamine-midazolam-butorphanol with propofol or gas anesthesia (isoflurane, sevoflurane) should be considered. chemical restraint protocols used in african wild dogs may be found in table 77 .2 and have been previously published. [32] [33] [34] drug combinations at higher dosages for free-ranging african wild dogs are available in the literature. 35, 36 reversal of alpha-2 agonists and opioids with atipamezole and naloxone decreases recovery time. to avoid dysphoria, it is advised to wait at least 60 minutes postinduction before administering reversals. recovery in a crate or nest box may reduce struggles to stand during recovery. telazol (tiletamine hcl and zolazepam hcl, zoetis, parsippany, new jersey) as a sole agent or used in combination with medetomidine is a reliable option during an emergency response but often results in a prolonged recovery time. 34 vascular access, intubation, and monitoring equipment are applied as in other canids. every wild dog anesthesia should include oxygen supplementation, electrocardiography (ecg), and monitoring of pulse oximetry, heart and respiratory rate, blood pressure, and temperature. as in other canids, the jugular, cephalic, and saphenous veins are commonly used sites for venipuncture. tables 77.3 maintenance caloric intake. specific recommendations for kilocalorie requirements may be found in the aza large canid care manual. 7 feeding african wild dogs a portion of their diet while separated from the pack aids in monitoring individual animals' food consumption. packs are generally fasted from their normal meat diet 1 day per week and may be provided with bones. african wild dogs are seasonally monoestrous obligate cooperative breeders with a brief copulatory tie. 18, 19 within a pack the alpha male and female produce the majority of surviving pups annually. 7, 20, 21 most successful reproduction occurs after 2 years of age, with senescence around 8-9 years. 8, 21 subordinate females may reproduce, but offspring typically do not survive. more often, subordinate females develop pseudopregnancy and may lactate in order to help care for the pups of the dominant pair. 1 females produce an average of six to eight pups 22,23 and up to 21 pups 8 in a den after a gestation of 69-71 days. [24] [25] [26] primiparous females have higher estrogen, which is reported to result in more male offspring. 24 hand-rearing is not recommended due to the extremely aggressive and social nature of these animals. 8 in zoos, the breeding pair is separated from the pack to prevent trauma to the pups. the group is gradually introduced when the pups begin to emerge from the nest box. a birth plan detailing responses to aggression toward the pups, large litter size, and other contingencies is recommended. newborn pups weigh about 300 g, open their eyes around 2 weeks of age, and emerge from the den to start taking solid food at 3 weeks. sex determination is similar to that for other canids. pups are weaned and start to follow the pack at 11-12 weeks of age. in free-range settings, all members of the pack raise the pups; they regurgitate food while the young are in the den and relinquish kills to the pups and yearlings. 1, 3 reproductive anatomy is similar to that of other canid species. owing to social dynamics, most reproduction has been natural; however, semen has been preserved and used for artificial inseminations. 15 captive lycaon pictus generally reproduce in the fall in the northern hemisphere. 8 estrus lasts 6-9 days and includes vulvar swelling and sanguineous discharge with interest from the male. attraction from the male may be observed for 1-2 weeks prior to tying. 14, 25 males show increased testicular development, spermatorrhea, and semen production; therefore the corresponding seasonal ability to collect sperm via electroejaculation is improved. 15 the progestin-based melengestrol acetate (mga) implant, previously used in canids, has been associated with uterine pathology. 27 the aza reproduction management center (formerly the wildlife contraception center) (www.stlzoo.org/ animals/scienceresearch/reproductivemanagementcenter) recommends gonadotropin releasing hormone (gnrh) agonists such as suprelorin (deslorelin acetate) implants or lupron depot (leuprolide acetate 4.7 mg implant, virbac extinction events. [38] [39] [40] [41] there is serologic evidence of exposure to canine parvovirus, canine distemper virus, adenovirus, rabies virus, coronavirus, rotavirus, and ehrlichia canis. 21, 42, 43 outbreaks of anthrax in kruger national park occur, but infected animals commonly survive. 43 contact with domestic dogs has been reported to increase exposure to some canid pathogens, but sylvatic viral strains also pose a significant threat. 5, 44 parasitic disease and infection has rarely been described. [44] [45] [46] toxocara canis, dipylidium caninum, spirometra sp., taeniidae, and ancylostoma spp., as well as two genera of canid protozoan gastrointestinal parasites, sarcocystis and isopora, were identified in fecal samples from free-ranging animals but were not associated with clinical disease. 44, 47 standard anthelmintics at canine dosages have been successfully used to treat internal parasites. it is recommended that african wild dogs be routinely tested and maintained on prophylactic heartworm preventative in endemic areas. over the last decade, valvular dysplasia of varying severity has been increasingly recognized as a significant concern in african wild dogs in north america. sibling groups and offspring have been affected over multiple generations, and 77.4 show normal hematologic and serum chemistry reference ranges for captive african wild dogs. 9, 37 diseases rabies and distemper have contributed to mortality in african dog populations, occasionally resulting in local neoplasia has not been extensively reported in the literature but appears to play an important role in the health of captive populations. 48 apocrine gland tumors have been documented in clinical settings, presenting as single or multiple dorsal cutaneous masses that can progress to large regionally invasive tumors (fig. 77.4) (agnew, personal communication, april 25, 2017) . cases from females are overrepresented in pathology reports submitted to the african wild dog ssp (kinsel, personal communication, april 10, 2017). surgical excision is recommended, although large tumors may require other forms of treatment. tumor growth results in ulceration and necrosis and negatively affects an animal's quality of life. other common neoplasias include hemangiosarcoma, peripheral odontogenic fibroma (fibromatous epulis), adrenocortical adenoma/carcinoma, and mammary and uterine neoplasia. other notable conditions diagnosed in african wild dogs include dental disease-particularly fractured teethpancreatitis, diabetes, spina bifida, syringomyelia, keratitis, snake bites, 49 and trauma from conspecifics (kinsel, personal communication, april 10, 2017). african wild dogs mask signs of illness and may have advanced disease by the time a change in behavior or appetite is observed. suggesting that the condition has a genetic, inheritable basis, as is well documented in domestic dogs. 8 cases range from minor to severe valvular insufficiency with congestive heart failure (fig. 77.3) . mildly affected animals exhibit no clinical signs, making the condition difficult to detect. the extent of this disease is uncertain, but it is highly probable that cardiac disease is underdiagnosed. incorporating preventative health, preshipment, and quarantine examinations should be conducted to determine an animal's health. a complete physical exam-including a dental examination, complete blood count, chemistry panel, heartworm testing, urinalysis, radiographs of the thorax and abdomen, and evaluation for endo-and ectoparasites-should be completed on a routine schedule as part of a preventive medicine plan. additionally, abdominal ultrasound examination or computed tomography in females and echocardiograms for both sexes are recommended owing to disease predilection in this species. athens, georgia) provided persistent protective titers. 57 a survey in 2006 showed that most african wild dogs maintained presumably protective titers after vaccination for canine distemper and rabies for 1 year; however, few dogs maintained titers for 2-3 years. 58 there is a paucity of scientific information regarding vaccination against canine parvovirus and leptospiral infection. protocols should be developed that take into consideration local environmental disease prevalence, animal health, and risk factors. select vaccination protocols for captive african wild dogs are listed in table 77 .5 (see also chapter 79). external and internal parasites should be treated according to domestic dog guidelines. fleas, ticks, and ear tip trauma from biting flies have responded well to products containing carbaryl or pyrethrins. good hygiene, removing standing water, fly traps, and premise sprays help control fly and mosquito populations. newly acquired animals should be quarantined away from the collection for a predetermined period based on a thorough risk assessment by the supervising veterinarian and have at least two negative fecal examinations. animals should be individually identified with microchip transponders placed subcutaneously between the shoulder blades or to the left of midline over the shoulder. currently there are no universal recommendations for vaccination protocols. the safety and efficacy of vaccines in african wild dogs have historically been unsatisfactory. vaccination strategies to conserve free-ranging populations have been reported and continue to be investigated. 50 modified live canine distemper vaccinations have failed to produce protective antibody levels in some cases 51 and have induced distemper resulting in mortality in other cases. 7, 45, 52, 53 vaccine-induced distemper can be avoided by using killed vaccines, and at least one vaccine (purevax ferret distemper, merial inc., athens, georgia) has been shown to produce measurable titers after a series of three injections at 2-to 3-week intervals. 54 subunit canine distemper vaccines (cdv-iscom, erasmus mc, rotterdam, the netherlands) stimulated appropriate titer formation, but titers did not endure relative to the purevax ferret distemper vaccine. vaccine recommendations for domestic dogs have been reduced from yearly to triennially; however, it is unknown whether nondomestic canids maintain titers in a similar manner. in one study, protective titers from the purevax vaccine persisted in 39%-85% of african wild dogs for a minimum of 1 year. 55 early studies have indicated that vaccination with killed rabies vaccines may not be sufficient for protection. 5, 50, 56 however, a recent study showed that a single intramuscular vaccination of dogs older than 14 weeks with imrab 3 (merial inc., the african wild dog: behavior, ecology and conservation canids: foxes, wolves, jackals and dogs. status survey and conservation action plan. iucn/ssc canid specialist group carnivora. in grzimek's animal life encyclopedia lycaon pictus (north africa subpopulation) birth order, estrogens and sex-ratio adaptation in african wild dogs (lycaon pictus) steroid metabolism and validation of noninvasive endocrine monitoring in the african wild dog (lycaon pictus) seasonal changes in steroid hormone profiles, body weight, semen quality and the reproductive tract in captive african wild dogs (lycaon pictus) in south africa naturally occurring and melengestrol acetate-associated reproductive tract lesions in zoo canids retrospective study of mortality of captive african wild dogs (lycaon pictus) in a french zoo cystic endometrial hyperplasia and pyometra in three captive african hunting dogs (lycaon pictus) control of reproduction and sex related behaviour in exotic wild carnivores with the gnrh analogue deslorelin: preliminary observations induction of contraception in some african wild carnivores by downregulation of lh and fsh secretion using the gnrh analogue deslorelin immobilization of wild dogs (lycaon pictus) with a tiletamine hydrochloride/ zolazepam hydrochloride combination and subsequent evaluation of selected blood chemistry parameters anesthesia of captive african wild dogs (lycaon pictus) using a medetomidine-ketamineatropine combination zoo animal and wildlife immobilization and anesthesia chemical restraint and immobilization of wild canids immobilization of freeranging african wild dogs (lycaon pictus) using a ketamine/ xylazine/atropine combination lycaon pictus, no selection by gender, all ages combined, 2013 cd.html in isis physiological reference intervals for captive wildlife: a cd-rom resource african wild dogs (lycaon pictus) endangered by a canine distemper epizootic among domestic dogs near the masai mara national reserve canine distemperrelated mortality among wild dogs (lycaon pictus) in chobe national park fatal canine distemper infection in a pack of african wild dogs in the serengeti ecosystem contact with domestic dogs increases pathogen exposure in endangered african wild dogs (lycaon pictus) diet of four sympatric carnivores in save valley conservancy, zimbabwe: implications for conservation of the african wild dog (lycaon pictus) aza canid tag: large canid (canidae) care manual population analysis and breeding and transfer plan: african painted (wild) dog (lycaon pictus) aza species survival plan yellow program general zims database reference: species360, zims.species360. org behavioural cues can be used to predict the outcome of artificial pack formation in african wild dogs (lycaon pictus) monitoring stress in captive and free-ranging african wild dogs (lycaon pictus) using faecal glucocorticoid metabolites evaluating adrenal activity in african wild dogs (lycaon pictus) by fecal corticosteroid analysis social stress and dominance reproductive hormonal patterns in pregnant, pseudopregnant and acyclic captive african wild dogs (lycaon pictus) studies of male reproduction in captive african wild dogs (lycaon pictus) enrichment options for african painted dogs (lycaon pictus) order carnivora hormonal and experiential factors in the expression of social and parenteral behavior of canids natural selection of the communal rearing of pups in african wild dogs (lycaon pictus) a molecular genetic analysis of social structure, dispersal, and interpack relations of the african wild dog (lycaon pictus) serosurvey for selected viral diseases and demography of african wild dogs in tanzania conservation role of captive african wild dogs (lycaon pictus) intrapartum uterine rupture with coincidental uterine adenomyosis in an african wild dog (lycaon pictus) vaccination strategies to conserve the endangered african wild dog (lycaon pictus) serum antibody levels before and after administration of live canine distemper vaccine to the wild dog (lycaon pictus) canine distemper in african hunting dogs (lycaon pictus) -possibly vaccine induced vaccine-associated canine distemper infection in a litter of african hunting dogs (lycaon pictus) comparison of oral and intramuscular recombinant canine distemper vaccination in african wild dogs (lycaon pictus) humoral immune response of african wild dogs (lycaon pictus) to novel canine distemper vaccines in proceedings of the attempts to reintroduce african wild dogs (lycaon pictus) into etosha national park, namibia single versus double dose rabies vaccination in captive african wild dogs (lycaon pictus) immunization and antibody persistence to canine distemper and rabies vaccination in captive african wild dogs (lycaon pictus rabies in african wild dogs (lycaon pictus) in the serengeti region serologic survey for selected microbial pathogens in african wild dogs (lycaon pictus) and sympatric domestic dogs (canis familiaris) in maasai mara an investigation into the health status and diseases of wild dogs (lycaon pictus) in kruger national park a survey of internal parasites in free-ranging african wild dogs (lycaon pictus distemperlike disease and encephalitozoonosis in wild dogs (lycaon pictus) molecular detection of babesia rossi and hepatozoon sp. in african wild dogs (lycaon pictus) in south africa african wild dog (lycaon pictus) and spotted hyaena (crocuta crocuta) in the luangwa valley, zambia multilobular tumor of bone in an african wild dog (lycaon pictus) successful snakebite treatment in three juvenile african wild dogs (lycaon pictus) with polyvalent antivenom: a namibian case report this chapter is dedicated to the researchers, veterinarians, biologists, and animal care staff that have contributed to our collective knowledge, helped conserve wild populations, and have improved the care of african wild dogs around the world. we thank drs. anneke moresco and mike kinsel for their contributions to reproduction, pathology, and disease presented in this chapter and drs. sathya chinnadurai and matthew lenyo for sharing their expertise and thoughtful review. key: cord-317787-6hz8dxsi authors: jaffey, jared a.; harmon, mark; masseau, isabelle; williams, kurt j.; reinero, carol title: presumptive development of fibrotic lung disease from bordetella bronchiseptica and post-infectious bronchiolitis obliterans in a dog date: 2019-10-10 journal: front vet sci doi: 10.3389/fvets.2019.00352 sha: doc_id: 317787 cord_uid: 6hz8dxsi a 7-month-old miniature poodle acquired from a pet store developed cough and subsequently respiratory distress compatible with bordetella bronchiseptica infection. partial but incomplete resolution of clinical signs and thoracic radiographic/computed tomographic imaging lesions were noted with use of susceptibility-guided antimicrobials. additionally, a concern for an infectious nidus led to left cranial lung lobectomy at 9 months of age. histopathology predominantly revealed polypoid and constrictive bronchiolitis obliterans (i.e., small airway disease). intermittent antimicrobial administration over the next 5 years failed to blunt progressive clinical signs. at 8 years, necropsy confirmed severe airway-centered interstitial fibrosis. this pattern of fibrosis was strongly suggestive of underlying small airway disease as the trigger. in retrospect, post-infectious bronchiolitis obliterans (pibo), a syndrome in young children caused by pulmonary infections but not yet recognized in pet dogs, likely initiated a pathway of fibrosis in this dog. in dogs with risk factors for community-acquired pathogens such as bordetella bronchiseptica, pibo is a differential diagnosis with development of severe, persistent respiratory signs incompletely responsive to appropriate antimicrobials. untreated pibo may lead to airway-centered interstitial fibrosis. future study is required to determine if targeted therapy of pibo could alter the course of end-stage pulmonary fibrosis. a 7-month-old miniature poodle acquired from a pet store developed cough and subsequently respiratory distress compatible with bordetella bronchiseptica infection. partial but incomplete resolution of clinical signs and thoracic radiographic/computed tomographic imaging lesions were noted with use of susceptibility-guided antimicrobials. additionally, a concern for an infectious nidus led to left cranial lung lobectomy at 9 months of age. histopathology predominantly revealed polypoid and constrictive bronchiolitis obliterans (i.e., small airway disease). intermittent antimicrobial administration over the next 5 years failed to blunt progressive clinical signs. at 8 years, necropsy confirmed severe airway-centered interstitial fibrosis. this pattern of fibrosis was strongly suggestive of underlying small airway disease as the trigger. in retrospect, post-infectious bronchiolitis obliterans (pibo), a syndrome in young children caused by pulmonary infections but not yet recognized in pet dogs, likely initiated a pathway of fibrosis in this dog. in dogs with risk factors for community-acquired pathogens such as bordetella bronchiseptica, pibo is a differential diagnosis with development of severe, persistent respiratory signs incompletely responsive to appropriate antimicrobials. untreated pibo may lead to airway-centered interstitial fibrosis. future study is required to determine if targeted therapy of pibo could alter the course of end-stage pulmonary fibrosis. keywords: pulmonary fibrosis, canine infectious respiratory disease complex (cirdc), pulmonary hypertension, pneumonia, interstitial lung disease, pibo background post-infectious bronchiolitis obliterans (pibo), a syndrome in children most commonly caused by mycoplasma pneumonia (1, 2) and adenovirus (1) and occasionally bordetella pertussis (3, 4) is associated with chronic inflammatory and fibrotic lesions of small airways leading to chronic airflow obstruction (5) . treatment is generally supportive therapy and frequently includes glucocorticoids (4) (5) (6) . in dogs, while many contagious respiratory pathogens cause tracheobronchitis and pneumonia, bronchiolar diseases including pibo are not well recognized as spontaneous clinical syndromes. importantly, severe damage to the lung can lead to end-stage and untreatable fibrosis, with most cases in dogs not having a recognizable trigger and thus being termed "idiopathic pulmonary fibrosis." this report describes a puppy developing pibo after bordetella bronchiseptica pneumonia with histologic evidence of small airway changes strongly supporting development of pulmonary fibrosis. recognizing addressable triggers of fibrotic lung disease could have important implications for delaying progression of end-stage lung lesions. a 7-month old male intact miniature poodle presented to the university of missouri veterinary health center with acute respiratory distress. previously the primary care veterinarian treated it for cough with successive 2-week courses of amoxicillin/clavulanic acid, doxycycline, and enrofloxacin with no improvement. it had been purchased 2 months prior from a pet store and was not current on vaccines or parasiticides and canine infectious respiratory disease complex (cirdc) was a top differential. upon presentation, the owner reported the puppy had increased respiratory effort, tachypnea, weakness, and hacking cough for 2 days. physical examination findings included heart rate of 120 beats/min, respiratory rate of 60 breaths/min, and pyrexia 103.8 • f (39.9 • c). cardiothoracic auscultation revealed crackles in all lung fields, with absence of murmur or arrhythmia. a blind bronchoalveolar lavage (bal) showed numerous intra-and extracellular bacteria consistent with septic suppurative pneumonia with light growth of bordetella bronchiseptica on enrichment broth. the reason for the pure growth of bordetella bronchiseptica only on enrichment broth could not be determined by the historic medical record; possible explanations could have been a recent unrecorded dose of an antimicrobial or improper sample handling prior to submission. a respiratory polymerase chain reaction assay on an endotracheal tube swab was negative for bordetella bronchiseptica, influenza a virus, adenovirus-2, distemper, herpesvirus, parainfluenza, respiratory coronavirus, and streptococcus equi subsp. zooepidemicus. in light of these results and sensitivity testing, the dog was treated with sulfamethoxazole/trimethoprim (12.5 mg/kg po q12 h); additionally, fenbendazole (23.4 mg/kg po q24 h) for 5 days was administered. ten days later the dog re-presented with respiratory decompensation despite antimicrobial therapy guided by susceptibility testing. physical examination findings included heart rate of 100 beats/min, respiratory rate of 60 breaths/min, temperature of 100.8 • f (38.2 • c), 5% dehydration, and cyanotic oral mucous membranes. cervical tracheal palpation elicited a hacking cough. cardiothoracic auscultation revealed crackles and soft wheezes in all lung fields. pulse oximetry yielded a hemoglobin saturation of 84%, (reference range, 95-100%) while breathing room air. supplemental oxygen via intranasal prongs was provided. thoracic radiographs indicated diffuse changes consistent with pneumonia including consolidation of multiple lung lobes and atelectasis of the left cranial lobe (figure 1 ). repeat bal revealed predominantly degenerate neutrophils and numerous extracellular/intracellular bacteria. culture of bal showed heavy growth of only bordetella bronchiseptica. enrofloxacin (10 mg/kg iv q24 h) was administered based on antimicrobial susceptibility. hypoxemia and pyrexia resolved but tachypnea and cough persisted. the dog was discharged after 8 days of hospitalization. treatment included orbifloxacin (6 mg/kg po q24 h) for 4 weeks, sterile saline nebulization and coupage every 12 h for 2 weeks. the dog showed improvement but not resolution of cough, respiratory rate/effort, and energy during 4 weeks of treatment. after discontinuation of orbifloxacin clinical signs worsened. orbifloxacin administered for 2 more weeks did not lead to improvement. thoracic computed tomography (ct, single slice ct scanner, picker pq 6000, phillips medical systems, north america, bothell, wa, usa) was performed to investigate an underlying cause for chronic cough and intermittent labored respiration (figures 2a,c ,e,g). severe reduction in left lung volume was compensated by hyperinflation of the right lung, leading to medial displacement of right cranial and accessory lung lobes. several end on gas-filled structures were seen in left lung lobes. tracheal bifurcation and left principal bronchus were slightly collapsed. no additional diagnostics were performed to look for evidence of an underlying immune defect. left-sided thoracotomy and left cranial lung lobectomy was performed because of failure of multiple antimicrobials, and in case this lobe was a nidus for infection. histopathology of the left cranial lung lobe demonstrated small airway disease consisting of intraluminal plugging of bronchioles with downstream honeycombing, air trapping and subpleural fibrosis ( figure 3a) ; the upstream bronchiolar lesions strongly supported small airways disease as the cause of the fibrosis. larger airways (bronchi) were similarly narrowed with epithelial and goblet cell hyperplasia, intraluminal mucus and mucosal and submucosal inflammatory cell infiltration. special stains failed to identify bacteria/microbes. interestingly, culture of lung tissue showed heavy growth of bordetella bronchiseptica. the absence of histologic evidence of infectious bronchopneumonia or positively staining bacterial organisms would strongly argue against bordetella as a pathogen at the time of biopsy. however, as small airway disease was not recognized as a clinical syndrome in dogs at that time, the positive culture of bordetella bronchiseptica was treated with marbofloxacin (5.2 mg/kg po q24 h) for 28 days in accordance with antimicrobial susceptibility. the dog was managed solely by his primary care veterinarian with courses of antimicrobials over the next 5 years; complete resolution of respiratory signs had never been achieved. cough and eventually exercise intolerance were progressive. at 6 years of age, it returned to the university of missouri veterinary health center for evaluation of acute hemorrhagic diarrhea syndrome. inspiratory crackles were noted on examination. arterial blood gas analysis revealed hypoxemia (p a o 2 = 41 mmhg; f i o 2 = 0.21). thoracic radiographs revealed an alveolar pattern and air bronchograms, bronchial thickening and unstructured interstitial pattern caudodorsally and occasionally in the dependent lung field (figure 4) . echocardiography revealed moderate pulmonary hypertension without left-sided cardiac disease (supplementary figure 1) . continuous-wave doppler assessment of peak tricuspid regurgitation was 3.49 m/s resulting in a tricuspid regurgitation pressure gradient between the right atrium and right ventricle of 48.7 mmhg (calculated using the modified bernoulli equation, p = 4v 2 ). thoracic ct with angiography, bronchoscopy, and bal were recommended but declined. in lieu of a definitive diagnosis the dog was treated with sildenafil (1.3 mg/kg po q12 h) and enrofloxacin (5.8 mg/kg po q 12 h) for pulmonary hypertension and possible bronchopneumonia, respectively. the dog clinically improved and was discharged 4 days later. the dog was treated with sildenafil indefinitely. approximately 2 years later the dog returned for evaluation of respiratory distress. in the intervening 2 years the dog's cough and exercise intolerance worsened substantially. pertinent physical examination findings included heart and respiratory rates of 180 beats and 98 breaths per min, cyanotic mucous membranes and diffuse inspiratory crackles. the dog was treated with 40% inhalational oxygen, furosemide (2 mg/kg iv once), pimobendan (0.32 mg/kg po once), and butorphanol (0.1 mg/kg iv once). the following day, the dog was breathing at 108 breaths/min in an orthopneic posture. because of progressively worsening respiratory distress and grave prognosis, euthanasia was elected. immediately before euthanasia (performed while the dog was under general anesthesia), thoracic ct (64-detector row toshiba aquilion, toshiba america medical systems, tustin, ca) was performed with owner consent. the dog was intubated and mechanically ventilated (engstrom carestation ventilator, ge healthcare). ventilator settings included inspired oxygen concentration of 40%, tidal volume 10 mls/kg, respiratory rate 10 breaths/min, and positive end-expiratory pressure (peep) 5 cm h 2 o. inspiratory and expiratory breath holds were ventilatorassisted and performed in tandem with ct scans; peep was set to 0 cm h 2 o for the expiratory breath hold. contiguous 2 mm transverse images were obtained from the level of c6 to l2 with the dog in sternal recumbency. the left caudal parenchyma was absent and replaced by small to large air-filled structures representing honeycombing. in contrast to the prior ct, the right lung now demonstrated patchy ground-glass opacities to consolidation. the latter was mostly distributed surrounding large to medium caliber airways, the majority of which were dilated, and accompanied by architectural distortion and traction bronchiectasis (figures 2b,d,f,h) . following euthanasia, lungs were removed with owner consent and fixed for histopathology. to prevent collapse and disruption of lung structures, each lobe was inflation-fixed with 10% formalin. histopathologic examination of left cranial, right caudal and accessory lung lobes was performed. in left cranial and right caudal lung lobes there was marked multifocal chronic polypoid and constrictive bronchiolitis obliterans with multifocal figure 2 | transverse (3 mm-thick) computed tomographic (ct) images of a miniature poodle obtained at 9 months (a,c,e,g; using a single slice ct) and 8 years (b,d,f,h; using a 64-detector row ct) of age displayed from the same level side by side. on the first study, the left cranial lung lobe (single opened arrow) is completely collapsed with almost complete absence of air in the airways. the caudal branch of the left cranial lobar bronchus is narrowed but filled with air (ventral to opened arrow on c). the left caudal lung lobe is also severely reduced in volume and thus, increased in attenuation with several air-filled end on airways (double arrows). the right lung is hyperinflated with the right cranial (rcr) and accessory (racc) lobes extending to the left of midline. mosaic pattern characterized by a well circumscribed hypoattenuating area is seen in the accessory lobe (arrowhead). the parenchyma is otherwise relatively normal. by 8 years of age and after undergoing left cranial lobectomy as a puppy, the entire hyperinflated right lung has developed severe patchy parenchymal lesions consisting of ground-glass opacification (black arrows) and consolidation (*). this latter is most severe surrounding medium-to large-caliber dilated airways. traction bronchiectasis (black arrowheads) in areas of architectural distortion is seen at the periphery of several of these airways, a common feature seen with pulmonary fibrosis. the mosaic pattern previously recognized in the accessory lobe (white arrowheads) is accentuated. several cystic air-filled structures (white arrows) of varying size occupy the left caudal lobe. frontiers in veterinary science | www.frontiersin.org left lateral projection showing several areas of increased opacity in the cranioventral, caudoventral and caudodorsal lung field (arrows). in the caudodorsal lung field, a large bronchus (arrowhead) is dilated and does not taper as it extends toward the periphery indicating bronchiectasis. in the more severe opacified areas, small air bronchograms are seen (white arrow). (b) right lateral projection revealing radiographic lesions (black and white arrows) similar to those on the left and most prominent in the caudodorsal lung field. they are less extensive in the ventral aspect of the thorax in comparison to the left lateral view. note that some lesions maybe less distinct due to motion artifact. (c) as expected following left cranial lobectomy, leftward mediastinal shift remains visible (black arrow) on the ventrodorsal projection. the borders of the left caudal lobe appear retracted (arrowhead) and the lung lobe is increased in opacity. lesions are most severe centrally and less extensive toward the periphery. similarly, radiographic opacity is increased (white arrows) surrounding major lobar structures (vessels and bronchus) of the right lung and gradually diminish in periphery of the lung. alveolar interstitial fibrosis (figure 3b ). this pattern of fibrosis radiating from and encompassing the diseased small airways is called airway-centered interstitial fibrosis and highlights bronchiolar pathology as the contributor to fibrosis (figure 3c) . pulmonary arterial medial hypertrophy and intimal hyperplasia were noted. the accessory lung lobe had severe bronchiolar ectasia with collapse of surrounding alveolar parenchyma and severe neutrophilic and macrophagic inflammation. there was no evidence of infection including with special stains. sustained damage to terminal and respiratory bronchioles postulated secondary to bordetella bronchiseptica possibly in combination with additional unidentified cirdc pathogens (e.g., adenovirus type 2 and/or mycoplasma spp.) early in life, led to biopsy-confirmed polyploid and constrictive bronchiolitis obliterans in an 9 month-old puppy. post-infectious bronchiolitis obliterans, a syndrome in children that can be caused by adenovirus, mycoplasma pneumonia, and less commonly bordetella pertussis, is treated after resolution of infection with supportive therapies and corticosteroids to target inflammation (1-3). as pibo is not a recognized syndrome in dogs, this dog failed to receive corticosteroid therapy, likely culminating in end-stage pulmonary fibrosis inclusive and distal to affected airways. airway-centered interstitial fibrosis in people is one of three distinct patterns of lung fibrosis and emphasizes the site of initial injury is the small airways (8) . histologic examination as a puppy and as an 8-year-old dog showed similar lesions of bronchiolitis obliterans and downstream fibrosis, with progressive, severe and more widespread lesions (especially fibrosis) from the latter time point. with awareness of pibo in dogs, an early trigger of pulmonary fibrosis could have potential for targeted therapy. direct treatment of a trigger causing eventual fibrosis represents a paradigm shift from the current strategy of attempting to treat fibrosis when it is end stage. bordetella bronchiseptica is the most common bacterial pathogen in cirdc, an umbrella term encompassing multiple bacterial and viral pathogens leading to acute respiratory tract infection (9) . most cases are self-limiting and confined to the trachea and bronchi, but when there is extension to the terminal bronchioles and alveoli, bronchopneumonia results (10) . bordetella bronchiseptica is the most common cause of community-acquired pneumonia in puppies (11) . although chronic respiratory disease is well documented in children having viral infections in infancy (12, 13) , chronic manifestations of cirdc are poorly described. chronic bronchitis (14) and bronchiectasis (15, 16) may be sequela. co-infections with cirdc are common. one recent study identified more than 1 respiratory pathogen in approximately 50% of dogs that were bordetella bronchiseptica-positive using real-time pcr on nasal and pharyngeal swab samples (17) . therefore, it is possible the dog in this report was infected with ≥1 respiratory pathogen including adenovirus type 2 and/or mycoplasma spp. while adenovirus type 2 was included in the initial respiratory pcr panel it is possible it was not identified because the sample was obtained from the endotracheal tube and not from balf or sampling was performed after the peak shedding (7-10 days) after the initial infection (18) . in addition, mycoplasma spp., was not included in the respiratory pathogen pcr assay nor was a culture specific for this pathogen pursued. the clinical syndrome bronchiolitis obliterans (bo; also known as obliterative bronchiolitis or constrictive bronchiolitis) targets small airways, defined as having internal diameters of <2 mm and lacking cartilage within their walls causing a spectrum of clinical signs (19) . while somewhat confusing, it is important to understand that "bronchiolitis obliterans" can be a clinical syndrome or a histologic descriptor. the former has not been characterized in pet dogs as it has been in humans and cats (19, 20) while the latter is fairly common. in humans, bronchiolar diseases can be classified as primary or secondary: in the former, pathology is anatomically limited to small airways and in the latter, disease extends either from large airways or from lung interstitium to small airways (19) . although a similar classification scheme has been adapted to cats, distinction between primary bronchiolar disease leading to pulmonary fibrosis and interstitial lung disease culminating in fibrosis with secondary involvement of small airways may be difficult to distinguish (20) . the dog in this case report with persistent and progressive clinical signs and paired ct imaging/histopathologic lung examination 7 years apart, provided support that pibo triggered pulmonary fibrosis. injuries linked to bo in people include respiratory infections, chronic gastroesophageal reflux, allergic reactions, collagen vascular diseases, organ transplantation and chronic exposure to air pollutants or toxic fumes (5, 21) . experimental canine bo can be induced by respiratory infections [adenovirus (22, 23) , mycoplasma (24) and co-infection of parainfluenza and bordetella bronchiseptica (25, 26) ], toxins (27) , and allogeneic hematopoietic cell transplantation (28) . chronic small airway obstruction from inflammation and fibrosis leads to cough, wheeze and dyspnea in people (21, 29) . disease may extend to alveoli and interstitium (30) . chronic bo can lead to pulmonary fibrosis, respiratory failure, and death in people (6, 31, 32) . a 1988 study showed pediatric diagnosis of bo was made postmortem in 37% of cases (31) ; however, by the following decade with improved socioeconomics, increased recognition, and use of thoracic high resolution ct scans, incidence dramatically declined (4, 32) . whether a similar pattern could occur in dogs given a high index of suspicion of bo remains to be seen. post-infectious bronchiolitis obliterans occurs in young children after severe pulmonary infections with measles, influenza, bordetella pertussis, mycoplasma pneumonia and adenovirus (5, 31, 32) . when cough and wheeze fail to resolve after acute respiratory infection, pibo is a strong consideration (30) . supportive criteria for pibo in lieu of lung biopsy includes history of infectious pneumonia within the first 3 years of life in otherwise healthy children; physical exam or lung function tests supporting airway obstruction; thoracic radiographic and especially thoracic ct compatible features, and exclusion of other chronic lung diseases (4, 33, 34) . the dog in our report had severe bordetella bronchiseptica pneumonia as a puppy, with persistent clinical signs despite appropriate antimicrobials. histopathology confirmed bo, at the time an unrecognized clinical condition, approximately 2 months after initial diagnosis of bronchopneumonia. honeycombing and subpleural fibrosis downstream of small airway lesions were present at the time of the initial biopsy and suggest a cause and effect relationship. results of thoracic radiography may be normal and are non-specific in bronchiolar diseases (5, 35) . thoracic radiography in children with pibo classically demonstrate hyperinflation/hyperlucency, atelectasis, patchy consolidation, airway wall thickening and bronchiectasis (4, 6) . as a puppy, radiographic changes reflected some changes reported in children. thoracic ct is more sensitive than radiography and airflow limitation can be captured with paired inspiratory/expiratory scans (36, 37) . common ct findings in children with pibo include a mosaic pattern, bronchiectasis, peribronchial thickening, atelectasis and/or consolidation (4, 36, 38) . the classic finding diagnostic for air trapping is accentuation of mosaic attenuation on expiratory series which can be missed on inspiratory series alone (36, 37) . the initial ct performed as a puppy occurred prior to a protocol for ventilator-acquired inspiratory and expiratory breath-holds at our facility and thus less well reflected air trapping. prior to euthanasia, ct lesions were dominated by fibrosis although patchy regions of air trapping were noted. histopathology of lung tissue approximately 7 years after initial lung biopsy showed marked multifocal chronic constrictive and obliterative bo with multifocal interstitial fibrosis. therefore, it is reasonable to suspect cirdc endured early in life contributed to chronic bo (i.e., pibo) and eventual interstitial fibrosis as an end-stage sequela in the dog reported here. however, we cannot rule out the possibility that the dog developed bo and interstitial fibrosis for reasons not yet explored in companion animals. empirical therapy of pibo in people includes glucocorticoids, bronchodilators, oxygen supplementation, mechanical ventilation or antimicrobials (4, 29) . surgical excision of bronchiectasis or segmental atelectic lung are performed if conservative therapy fails to achieve clinical improvement (4). interestingly, our dog clinically improved after removal of the atelectic left cranial lung lobe as a puppy. the dog reported here was treated with numerous courses of various different antimicrobials by the primary care veterinarian over the course of 5 years without clinical improvement. therapy over this time was not guided by confirmation of pathogenic organisms from airway sampling (e.g., balf cytology, culture, or pcr). the lack of clinical improvement with multiple types and courses of antimicrobials makes repeated bacterial infections unlikely, but not impossible. therefore, chronic-intermittent respiratory bacterial infections could not be ruled out as contributory to the development of fibrotic lung disease in this dog. pulmonary fibrosis (fibrotic interstitial lung disease), represents a heterogeneous group of syndromes originating from diverse injuries culminating in scar tissue and an endstage lung (39) . fibrosis attempts to repair tissue and protect against subsequent injury by increasing tissue strength; however, repetitive injury may lead to dysregulated fibrosis that is maladaptive and detrimental (40) . in dogs, fibrosis may originate secondary to genetic contributors (e.g., west highland white terrier) or drugs/toxins, with most cases failing to have an identified trigger (39) . the dog in this report strongly suggests pibo can progress to widespread airway-centered interstitial fibrosis. it is unknown if targeted therapy (e.g., glucocorticoids) could have prevented or slowed fibrosis by diminishing inflammation leading to aberrant repair. in dogs, pibo should be a differential diagnosis for otherwise healthy young dogs developing cirdc pneumonia and respiratory signs persisting despite appropriate antimicrobials and with compatible thoracic imaging or histologic changes. all datasets generated for this study are included in the manuscript/supplementary file. jj, cr, mh, im, and kw contributed to writing of the manuscript and literature review. kw contributed to the histopathological interpretation and description. im and cr contributed to interpretation and description of the imaging findings. mh was responsible for interpretation of echocardiography imaging, interpretation, and description. all authors contributed to the final review of the manuscript. post-infectious bronchiolitis obliterans in children: a review of 42 cases post-infectious bronchiolitis obliterans: clinical, radiological and pulmonary function sequelae macleods's) syndrome following pertussis infection in an infant post infectious bronchiolitis obliterans in children postinfectious bronchiolitis obliterans in children: clinical and radiological profile and prognostic factors clinical features of postinfectious bronchiolitis obliterans in children undergoing long-term nebulization treatment fluoroscopic and radiographic assessment of tracheal height variations between inspiration and expiration in apparently healthy adult small breed dogs update on pulmonary fibrosis: not all fibrosis is created equally etiologic study of upper respiratory infections of household dogs kennedy and palmer's pathology of domestic animals community-acquired infectious pneumonia in puppies: 65 cases chronic pulmonary sequelae of adenovirus infection respiratory disease in young adults: influence of early childhood lower respiratory tract illness, social class, air pollution, and smoking chronic respiratory disease in the dog demographic, clinical, and radiographic features of bronchiectasis in dogs: 316 cases bronchoscopy, imaging, and concurrent diseases in dogs with bronchiectasis detection of respiratory viruses and bordetella bronchiceptica in dogs with acute respiratory tract infections canine respiratory viruses bronchiolar disorders perspectives in veterinary medicine: description and classification of bronchiolar disorders in cats obliterative bronchiolitis changes in lung mechanics and reactivity with age after viral bronchiolitis in beagle puppies bronchiolitis obliterans and pneumonia induced in young dogs by experimental adenovirus infection spontaneous bronchopneumonia in laboratory dogs infected with untyped mycoplasma spp bronchoalveolar lavage fluid cytology reflects airway inflammation in beagle puppies with acute bronchiolitis canine parainfluenza type 2 bronchiolitis increases histamine responsiveness in beagle puppies pulmonary function tests in the detection of small airway obstruction in a canine model of bronchiolitis obliterans a canine model of chronic graft-versus-host disease clinical features of post-infectious bronchiolitis obliterans in children undergoing longterm azithromycin treatment bronchiolitis obliterans in children: clinical profile and diagnosis obliterative bronchiolitis in children bronchiolitis obliterans in the 1990s in korea and the united states postinfectious bronchiolitis obliterans in children: clinical and pulmonary function findings clinical prediction rule to diagnose post-infectious bronchiolitis obliterans in children imaging of small airways and emphysema postinfectious bronchiolitis obliterans: can ct scan findings at early age anticipate lung function? characterization of severe small airway disease in a puppy using computed tomography oleic acid-associated bronchiolitis obliterans-organizing pneumonia in beagle dogs interstitial lung diseases in dogs and cats part i: the idiopathic interstitial pneumonias diagnostic approach to advanced fibrotic interstitial lung disease: bringing together clinical, radiologic, and histologic clues the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2019.00352/full#supplementary-material key: cord-288202-r3r2bc7v authors: morel, noelia; lassabe, gabriel; elola, susana; bondad, mauricio; herrera, silvia; marí, carlos; last, jerold a.; jensen, oscar; gonzalez-sapienza, gualberto title: a monoclonal antibody-based copro-elisa kit for canine echinococcosis to support the paho effort for hydatid disease control in south america date: 2013-01-10 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001967 sha: doc_id: 288202 cord_uid: r3r2bc7v cystic echinococcosis is still a major concern in south america. while some regions show advances in the control of the disease, others have among the highest incidence in the world. to reverse this situation the pan american health organization (paho) has launched a regional project on cystic echinococcosis control and surveillance. an early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. under this premise, we have developed a new copro-elisa test after extensive screening of a large panel of monoclonal antibodies (mabs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. the key component of the test, mabeg9 has a convenient igg isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. the test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. this characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion. cystic echinococcosis, caused by infections with echinococcus granulosus, is one of the main zoonoses related to dogs and is affecting most parts of the globe [1] . different control programs aimed to decrease the impact of the disease have been instrumented in many countries. although the rate of success has been highly variable, it has become evident that a tight control of dog infections is the key element to arrest the life cycle of the parasite. this has created a demand for reliable diagnostic tests for canine echinococcosis as a tool to obtain epidemiological baseline information and help in the surveillance of hydatid control [2] [3] [4] [5] . however, accurate diagnosis of dog infection is complex and challenging, and other than careful necropsy of dogs, there is no perfect gold standard [6] . parasitological examination of eggs is unsafe and not very useful because e. granulosus are morphologically difficult to distinguish from other taeniae eggs, and appear late (after the first month) of infection. traditionally, screening of dogs for e. granulosus has been done by arecoline purgation, followed by examination of the purge for parasites by trained personnel. the method is highly specific, but it is tedious, biohazardous, unpopular among dog owners, and its sensitivity is modest, particularly when the parasite burden is low and bowel evacuation is incomplete [7, 8] . as an alternative, different laboratory tests for ante-mortem diagnosis of canine echinococcosis have been developed, including detection of antibodies in serum, polymerase chain reaction (pcr) amplification of parasite dna and immunological detection of antigens (coproantigens) in fecal samples. different studies have been carried out to explore the potential use of dog serology to diagnose the disease. however, the systemic immune response to the parasite is poor and consequently the sensitivity attained is also low [4] . parasite dna excreted with eggs, proglottids or cells has been detected in fecal specimens by pcr amplification using specific primers derived from mitochondrial dna [9, 10] . the method has provided the high specificity of pcr, but due to the presence of inhibitors the dna extraction procedure is cumbersome and the technique requires expensive reagents and specialized laboratories [11] . in addition, the pre-patent period of the infection, when there is no egg production, is a critical time-window that challenges the sensitivity of the test. introduced at the beginning of the 1990's, the detection of parasite antigens in fecal samples by immunoassays became a widely accepted diagnostic test [12, 13] . the major attractive features of the method include its simplicity, the possibility of detecting parasite components even in the prepatent period, and the fact that the target antigens (coproantigens) are highly stable. samples can be collected in 1% of 40% formaldehyde and kept at room temperature for extended periods of time, facilitating the logistics of large-scale studies in remote areas [7, [14] [15] [16] . the study of a large group of infected dogs necropsied at the end of the pre-patent period demonstrated a superior sensitivity for the copro-elisa (83%), when compared to copro-pcr (26%) or arecoline purgation (43 and 77% after one or two doses, respectively). most failures to detect positive infected dogs occurred when the number of worms was less than one hundred [5] . despite its proven utility and the numerous reports of copro-elisa developments, the availability of commercial or homemade tests is often a major obstacle to the implementation and evaluation of echinococcosis control programs. the need to overcome this difficulty has been recognized as an early goal of the southern cone sub-regional project on cystic echinococcosis control and surveillance: argentina, brazil, chile and uruguay, and paho (pan american health organization), a joint and collaborative effort to battle the disease in the region. in this framework, we have developed an immunoassay for e. granulosus copro-antigen detection under the premise that in addition to performing with high standards of proven sensitivity and specificity, it had to be robust, standardized and developed in a kit format to be available for its use in regional programs for the control of the disease. all activities involving animals were performed with special care to establish high standards of biosafety and assure animal welfare. all protocols were performed according to the international guiding principles for biomedical research involving animals, (cioms) and were approved by the comisión nacional de zoonosis and the research department of the ministry of health of the province of chubut. nineteen adult dogs, of mixed breeds and sex, were vaccinated against distemper encephalomyelitis, parvovirosis, rabies, leptospirosis, hepatitis and coronavirus (merial, france), treated orally with praziquantel 5 mg/kg, pyrantel embonate 15 mg/kg and febantel 15 mg/kg (disper, uruguay) to eliminate worm parasites, and topically with a solution of fipronil and methoprene (merial) to arrest the development of ectoparasites. dogs were purchase from local suppliers and maintained on commercial dog food and water ad libitum. nineteen fecal samples (n1-n19), corresponding to non-infected dogs, were collected two days after deworming. then, ten dogs (identified as p1-p10) were infected orally with 5,000-150,000 viable (.80%) protoscoleces from ovine fertile hydatid cysts. two additional dogs (identified as th1 and th2) were infected orally with 1 and 3 larvae of taenia hydatigena, respectively. parasite material was obtained from sheep slaughter in local abattoirs in paysandú, uruguay. stool samples from each dog were collected daily during the experimental period, at the end which (27-30 days post-infection (dpi)), the animals infected with e. granulosus were euthanized for necropsy diagnosis by intramuscular injection of acepromazine maleate (0.11 mg/kg) and ketamine (20 mg/kg) (laboratorio holliday, argentina), following by an intravenous overdose of sodium thiopental (laboratorio crusur vet, uruguay). the dogs infected with t. hydatigena were purged at 70 dpi with arecoline bromhydrate (crescent chemical co. inc., new york, usa), following by treatment with anti-parasitic drugs. three additional samples from dogs experimentally infected with e. granulosus (26 dpi) and two with t. hydatigena (about 55 dpi) from previous studies [14, 16] were obtained as a kind gift from dr. carlos carmona. in addition, 85 unwanted dogs from the province of chubut, argentina, and 11 unwanted dogs from junin, peru, were euthanized following the procedure described above, and the small intestines were removed post mortem, opened longitudinally and examined directly for the presence of parasites following the recommendations of the who/oie [17] . all activities were performed following the local legislation and the international guiding principles for biomedical research involving animals (http://www.cioms.ch/). stool samples from each dog were collected daily during the experimental infection period, or from the rectum upon necropsy, in 1% of 40% formaldehyde, phosphate buffered saline (pbs), in a 1:4 v/v ratio. samples were vigorously shaken to obtain homogeneous slurries, and were then boiled for 20 min in a water bath, to eliminate their biological risks. after centrifugation for 10 min at 2,2006 g the supernatants were aliquoted and kept frozen at 220uc until used. the parasite antigens were prepared essentially as described by casaravilla et al. [16] . cystic echinococcosis, caused by infection with the larval stage of the echinococcus granulosus tapeworm, is a lifethreatening zoonosis of worldwide distribution. the adult worm parasitizes the small intestine of dogs, which become infected after eating offal of an animal contaminated with the parasite, and releases eggs into the environment that can be accidentally ingested by domestic animals or humans, maintaining the life cycle of the parasite. deworming of dogs is a major component of control programs, and simple and reliable methods are needed to monitor the base-line infection in the canine population. the lack of these tests was recognized as a major obstacle to the paho effort to control the disease in south america. this paper describes the development of a diagnostic assay that detects parasite antigens in dog feces. the key component is a monoclonal antibody carefully selected to attain high levels of sensitivity and specificity, which were established with a large panel of field fecal samples obtained from animals diagnosed by necropsy. several aspects of the long-term stability of the test were optimized to facilitate its shelf-life and transference to other laboratories. unit with an ym-10 membrane (millipore, usa), followed by dialysis against pbs. e. granulosus somatic antigen (sm) was obtained by sonication of adult parasites in pbs supplemented with ultra protease inhibitor tablets (roche, indianapolis) on ice, centrifugation and filtration (0.22 mm). antigens from t. hydatigena were similarly prepared. protein content was determined using a bca kit (pierce, rockford, illinois). to prepare polyclonal antibodies, 100 mg of sm or e/s antigens were dissolved in 250 ml of pbs and vigorously mixed with 250 ml of freund's complete adjuvant (pierce, illinois) to form a thick emulsion. this emulsion was then injected subcutaneously into several points on the back of new zealand white rabbits. after 4 and 8 weeks, the animals were immunized intramuscularly with additional doses of 100 mg of antigen emulsified in freund's incomplete adjuvant. ten days after the final booster the animals were bled. the igg fraction was prepared by precipitation of the sera with ammonium sulfate, affinity purified on protein g agarose (amersham, uppsala, sweden), and kept at 220uc until used. for monoclonal antibody (mab) preparation, balb/c mice were primed intraperitonally with 40 mg of e. granulosus sm or e/s antigen in freund's complete adjuvant, and boosted after 3 and 6 weeks with the corresponding antigen using freund's incomplete adjuvant. three days after the last booster mice were sacrificed and the splenocytes fused with sp2/0 cells using standard procedures. cultures producing mabs reactive with the corresponding antigen were selected by elisa. we initially performed a fusion experiment using mice immunized with alternate doses of e/s and sm antigens, and additional selection in a sandwich format using fecal samples. mab cpr39 was selected due to its convenient isotype and performance. several fusion experiments were performed to produce a large panel of monoclonal antibodies. the screening of the monoclonal antibodies was initially performed as described before, and then each of the double-positive supernatants (13 clones) was tested in a sandwich format in combination with the different polyclonal antibodies. however, this time instead of using fecal samples from heavily infected dogs, each supernatant was tested against the following: i) a negative sample from a non-infected dog, ii) a sample collected from a low-burden dog at 28 dpi (dog p9, 74worms), iii) a sample from a heavily infected dog taken at an early stage (10 dpi) of the pre-patent period (dog p8, 3459 worms), and iv) a sample from dog th2 taken at 56 dpi that was positive in the cpr39 copro-elisa. out of this complex screening clone mabeg9, in combination with polyclonal antibody pabc4 (prepared from a rabbit immunized with e/s), was chosen because this antibody pair produced high signals with samples (ii) and (iii), and low readings with samples (i) and (iv). parasite preparations were resolved by sds-page 12% under reducing conditions and then transferred onto nitrocellulose sheets (bio-rad, hercules, california, usa). the nitrocellulose was blocked with 3% non-fat milk powder in pbs 1 h at 37uc and was incubated for 1 h at 37uc with a 1:500 dilution of the rabbit antisera in pbs containing 0.05% of tween 20 (pbs-t), 3% non-fat milk, or directly with the culture supernatants of the mabs antibodies. the membranes were then incubated for 1 h at 37uc with alkaline phosphatase-conjugated to anti-rabbit igg or antimouse igg (pierce) (diluted 1/2500). after extensive washing, a substrate solution containing 5-bromo-4-chloro-39-indolylphosphate p-toluidine salt (bcip) and nitro-blue tetrazolium chloride (nbt) was added according to the manufacturer's instructions (sigma). five mg/ml of polyclonal igg (100 ml/well) (pab cg10 or pabc4 for the mab cpr39 or mabeg9 copro-elisa, respectively) was dispensed into microtitration plates (greiner, germany) and incubated overnight at 4uc. the plates were then blocked with 5% non-fat milk (pbs) and washed with pbs-t. alternatively, the plates were further treated with pbs, 0.1% bovine serum albumin (bsa), 5% sucrose, 0.02% sodium azide, flapped repeatedly against adsorbent paper, dried in a 40% relative humidity chamber for 4 h, and kept in sealed aluminum foil bags containing adsorbent packets (sigma) until used. samples were analyzed in triplicates, after a 1:2 dilution in pbs; 100 ml/well were incubated for 1 h at room temperature, the wells washed and loaded (100 ml/well) with a 1:50 dilution of the mab supernatants and incubated for 1 h at room temperature. finally, the wells were incubated (1 h upon necropsy at 27-30 dpi, 9 of the experimentally infected dogs p1-p10 dogs harbored e. granulosus worms. the number of worms recovered from each dog varied widely, from 74 to 8,500, and showed no correlation with the initial number of protoescoleces used for infection. no other parasites were observed in addition to the e. granulosus worms. adult worms (typically less than 1 m long) were recovered from dogs th1 and th2 infected with t. hydatigena. after careful washing, the recovered parasites were kept in culture to prepare excretion/secretion antigens, or were sonicated as described to obtain somatic antigens. the sds-page analysis of these antigens is shown in figure 1a ; a protoescoleces extract was also included as a reference antigen. all preparations possessed a complex composition and the sds-page profile did not reveal any obvious similarities among the individual components of the different preparation. initially, the reactivity of polyclonal antibody pabcgb10 raised against the sm antigen was characterized, figure 1b . there was a strong response to a large number of components of the immunizing antigen, as well as to high molecular weight components of ege/s and t hydatigena. destruction of sugar epitopes by periodate treatment significantly decreased the reactivity of this antibody with the somatic antigen, as has been observed before [2, 19] , but had little effect in the case of the e/s preparation. the marked cross-reactivity of the polyclonal antibodies with e/s components of t. hydatigena evidences how difficult it is to obtain a specific assay using these antibodies as reagents. for that reason we concentrated our efforts in the preparation and selection of monoclonal antibodies after initial screening against e/s and sm and further selection using pab cgb10 for capture and the hybridoma supernatants for detection, four clones providing the best signal/noise ratios with fecal samples were selected. among these, clone mabcpr39, secreting an igg2b, was chosen. this elisa was used to examine fecal samples obtained at the end of the infection, days 27-30 for e. granulosus or at selected dpi (highest cross-reactivity) for t. hydatigena ( figure 2) . the assay performed with negligible reactivity with samples from noninfected dogs and moderate cross-reactivity with t. hydatigena infected animals. all e. granulosus infected dogs were positive, however, the readouts of the samples corresponding to dogs with small parasite burdens were significantly higher, but too close to the mean value of the negative dogs to give unequivocal results. the assay was then used to study the time course of coproantigen excretion during the experimental infection period ( figure 3) . using a cut-off derived from the analysis of field samples (see below), we found that dogs with high parasite burdens became positive after two weeks, and in spite of some fluctuations remained so thereafter. on the other hand, dogs with less than 200 worms at necropsy, presented low readouts throughout the experiment, becoming positives only at the end of the experimental infection. along the time course of the examined period, the dogs harboring t. hydatigena worms presented very low values (below the cut-off), showing positive values only in very few days after 40 dpi (not shown). due to the limited sensitivity of the cpr39 copro-elisa, new antibodies were prepared. the best combination resulted when pab c4 was used as capture antibody in combination with mab eg9. the reactivity of pabc4 with different parasite antigens in western blot was similar to that of pabcgb10 (figure 1b) and it is not shown. mabeg9 is an igg; its reactivity with different parasite extracts is displayed in figure 1c . the antibody reacted with a large number of the egsm bands, mostly in the middle to high molecular weight range. two components of 100 and 160 kda are the most prominent immunoreactive bands of the e/s antigens, which appear to be also present in the sm preparation. the crossreactivity with th e/s antigens was negligible (as was also the case with protoscoleces antigens, not shown). treatment with periodate had no effect upon the reactivity of mabeg9 with any of the antigens, which was unexpected because previous studies have shown that the sugar epitopes have a major role in the immune response against e. granulosus coproantigens [2, 19] . to rule out technical artifacts, the efficacy of the periodate treatment was tested in parallel using the monoclonal antibody e492/g1 that defines a sugar epitope highly expressed in e. granulosus protoscoleces preparations [20] , figure 1d . when the experimentally-infected dog samples were analyzed with the new assay, it was evident that the eg9 copro-elisa produced stronger signals with samples from dogs with small numbers of worms, allowing a better discrimination between noninfected and infected dogs ( figure 4) . this is an important improvement because most of the reported tests failed to detect these kind of samples. cross-reactivity with t. hydatigena coproanti-gens did not seem to be a major problem when the time course of coproantigen excretion was study with the eg9 copro-elisa ( figure 5 ). using the cut-off estimated by the receiver operating characteristic (roc) curves from the analysis of field samples (see below), only on very few occasions and after 40 dpi were some of the samples from t. hydatigena infected dogs positive. the eg9 copro-elisa allowed an earlier detection of infected dogs. animals with 200 or more parasites became positive between 12-15 dpi and by dpi 20 all infected dogs were positives and remained so thereafter. curiously, coproantigens in dogs p7 and p8 samples could be detected very early. this is more remarkable in the case of dog p7 that harbored a rather small number of worms (128) at necropsy, and may be the result of the spontaneous expulsion of worms a few days after infection. the cut-off and cross-reactivity of the assays were evaluated with field samples collected from 96 unwanted dogs in the provinces of chubut, argentina and junin, peru. upon necropsy, 9 of 85 dogs from chubut, and 6 of 11 dogs from junin were positive for e. granulosus, 48 were infected with other parasites and 33 were found negative, table 1. fecal samples from these dogs were tested using the cpr39 and eg9 copro-elisas and the cutoff selected from receiver operating characteristic (roc) curves [18] to have a convenient balance between sensitivity and specificity as follows: cpr39 elisa = 0.224 au and eg9 elisa = 0.340 au. using these values, the sensitivity of the tests were 80.0 and 86.6% respectively, which is in agreement with the results obtained with the experimental infection of dogs, figure 4 . the specificity of the eg9 test was also superior 86.4% versus 82.7% for the cpr39 elisa. in addition, the cpr39 test produced readings close to the cut-off for an important number of samples, figure 6 , which may be problematic for the use of a cutoff internal calibrator, because small inter-assay variations in the reading of the calibrator may have an important effect upon the number of samples that are classified in either category. actually, if samples that fall between 610% of the cut-off are considered as undetermined (a common practice), none of the samples analyzed with the eg9 test would fall in this category, while four of the samples analyzed with the cpr39 would. while it was not possible to obtain an exact counting of parasites in the dogs from the province of chubut, this was feasible in the case of the animals necropsied in junin. although most dogs had a large number of worms, two of them did not, and these two dogs were exaustively examined. we found only 9 e. granulosus and 2 ascaris lumbricoides, and 47 e. granulosus and very few dipylidium caninum worms in the first and second dogs, respectively. interestingly, the average absorbance readings of the fecal samples obtained from these animal were 1.16 and 2.99 au, which represent strong positive results, indicating the sensitivity of the test and confirming the capacity of the assay to detect small numbers of parasites that had been observed in the experimentally infected dogs. in this regard, when the e. granulosus positive dogs from the experimental infection are included, the overall sensitivity of the test rises to 92.6%. despite the fact that mabeg9 was selected for its reduced cross-reactivity with the thsm antigen, most of its cross-reactivity was against dogs infected with t. spp (mostly hydatigena) alone or together with other parasites. only one dog infected with t. canis (1.44 au) and one of the non-infected dogs (0.694 au) from the province of chubut were classified as positive with the eg9 elisa, figure 6 . the relatively high readings of these two samples are unexpected. based on the results of the experimental infection of dogs and the fact that all other samples (19) from dogs harboring t. canis alone or together with dipylidium caninum showed very low readings, a misdiagnosis at necropsy can not be discarded. it is worth noting that cross-reactivity was essentially against taenia. despite the fact that we could not identify to species all field samples, the great majority of samples from chubut and all from junin corresponded to t. hydatigena; hence most false positive results will be related to dogs eating offal. development of the elisa test in a kit format and interlaboratory analysis of blind samples the eg9 test was then formulated in a kit format to facilitate its use and transference to other laboratories. initially, we studied different conditions for coating and drying the plates and found that using sugars as additives during drying have the best effect on the stability of the capture antibody. trehalose and sucrose showed the best results and the latter was chosen because of its much lower cost. figure 7a shows that dried plates showed similar capacity as fresh ones to discriminate between weak positive and negative samples over a one year period of storage at 4uc. similar results were obtained after 2 years of storage (not shown). to set up the value of the cut-off an internal standard was prepared by adjusting the dilution of the e. granulosus sm antigen in order to produce a readout equal to the value of the cut-off. the stability of this cut-off calibrator solution and other components of the assay (in their ready-to-use dilution) were tested using different diluent buffers at 4uc, room temperature, and 37uc, and some representative results are shown in figure 7b-d. different formulations of the calibrator solution were stable over a 6 month period, even at 37uc; only the results for the sm antigen diluted in pbs, 5% glycerol, 0.1% kathon (dow chemicals, midland, michigan) are shown. the reactivity of mab eg9 started to diminish after a few days at 37uc, but was highly stable over the 6 month period at 4uc (not shown) or at room temperature, figure 7c . the most critical component was the secondary antibody. for several different formulations (only two shown in figure 7d ) all of them rapidly lost the enzymatic activity at 37uc, but using pbs, 0.1% bsa, 0.1% kathon, 0.1 mm tmb, the conjugate remained stable for up to 150 days at room temperature (or 4uc, not shown). overall, we can conclude that even at room temperature the kit has a safe shelf life of 3 months, which can surely be extended upon refrigeration, which is a convenient advantage for the shipment and use of the kit in remote places. due to the availability of commercial ready-to-use tmb substrate solutions, the preparation of this kit component was not pursued in this study. in case of high demand of the kit, it will be necessary to prepare new pab. in our experience, this is a critical component of the test, but we have selected similarly performing pabs from a panel of 3 to 4 immunized rabbits, after initial selection with fecal samples from dogs harboring low number of e. granulosus parasites and counter selection with samples from t. hydatigena infected dogs. the reproducibility of the results obtained with the copro-elisa kit and the feasibility of its transference were demonstrated by the analysis of blind samples. to this end, 52 fecal samples collected in the province of junin, peru were processed as described, and aliquots were distributed to two laboratories (ins and digesa) in lima, peru, where the copro-elisa kit had been transferred, and to our laboratory in uruguay (facultad de química). the samples were independently analyzed by the three laboratories and classified as positive, negative or uncertain if their readout felt within the calibrator absorbance reading 610% range. twenty four samples were positive, indicating a prevalence of 46.1% in the studied areas (canchayllo and jauja). the kit performed with excellent reproducibility and all individual samples were equally classified by the three laboratories with the exception of two of the samples with readings close to the cut-off, one of them categorized as negative by two of the labs and uncertain by the third one, and another sample classified as (weak) positive by two of the labs and uncertain by the remaining one. two tests were developed and the difference in their performance highlights the importance that careful selection of antibody pairs has to attain a high standard of sensitivity and specificity. the eg9 test performed with high sensitivity (92.6%) and good specificity (86.4%). these parameters are closely similar to previously reported values for other tests. however, it is very important to keep in mind that the only trustworthy comparison of tests is that obtained with a common panel of samples assayed under identical conditions, which highlights the need of interlaboratory studies to compare the performance of existing test. the test cross-reactivity was low, and in addition, since it was basically restricted to t. hydatigena, most false positive results will still indicate access of dogs to offal. a major contribution of our study is the use of a well-characterized mab that assures availability and batch-to-batch reproducibility, as well as the formulation of the assay in a kit format with extended shelf life. the eg9 test is being shared with other control programs in the region and we hope that it will help to monitor the progress of control programs and standardize the epidemiological baseline information in the region. checklist s1 stard checklist for the eg9 copro antigen test. flowchart s1 stard flow chart detailing the method used to assess the diagnostic performance of the eg9 copro antigen test. (docx) partial characterisation of carbohydrate-rich echinococcus granulosus coproantigens control of hydatidosis echinococcosis: disease, detection and transmission screening for echinococcus granulosus in dogs: comparison between arecoline purgation, coproelisa and copropcr with necropsy in pre-patent infections echinococcosis: diagnosis and diagnostic interpretation in population studies coproantigens in taeniasis and echinococcosis detection of echinococcus granulosus coproantigens in australian canids with natural or experimental infection copro-diagnosis of echinococcus granulosus infection in dogs by amplification of a newly identified repeated dna sequence polymerase chain reaction for detection of patent infections of echinococcus granulosus (''sheep strain'') in naturally infected dogs copro-dna tests for diagnosis of animal taeniid cestodes coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans detection of echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes coproantigen detection in dogs experimentally and naturally infected with echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay detection of echinococcus granulosus coproantigens in faeces from naturally infected rural domestic dogs in south eastern australia production and characterization of monoclonal antibodies against excretory/secretory products of adult echinococcus granulosus, and their application to coproantigen detection manual on echinococcosis in humans and animals:a public health problem of global concern the meaning and use of the area under a receiver operating characteristic (roc) curve echinococcus granulosus coproantigens: chromatographic fractionation and characterization modulation of the cellular immune response by a carbohydrate rich fraction from echinococcus granulosus protoscoleces in infected or immunized balb/c mice we thank dr. carmona and dr. cecilia casaravilla for their advice and provision of samples. we are also grateful to the medical veterinarians and staff of the comisión nacional de zoonosis, uruguay, for help with the experimental infection of dogs. the personnel and medical veterinarians from the dirección regional de salud de huancayo, peru, are also acknowledged for their help with the pilot study of dog infection. key: cord-034471-enmtckpe authors: tuckel, peter s.; milczarski, william title: the changing epidemiology of dog bite injuries in the united states, 2005–2018 date: 2020-11-01 journal: inj epidemiol doi: 10.1186/s40621-020-00281-y sha: doc_id: 34471 cord_uid: enmtckpe background: in 2018, the most recent year for which data are available, dog bites ranked as the 13th leading cause of nonfatal emergency department visits in the united states. as dog ownership spirals upwards in the united states, it is important to continue to monitor the epidemiology of dog bite injuries. this study provides contemporary data on the incidence of dog bites injuries in the united states and in new york and profiles individuals who have been treated for dog bites in emergency departments. the study also examines the demographic correlates of the rate of injuries at the neighborhood level in new york city and maps the rate in each neighborhood. methods: at the national level, the study examines longitudinal data on dog bite injuries from 2005 to 2018 gathered by the centers for disease control and prevention. for new york, the study analyzes data for 2005–2018 collected by the new york state department of health. a negative binomial regression analysis was performed on the state data to measure the simultaneous effects of demographic variables on the incidence of dog-related injuries. a thematically shaded map of the rate of dog bite injuries in new york city’s neighborhoods was created to identify neighborhoods with higher-than-average concentration of injuries. results: in both the united states and new york the rate of dog-bite injuries increased from 2005 to 2011 and then underwent a significant decline. injuries due to dog bites, however, still remain a sizable public health problem. injuries are more prevalent among school-age children, inhabitants of less-densely populated areas, and residents of poorer neighborhoods. in new york city, poorer neighborhoods are also associated with fewer dogs being spayed or neutered. conclusions: to reduce the rate of dog bite injuries, prevention programs – particularly those which center on teaching the dangers of canine interactions with humans – should be targeted at children. dog bite injuries tend to be clustered in identifiable neighborhoods. dog bite prevention programs and stricter enforcement of dog laws can target these neighborhoods. while the appellation attached to dogs is "man's best friend," dog bite injuries are a common occurrence. data covering the years 2001-2003 revealed that approximately 4.5 million individuals in the united states were bitten by dogs each year (gilchrist et al. 2008) . of these, 19% necessitated medical attention. between 2005 and 2013, there were an average of 337,103 visits to emergency departments (eds) per year for dog bites (loder 2019) . in 2018, almost 27,000 individuals required reconstructive surgery owing to dog bites (american society of plastic surgeons 2018). the morbidity associated with dog bites is particularly pronounced among children. one study estimates that approximately one-half of all children aged 12 and younger have been bitten by a dog (beck and jones 1985) . of nine causes of injury resulting from activities children frequently engage in (e.g., baseball, playground accidents, etc.), dog bites rank second in terms of annual visits to an ed (u.s. product safety commission 1996) . most dog bite injuries in the us are inflicted by owned pet animals and not strays. three of five bite victims were bitten by the family dog or one living in the neighborhood (overall and love 2001) . significantly, the number of dogs in the united states has steadily increased over the last two decades. in 2000, approximately 68 million dogs were owned as pets in the united states. by 2017, the number of dogs had climbed to 89.7 million (appa 2017). the total population of the united states in 2017 numbered 325 million. in terms of household penetration, 63.4 million households (49.3%) now own a dog (insurance information institute 2019). as the number of dogs has spiraled upwards, both the demographics of dog owners and the characteristics of the dogs themselves have undergone a noticeable change. during the past decade, the rates of dog ownership have risen sharply among older adults, hispanics, and residents of large metropolitan areas. correspondingly, there has been a decrease of dog ownership among families with young children (marketresearch.com 2019). the share of dogs which are smaller (weigh under 25 pounds) has increased as well in this time span and this trend is expected to continue (petfoodindustry.com 2015) . with the growth in the ownership of dogs in the united states and the shift in the characteristics of both owners and dogs, it is important to continue to monitor the epidemiology of dog injuries and implications for public health. this study has five objectives: (1) to provide contemporary data on the incidence of dog bites in the united states and in new york, (2) to furnish a detailed profile of individuals who have been treated for dog bites in new york to describe individuals who are most at risk, (3) to present the socio-demographic correlates of the rate of dog bite injuries at the neighborhood level in new york city which can help to identify the characteristics of neighborhoods with a higher incidence of dog bite injuries, (4) to map the incidence of dog bite injuries at the local level which can be used to target neighborhoods which have a disproportionately large number of dog bite injuries, and (5) to provide data on the changing composition of the dog-owning population to help explain the epidemiological findings. the analyses conducted in this study rest principally on data collected from ed visits in the united states and new york. the national-level data are derived from the web-based injury statistics query and reporting system (wisqars) (centers for disease control and prevention 2019). wisqars is an online, interactive database which provides national estimates of both fatal and nonfatal injuries. the present study utilizes the nonfatal injury data which comes from the national electronic injury surveillance system-all injury program (neiss-aip), sponsored by the u.s. consumer product safety commission and the cdc's national center on injury prevention and control. the neiss-aip is based on a sample of 66 hospitals randomly selected from all hospitals in the united states which have a 24-h ed and a minimum of six beds. the sample is stratified by hospital size measured in terms of the number of ed visits each year. the nonfatal injury data provide estimates of injuries treated in eds by cause of injury (e.g., dog bites), race/ethnicity, gender, and disposition of the patient after being released from the ed. in addition to the wisqars database, this study examines individual-level patient records from new york. these patient records include a large number of demographic, diagnostic, and treatment variables. the patient records also include more detailed information concerning the racial and ethnic characteristics of patients than is contained in the national data sets. importantly, the new york patient records include geographic identifiers such as the county or the 5-digit zip code in which the patient resides. the data for new york come from the statewide planning and research cooperative system (sparcs), which is under the auspices of the new york state department of health (2019). sparcs assembles data on outpatient, inpatient, and ambulatory surgery patients treated in all hospitals in new york state. this study also draws upon data gathered by new york city's department of health and mental hygiene (dohmh) (2019). the database consists of dog bites which are reported via online, fax, or phone to the city's dohmh animal bite unit. each record in the database provides information on: (1) the date of the bite, (2) the breed, age, and gender or the dog, (3) whether the dog was spayed or neutered, and (4) the zip code and borough of the person who was bitten. altogether, there were 10,280 records spanning the years from 2015 to 2017. this tally clearly underrepresents the incidence of dog bite injuries in the city. the comparable number of dog bite injuries which were treated in eds in the city during the period 2015 to 2017 totaled 44,947. most likely, this disparity was because individuals who were bitten both had to know to contact and take the initiative to contact dohmh. it should be noted, too, that the breed of the dog was missing on 15.4% of the cases and the zip code of the person who was bitten was missing on 26% of the cases. injury code. for both the national and state data sets, identification of patients who were treated for a dog bite was based on two separate injury codes. the international classification of diseases, ninth revision (icd-9) external cause of injury code (e-code) e906.0 -dog bitewas utilized for the years prior to 2015. both the icd-9 e-code e906.0 and the icd-10cm e-code w54.0xxa -bitten by dog (initial encounter)were utilized for the year 2015. just the icd-10cm e-code w54.0xxa was used for the years 2016-2018. sociodemographic characteristics. both the wisqars and sparcs data sets furnished information about the age and gender of patients. the sparcs data sets also included two separate variables about the race and ethnicity of patients. a typology was created from these two variables with the following five values: "white, non-hispanic," "black, non-hispanic," "asian, non-hispanic," "other, non-hispanic," and "hispanic." importantly, the sparcs database included the patient's county of residence and his/her 5-digit zip code. to measure the combined effects of year, background characteristics (i.e., gender, age, race/ethnicity), and geographic location on the incidence of dog bites, we conducted a negative binomial regression analysis using the patient records from new york. a negative binomial regression analysis was performed instead of a poisson regression due to overdispersion of the data. the population-based counts of both the number of outpatients and inpatients who were bitten by a dog served as the dependent variable in this analysis. the predictor variables comprised the year, geographic location, and the demographic characteristics of the patients. year was measured as an interval-level variable ranging in values from 1 (corresponding to the year 2005) to 14 (corresponding to the year 2018). to capture possible curvilinear effects of year on the incidence of dog bites, a multiplicative term created by squaring the year variable was also incorporated into the analysis. geographic location was a dichotomous variable with a value of 1 indicating new york city and a value of 0 indicating new york state omitting new york city. gender was also a dichotomous variable with a value of 1 indicating male and a value of 0 indicating female. the age variable consisted of 7 categories: under 5, 5 to 9, 10 to 14, 15 to 19, 20 to 44, 45 to 64, and 65 and older. the racial-ethnic background of patients was made up of 5 groups as mentioned above: non-hispanic white, non-hispanic black, non-hispanic asian, non-hispanic other, and hispanic. since it can be assumed that the risk of being bitten by a dog varies by population sizes, an offset variable was introduced into the analysis. the offset variable was created in two steps. first, population counts were tallied for each combination of year, geographic location, gender, age group, and racial-ethnic category. so, for example, one count might comprise non-hispanic asian females between the ages of 10 to 14 living in new york city in 2014. altogether, this step yielded 1960 different counts. next, natural log transformations were carried out on each of these counts. to measure the demographic correlates of the rate of dog bite injuries at the county level in new york state (n = 62), a three-step process was undertaken. first, the number of both outpatients and inpatients were combined for each county for the year 2018 (the most recent year for which data are available). second, these figures were divided by the population of each county to obtain an injury rate. finally, the rates were correlated with an array of socio-demographic variables at the county derived from the american community survey 2014-2018 (5-year estimates) (u.s. census bureau 2018). these variables consisted of the following: (1) population density per square mile, (2) the racial-ethnic composition of the county, (3) median family income, (4) per capita income, (31) percent of families with income below the poverty level, (6) percent of the population 25 and over with a b.a. degree or more, (7) percent of the population with no health insurance, and (8) percent of the insured population with public health insurance. a similar procedure was conducted to examine the socio-demographic correlates associated with dog bite injuries at the neighborhood level in new york city. for this analysis, the number of outpatients and inpatients were combined for each 5 digit zip code in new york city (n = 179). next these figures were aggregated up to the united health fund (uhf) level (n = 42) and divided by the population of each uhf district to obtain an injury rate. these rates were then correlated with the same set of socio-demographic variables described above calculated for each uhf district. to determine the geographic distribution of patients injured by dog bites at the neighborhood level in new york city, a thematically shaded map of the injury rate by the united health fund (uhf) district in which the patient resided was created. a global moran's i was computed to assess whether the spatial distribution of the residences of the patients was geographically clustered or dispersed. the rates of dog bite-related injuries by age and sex for the period 2005-2018 are presented in table 1 . consistent with previous research findings, the data show that age is strongly related to the rate of dog bite injuries (gilchrist et al. 2008; weiss et al. 1998; hoff et al. 2005; quirk 2012; holzer et al. 2019) . the modal category is the age group 5 to 9, followed by the age groups 0 to 4 and 10 to 14. from the age group 15 to 19 and older, injuries taper off considerably. the data also reveal that gender is associated with dog bite injuries. through the age of 14, the rate of males exceeds that of females by a wide margin. for the older age groups, the disparity between the gender groups narrows. table 2 displays the annual estimated frequency and rates of dog bite injuries resulting in an ed visit in the united states from 2005 to 2018. figure 1 graphs the estimated annual rates. the data show that the rates of injuries tended to increase until 2012 and then underwent an overall decline thereafter (p = .046 for the curvilinear relationship). noteworthy is that the relationship between the incidence of dog bite injuries varies by age group over time. figure 2 exhibits the rates of dog bite injuries resulting in a visit to an ed by age group during the time period 2005 to 2018. paralleling the overall trend for the country as a whole, the rates of the two youngest age groups (0 to 9, 10-19) initially increase up to 2012 and then undergo a steep decline. the opposite pattern prevails for the two older age groups (20-44, 45 plus). here the rates of these two groups increase over the course of the 13 year span. the results of the negative binomial regression analysis examining the simultaneous effects of time and key demographic variables on the incidence of dog bites treated in an ed are displayed in table 3 . the analysis is confined to two geographic locations -new york city and new york state excluding new york city. the predictor variables consist of year, year squared, place of residence, gender, age group, and the racial-ethnic background of patients. the effects of year and the multiplicative term of year squared are both significant. a graphic display of these terms indicates that from 2005 to 2012 the frequency of dog bite injuries increased and then from 2013 to 2018 decreased, controlling for the other variables in the analysis. the same general pattern emerges if the injury rate of just individuals who were admitted as inpatients serves as the dependent variable. both trends mirror the results observed at the national level. inspection of table 3 indicates that residents of new york state outside of new york city are more likely to be treated in an ed for a dog bite than residents of the city. this finding reflects the greater prevalence of dog bite injuries in less densely populated areas. coinciding with the findings from the national data discussed above, the table also shows that there is a significant gender gap in the incidence of dog-related injuries. males are 1.12 times more likely to visit an ed due to a dog bite than females. as expected, age is a major determinant of the risk of injury from a dog bite. compared with patients who are 65 and older (the reference category), patients aged 5 to 9 are 2.7 times more likely to incur a dog bite injury and patients aged 10 to 14 are 2.3 more likely to sustain an injury. individuals in the other age categories (0 to 4, 15 to 19, 20 to 44, and 45 to 64) are also significantly more likely to be injured by a dog bite than those in the reference category. finally, the data reveal that non-hispanic asians are considerably less likely to be treated in an ed for a dog bite than hispanics (the reference category). the odds ratios for the other racial-ethnic groups are not statistically significant. table 4 displays the relationship between key sociodemographic variables and the rate of injuries due to dog bites at both the county level in new york state and the neighborhood level in new york city. the data indicate that the rate of injuries due to dog bites is negatively associated with population density. this relationship between injury rate and population density is most pronounced at the county level. the data also show that at the county level, the injury rate is positively associated with the percent of the population which is non-hispanic white, reflecting the relationship between urbanicity and racial-ethnic composition. significantly, at both the county and uhf levels, there is a strong negative association between the injury rate and a number of economic variables. injuries are more prevalent in counties or neighborhoods with lower median family income, per capita income, or proportion of the population which is not college-educated. table 5 presents the results of an analysis performed on self-reported incidents of dog bites in new york city's united health fund districts for the years 2015 to 2017. the table shows the socio-demographic correlates of both the percent of dogs which were spayed/neutered and the percent of dogs which were pit bulls in the 42 uhf districts. of the breeds identified in the data set (84.6%), pit bulls were the most numerous (33.6%), followed in order by shih tzu (5.3%), chihuahua (5.2%), german shepherd (4.1%), and yorkshire terrier (3.1%). this finding is consistent with previous research showing that pit bulls are responsible for more bites than any other dog breed (mcreynolds 2019). of the selfreported cases 29.1% were classified as spayed or neutered. the results reveal that poorer neighborhoods were associated with a higher proportion of dogs which had not been spayed/neutered and also a higher proportion of dogs which were pit bulls. coinciding with expectations, the rates of dog-bite injuries are not uniformly distributed across the uhf districts. a choropleth map of the rates shows that the hunts point-mott haven neighborhood in the bronx, east harlem neighborhood in manhattan, the sunset park neighborhood in brooklyn, and the port richard and stapleton-st. george neighborhoods in staten island have notably higher rates than other uhf districts (see fig. 3 ). the moran's i index yields a value of .356 (p < .001), indicating a pattern of spatial clustering . surveys of dog owners during the last decade reveal significant changes in their demographic characteristics (table 6 ). the data in the table shows that the age distribution of dog owners has skewed upwards in the past decade. in 2008, 26.1% of dog owners fell into the age category of 55 to 74; by 2018, the number of owners in this age category rose to 31.5%. in this same 10-year span of time, dog owners were also more likely to be hispanic, reside in larger metropolitan areas, and have higher levels of education. another noteworthy change is the reduction in the number of younger children living in the household. the number of children in each of the age brackets under 6, 6 to 11, and 12 to 17 all dropped in the years from 2008 to 2018. this study has found that the rate of dog bite injuries has been declining in recent years. this decline is in evidence at both the national and state levels of analysis. the decline has been most visible among those under the age of 19particularly children under the age of 9. one explanation for this downward trend might be that it is simply an artifact of the methodology employed in this study. most of the findings contained in this study are based on dog bite injuries treated in emergency room departments. it may be the case, though, that in recent years individuals bitten by dogs have increasingly sought treatment in other venues such as private physicians' offices or urgent care centers. while this may be a factor associated with the downward trend in dog bite injuries, another finding uncovered in this study --a decline in the number of inpatients treated for dog bites in new york --does not lend support to this explanation. a second explanation for the recent decline in dog bite injuries centers on the change in the profile of dog owners and the characteristics of the dogs themselves. survey data presented in this study indicates that there has been a decline in the presence of young children in dog-owning households over the past decade. since young children are the most likely age group to be bitten by dogs and the overwhelming majority of injuries in the united states occur in the home, the reduction in the number of younger-aged children living at home would help to explain the drop off in dog-related injuries (gilchrist et al. 2008; weiss et al. 1998; quirk 2012; overall and love 2001) . the survey data further shows that dog owners are increasingly residing in larger metropolitan areas where dog sizes tend to be smaller. also, the dogs are more likely to be confined indoors or in a yard, and kept on a leash (marketresearch.com 2019). as research has demonstrated, small and medium size dogs pose less of a danger to humans and dogs on a leash lower the risk of unwanted contacts with humans. the survey data also reveals that over the past decade owners have become older and better educated. this shift in age and education is consistent with the notion that the role of the dog has changed from being less of a guard dog and more of a pet or family member. a nationwide harris poll of adult americans buttresses this notion. the poll found that among dog owners, 92% view their dogs as members of their family (harris poll 2011). significant numbers report that they "allowed my pet to sleep in the bed with me" (70%), "bought my pet a holiday present" (60%), thought "it was a good idea to have dogs in long-term care facilities" to reduce stress (89%). the surge in the number of dog parksdesigned to better meet the physical and emotional needs of dogs --is another indicator of the changing role of the dog in american life. in the last decade there has been a 40% increase in dog parks, according to the trust for public land (lowrey 2020) . thus, it is likely that the changing role of the dog in u.s. also accounts for the lower incidence of dog bite injuries. while there has been a diminution in the rate of injuries due to dog bites in recent years, dog bites still remain a leading cause of nonfatal injuries in the united states. for the year 2018the most recent year for which nationwide data are available --there were a total of 344, 201 nonfatal injuries treated in an ed due to dog bites. as this study has noted, there is a distinctive sociodemographic profile of individuals who suffer an injury from a dog bite. sizable age disparities exist, with younger individuals considerably more likely to be treated for a dog bite than older individuals, especially school-age children. numerous reasons have been given to account for the greater susceptibility to dog bites on the part of young children. children may lack the maturity to understand the "signaling behavior" of dogs, misinterpreting the cues dogs emit when in an agitated state. children may also make sudden body movements or high-pitched sounds which can frighten dogs and precipitate an aggressive response (overall and love 2001) . in addition, because of their "innate curiosity," children may more readily approach strange dogs (cohen-manheim et al. 2018) . aside from age, significant associations also exist between dog bite injuries and the place of residence and the economic background of patients. dog bite injures are more prevalent among inhabitants of less densely populated areas and poorer neighborhoods. the negative relationship between the incidence of dog bites and the socioeconomic status of the neighborhood could be due to several factors. first, dogs might lack proper training or be taught to act aggressively to protect the household. second, they may lack the necessary supervision by being chained outdoors for lengthy periods of time or being allowed to run loose. third, as the data in this study has demonstrated, the owners may not be compliant with the licensing requirements such as spaying or neutering their dogs. though dog bites remain a sizable problem, it is one which is largely preventable. in this study we have found that dog bite injuries tend to be clustered in identifiable neighborhoods. dog bite prevention programs as well as stricter enforcement of dog laws can target these neighborhoods to significantly reduce the incidence of injuries. some limitations attached to this study should be noted. one is that population counts of dogs and specific breeds within new york city neighborhoods are not available. thus, we cannot determine the degree to which the varying prevalence of dog bite injuries in new york city's neighborhoods is due to the differing number of dogs in these neighborhoods. nor can we determine whether pit bulls rank as the most dangerous breed in new york city because of their attributes, or because of their numerical representation in the city. a further limitation pertains to the self-reported data on dog bites in the city. dohmh states on their web page that "data on breed, age, gender and spayed or neutered status have not been verified by dohmh and is listed only as reported to dohmh". a third limitation pertains to the underlying reasons for the variability in the annual rate of dog bite injuries in the united states, particularly in the last several years. while we believe that this variability reflects a real diminution in the rate of dog bite injuries, we cannot dismiss the possibility that some of this variability might be due to data quality issues. this study has found that the rate of dog bite injuries in the united states has decreased in recent years. we attribute this decline mainly to a shift upwards in the age distribution of dog owners and to the changing role of the dog in american families from being less of a guard dog to being more of a companion. as dog ownership continues to spiral upwards (a trend which has been accelerated by the coronavirus and subsequent lockdowns), it will be important to monitor the frequency of dog-bite related injuries to see if this positive trend persists. though dog bite injuries have declined in recent years, the extent of these injuries still constitutes a major health problem. young children especially are vulnerable to being bitten by a dog. prevention programsparticularly those which center on teaching the dangers of canine interactions with humansshould be targeted at this age group. this study also has noted that residents of poorer neighborhoods in urban areas are more susceptible to being injured than residents of more affluent neighborhoods. future research needs to be conducted to increase our understanding of why there is a negative association between a neighborhood's socioeconomic status and injury rates from dog bites. hopefully this greater understanding will lead to a reduction in the disparity of these rates. american society of plastic surgeons. national plastic surgery statistics number of dogs in the united states from unreported dog bites in children national center for injury prevention and control. web-based injury statistics query and reporting system (wiaqars) epidemiology of hospitalizations due to dog bite injuries in israel dog bites: still a problem? injury prevention americans-have-always-had-interesting-relationships-with-their-pets-whetherthat-pet-is-a-cat-dog-parakeet-or-something-else-the-pet-industry-is-thrivingand-for-good-reason-more-than-three-in-f emergency department visits and hospitalizations resulting from dog bites dog bite injuries in the usa: prevalence, correlates and recent trends statistics#:~:text=pet%20insurance,-the%2 0pet%20insurance&text=the%20total%20number%20of%20pets,insurance%2 0companies%20in%20north%20america the demographics of dog bites in the united states new york times. 2020. section b, 9. marketresearch.com. topline pet demographics. in: private correspondence new study identifies most damaging dog bites by breed: newstat dog bites to humans-demography, epidemiology, injury, and risk ownership of small dogs is on the rise non-fatal dog bite injuries in the usa american community survey product safety commission. injuries associated with selected sports and recreational equipment treated in hospital emergency departments, calendar year 1994 incidence of dog bite injuries treated in emergency departments publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank marketresearch.com for furnishing us with information on the changing demographics of dog owners in the united states, and for their helpful comments concerning the interpretation of the data. the authors would also like to thank yonatan gorin, master's student in business administration at touro university worldwide, for the technical assistance he lent to this study. we also would also like to acknowledge the many helpful comments provided by the anonymous reviewers. pst conceptualized and designed the study; collected and analyzed the data; and wrote the initial draft of the manuscript. wm collected and analyzed the data and revised the manuscript. both authors approved the manuscript for publication. funds to acquire some of the data used in this study were obtained through hunter college's president's fund of faculty advancement. the wisqars database can be accessed online (https://www.cdc.gov/injury/ wisqars/nonfatal.html). information about obtaining the sparcs data can be obtained by contacting the new york state department of health (https:// www.health.ny.gov/statistics/sparcs/). the dohmh data can be retrieved online https://data.cityofnewyork.us/health/dohmh-dog-bite-data/rsghakpg. this study uses publicly available aggregate data at the national, county, and local levels and employs de-identified individual-level data for state and local-level analyses. we are therefore exempt from securing human subjects review from our college's institutional review board. not applicable. the authors have no conflict of interest to declare. key: cord-316500-vik30moa authors: cardillo, lorena; piegari, giuseppe; iovane, valentina; viscardi, maurizio; alfano, flora; cerrone, anna; pagnini, ugo; montagnaro, serena; galiero, giorgio; pisanelli, giuseppe; fusco, giovanna title: lifestyle as risk factor for infectious causes of death in young dogs: a retrospective study in southern italy (2015–2017) date: 2020-06-05 journal: vet med int doi: 10.1155/2020/6207297 sha: doc_id: 316500 cord_uid: vik30moa infectious diseases are a common cause of death in young dogs. several factors are thought to predispose young dogs to microbiological infections. identifying the cause of death is often a challenge, and broad diagnostic analysis is often needed. here, we aimed to determine the infectious causes of death in young dogs aged up to 1 year, examining how it relates to age (under and over 6 months), lifestyle (owned versus ownerless), breed (purebred and crossbreed), and gender. a retrospective study was conducted in a 3-year period (2015–2017) on 138 dead dogs that had undergone necropsy and microbiological diagnostics. enteritis and pneumonia were the most commonly observed lesions. polymicrobism was more prevalent (62.3%) than single-agent infections and associated with a higher rate of generalised lesions. ownerless dogs showed over a three-fold higher predisposition to viral coinfections than owned dogs. above all, canine parvovirus was the most prevalent agent (77.5%), followed by canine coronavirus (31.1%) and canine adenovirus (23.9%); ownerless pups had a higher predisposition to these viruses. escherichia coli (23.9%), clostridium perfringens type a (18.1%), and enterococcus spp. (8.7%) were the most commonly identified bacteria, which mostly involved in coinfections. a lower prevalence of cdv and clostridium perfringens type a was observed in puppies under 6 months of age. in conclusion, this study is the first comprehensive survey on a wide panel of microbiological agents related to necropsy lesions. it lays the groundwork for future studies attempting to understand the circulation of infectious agents in a determined area. a correct and complete determination of the cause of death in young dogs is a challenge for veterinarian practitioners. a mere anatomopathological examination is often not sufficient to define a lesion's etiology. puppies are susceptible to several viral and bacterial pathogens owing to the incomplete ability of their immature immune system [1] . in the first days of life, bacterial infections are described to be the prevalent cause of neonatal disease and death [2] ; in contrast, at other ages, many factors have been attributed for outbreaks of viral diseases, including age, vaccination status, breed [3, 4] , habitat [5] , and seasons [6] . stressful conditions due to overpopulation, high environmental contamination [7] , lengthy travel for illegal importations, and lack of vaccination can create immune deficiency [8] . in this context, viral infections and bacterial superinfections can occur, and mixed infections are frequently detected [9, 10] . polymicrobial infection recognises several etiopathogenetic mechanisms: (1) universally recognised virus-induced immunosuppression created by some agents such as canine parvovirus (cpv) leukopenia and disruption of the gastrointestinal barrier [1] or canine distemper virus (cdv) lymphopenia [11] , which create a niche for the growth of other opportunistic pathogens; (2) the so-called primary with secondary infections, where the first agent creates the ideal condition needed for the colonisation and replication of the second one, e.g., "kennel cough," where some respiratory viruses such as cdv, canine adenovirus type 2 (cav-2), canid herpesvirus-1 (chv-1), and others precede the secondary bacterial infection [12, 13] ; (3) a condition characterised by concurrent infection of multiple agents to induce the disease [8] . in addition, an immune-compromised system can play a key role for systemic spread of localised infections in various ways, including extraintestinal diffusion of enteric pathogens, because of gut dysbiosis, either through the choledochus or via bacterial translocation from the lymphhematic route [14] [15] [16] , as described for coliform septicemia in dogs with viral-induced damage of intestinal epithelial cells during parvoviral infections [17] . in many cases, coinfectants can exacerbate clinical signs; thus, normally mild pathogens can cause severe diseases [18, 20] . although vaccines for some pathogens that cause high mortality in pups have been produced, a failure of vaccination can occur, due to interference of high titres of maternal-derived antibodies (mdas), incorrect vaccination protocols, high environmental contamination, or stressful conditions [21, 22] . e identification of specific causes of death has a fundamental epidemiologic role. several studies have been conducted in the past, based only on anatomopathological lesions [23] [24] [25] [26] , and, more recently, a retrospective study was reported in the province of rome (italy), based on anatomohistopathological examinations and collateral exams [27] . to the authors' knowledge, there are no epidemiologic surveys on the causes of death for infectious diseases in young dogs performed in southern italy. us, the aim of this study was to identify infections and coinfections associated with macroscopic lesions in deceased dogs under 1 year of age, related to their age and lifestyle. our study is a retrospective survey carried out at the istituto zooprofilattico del mezzogiorno (izsm) of portici, naples (southern italy), as part of our routine diagnostic activities aimed to verify the causes of death in owned and ownerless young dogs. from 511 dead dogs brought to the izsm by veterinary practitioners, owners, or law enforcement officers in order to assess the cause of death, 138 fulfilled the criteria to be included in the study. ese criteria included signs of death due to infectious diseases, confirmed by anatomopathological and chemical exams, complete panel for necropsy, and microbiological examinations; ages ranged from 0 to 12 months. data included signalment, anamnesis, necropsy, and microbiologic reports, which were carried out over a 3-year period (january 2015 to december 2017) from the informative system for analysis laboratory management (s.i.g.l.a.) database of the izsm. for the content of this survey, the authorisation of the ethical committee was deemed unnecessary, according to national regulations. diagnosis of cause of death. all necropsies were performed in the necropsy room in the izsm with a standard necropsy protocol codified by piegari et al. [28, 29] . approximatively, 2 cm 2 of tissue samples and swabs were collected from deeper structures of the liver, lungs, brain, heart, and small intestine. fecal swabs were collected from rectal ampulla. specimens were processed for microbiological examinations as soon as possible. approximately, 2 mg of each tissue sample was collected and suspended in 2 ml of sterile phosphate buffer saline (pbs) in 2-ml tubes, homogenised with glass beads with the tissuelyser (qiagen), and clarified by centrifugation for 5 min at 4000 rpm. fecal specimens were subjected to a previously described treatment, in which 100 mg of feces was suspended in 900 µl of tissue lysis buffer (atl), vortexed for 1-2 min, and incubated for 10 min at room temperature to obtain sedimentation of greater particles; finally, 600 µl of supernatant was transferred to a 1.5-ml tube and incubated at 70°c for 10 min in a thermomixer (eppendorf ). aliquots of 200 µl of both tissue and fecal supernatants were collected for nucleic acid extraction using the qiasymphony automated system (qiagen) and processed according to the manufacturer's protocol, eluted in 60 µl, and stored at −80°c until use. samples and fecal swabs were tested for the most common viruses that cause diarrhea, pneumonia, hepatitis, encephalitis, and myocarditis in young dogs, using pcr protocols reported in table 1 . for molecular detection and characterisation of cpv variants (2a-2b-2c), real-time pcr was performed using different sets of primers and probes to amplify fragments encoding for capsid protein vp2, as described previously [30] . for ccovs, the molecular detection and genotyping of ccov-i and ii were performed using a set of primers and probes that amplify fragments of orf5 as described by decaro et al. [32] , and the 2 subtypes ccov-iia and iib were characterised by the amplification of the gene encoding the spike (s) protein using the rt-pcr protocol reported by decaro et al. [33] . finally, for cavs, the protocol used was based on a duplex real-time pcr assay for the simultaneous detection of types 1 and 2, amplifying a fragment of the hexon gene, as described by dowgier et al. [40] . e characterisation of cdv samples was performed on the hemagglutinin (h) gene sequence, according to an et al. [41] . next, 1 µl of the amplification product was applied to the tapestation 2200 (agilent technologies) using d1000 screen tape and reagents (agilent technologies). e pcr products were purified using the minielute reaction cleanup kit (qiagen) according to the manufacturer's protocol. reactions were subjected to sanger sequencing carried out by bigdye terminator cycle sequencing kit v.1.1 (applied biosystems). e amplification conditions used for the sequencing reactions included an initial denaturation at 95°c for 1 min, followed by 25 cycles of denaturation at 96°c for 10 s, annealing at 50°c for 5 s, and extension at 60°c for 4 min. amplicons were then cleaned up using the dyeex 2.0 spin kit (qiagen) following the manufacture's indications. e sequencing reactions were applied to a 3500 genetic analyzer capillary electrophoresis system (applied biosystems). e forward and reverse sequences were assembled using the geneious r9 software package (biomatter) and compared to analogous sequences in the blast genetic database (http://www.ncbi.nlm.nih.gov/blast.cgi). bacteriological analyses were performed by validated standard methods. briefly, swab samples from the first isolation of the liver, lungs, brain, and small intestine were plated on macconkey agar, aerobically incubated at 37 ± 1°c for 24-48 h and 5% blood sheep agar, incubated at 37 ± 1°c for up to 72 h in an aerobic or anaerobic atmosphere. for campylobacter spp., skirrow agar was used, and plates were incubated at 42 ± 1°c for 24-48 h in microaerophilic atmosphere, containing 6% o 2 , 10% co 2 , and 84% n 2 . anaerobic atmosphere was created by adding a 2.5-l oxoid anaerogen sachets to an oxoid anaerojar (2.5 l, ermo fisher scientific). when appropriate, enrichment broths were used. salmonella spp. were isolated using preenrichment müller-kauffmann tetreathionatenovobicin broth (mkttn) incubated at 37 ± 1°c for 24 ± 3 h and rappaport-vassalidis soy broth (rvs) at 41.5 ± 1°c for 24 ± 3 h and subsequently plated on xylose lysine desoxycholate agar (xld) and brilliance salmonella agar base (bsa), both incubated at 37 ± 1°c for 24-48 h. single colonies were selected and plated on specific media according to the findings. for biochemical identification, the vitek2 system was used (biomérieux). finally, pcr methods were used in order to characterise c. perfringens toxins [42] . according to the identified etiologic agent, 5 categories were created: no agent detected, pure viral infections, mixed viral infections, bacterial infections, and mixed viral-bacterial infections. univariate models were used. e variables considered were lifestyle, age, gender, and breed. data were analyzed with an online software for statistical computation (vassarstats, vassar college). prevalence was calculated at a 95% confidence level. a chi-squared test of association was used to obtain the statistical significance level between groups, and risk assessment was performed with the odds ratio (or) test to confirm the difference between groups. results were considered statistically significant with a p value <0.05 and an or > 1.0. from january 2015 to december 2017, 511 necropsies and microbiological analyses were recorded. data were obtained from the izsm database, and a total of 138 dogs were identified to fulfil the inclusion criteria. ese dogs were under 1 year of age and comprised of 53 females (38.4%), 78 males (56.5%), and 7 of unknown sex (5.1%). ey belonged to 30 different breeds: 37 crossbreeds (26.8%) and 93 purebreds (67.3%). of the purebreds, the following breeds were recorded: 12 pomeranians (8.7%), 11 maltese (7.9%), 8 chow chows (5.8%), 6 each english bulldogs and chihuahuas (4.3% for each), 5 each labrador retrievers and yorkshire terriers (3.6%), 4 neapolitan mastiffs (2.9%), 3 each for fox terriers, german shepherds, husky, italian mastiffs, pointers and poodles (2.1%), 2 each for cavalier king charles spaniel, pinscher and shiba inu (1.4%), 1 each for maremma shepherd, pitbull, jack russell terrier, great dane, english setter, beagle, dogue de bordeaux, argentine mastiff, akita inu, and pug and dachshund (0.7%). for 8 reports (5.8%), however, no information about the breed was available. four variables were examined: "lifestyle," which included 70 ownerless dogs (50.7%), 33 strays that were housed in shelters (47.1%), and 37 that were confiscated by law enforcement for illegal importation (52.8%), and 68 dogs who lived in a house with their owner (49.2%). e "age" variable included 109 dogs under 6 months (78.9%) and 29 dogs from 6-12 months old (21%). real-time pcr, screening cpv/fplv [30] real-time pcr, cpv-2 based vaccine/cpv field strain [31] real-time pcr, antigenic variants cpv-2a/2b and cpv-2b/2c [32] canine coronaviruses (ccovs) real-time rt-pcr, screening ccovs [33] real-time rt-pcr, genotypes ccov-i/ccov-ii [34] real-time rt-pcr, subtypes ccov-iia/ccov-iib [35] canid herpesvirus 1 (chv-1) real-time pcr [36] canine distemper virus (cdv) real-time rt-pcr [37] rt-pcr [41] rotavirus (rv) real-time rt-pcr [38] canine adenoviruses (cav-1 and cav-2) pcr [39] real-time pcr [40] veterinary medicine international 3.1. necropsy examination. evaluation of the necropsy examination reports showed that the most frequently observed lesion was enteritis, which was found in 115 cases (83.3%), followed by pneumonia in 101 cases (73.1%), hepatitis in 65 cases (47.1%), encephalitis in 46 cases (33.3%), and finally myocarditis in 8 cases (5.8%). multiorgan lesions were found to be more prevalent than those on a single organ; these lesion types were observed in 116 (84%) and 16 cases (11.6%), respectively ( figure 1 ). in 6 cases, no lesions were observed (4.3%). e organs were submitted to a broad microbiological analysis, and the chi-square analysis showed that there was a different trend in the distribution of the lesions in the organs related to microbiologic categories (p < 0.0001). us, while in single-organ lesions there was a higher prevalence of pure viral infections (43.7%), for cases with lesions in 2 or more organs, mixed viral-bacterial infections were more prevalent. e distribution of the lesions for the variables-lifestyle, age, gender, and breed-was investigated too (table 2) , and a different trend was observed between ownerless and owned dogs (p � 0.013) as well as between crossbreed and purebred dogs (p � 0.0026). cases in which one or two organs were affected were more frequently observed in owned dogs, whereas instances with three-or-four-organ lesions were prevalent in the ownerless group of dogs. regarding the breed variable, in crossbreeds, two organs were more commonly affected (56.7%); in contrast, purebreds had more diffuse lesions on three (30.1%) and four organs (21.5%). no difference was observed for age (p � 0.1936) or gender variables (p � 0.2328). a univariate analysis of the correlation of microbiological categories with the four variables considered in this study was conducted (table 3) . a significant difference was observed in ownerless dogs, which were approximatively 3fold more predisposed to viral mixed infections (or � 3.24; p � 0.0117). in contrast, ownerless dogs were less frequently affected by bacterial infections than owned dogs (or � 0.2; p � 0.0120). for the breed variable, a statistically significant difference was observed in pure viral infections; thus, purebreds exhibited lower rates of pure viral infections than crossbreeds (or � 0.22; p � 0.0012). no difference was observed in the age or gender variables for any of the microbiological categories that were examined. our analysis of pathogens showed high prevalence of cpv (107/138; 77.5%), and it was frequently the causative agent of pure viral infections (27/33; 81.8%). furthermore, it was also the most prevalent virus involved in viral coinfections (26/26; 100%) and viral-bacterial coinfections (55/ 58; 94.8%). high prevalence of canine coronaviruses was also observed (43/138; 31.1%). although it was rarely observed in pure viral infections (4/33; 12.1%), canine coronaviruses were the second most prevalent agent involved in viral (18/ 26; 69.2%) and viral-bacterial (21/58; 36.2%) coinfections. a frequent association of this virus with cpv was observed in 79% (34/43) of ccovs-positive samples. a modest circulation of cavs and cdv was found in 23 (16.6%) and 19 (13.7%) pups, respectively. in contrast, chv-1 and canine rotavirus (rv) had a very low prevalence: 5 (3.6%) and 2 cases (1.4%), respectively. bacterial infections were observed in the 52.9% of cases (73/138), mainly associated with viral pathogens (58/138; 42%). e following bacteria were found to be most prevalent: escherichia coli was detected in 33 cases (23.9%), clostridium perfringens type a (cpa) in 25 cases (18.1%), and enterococcus spp. in 12 cases (8.7%). ey were rarely identified as single pathogens but were often involved in mixed infections, mostly associated with cpv. us, e. coli, cpa, and enterococcus spp. showed mixed infections with cpv in 75.7% (25/33), 80% (20/25), and in 100% of the cases, respectively. a very low prevalence was detected for other bacteria, which were mostly identified as opportunistic agents. after examining bacterial and viral prevalence, infection risk was examined for the most frequently detected pathogens related to the four variables considered in the study (table 4) . ownerless dogs showed a higher risk of infection to viral pathogens than owned dogs. e highest hazard risk was due to canine coronaviruses, with an odds ratio of 14.96 (p < 0.0001). when considering age as the variable, younger dogs, under 6 months, were less predisposed to cpv, cdv, and cpa than older dogs (with p values of 0.0444, 0.0007, and 0.0470, respectively). finally, purebred dogs showed a higher prevalence for e. coli infections than crossbreeds (or � 4.88; p � 0.0137). e relationship between pathogen and single/multiple organ (s) being affected was also investigated ( table 5 ). in single infections, cpv and ccovs were associated with a higher degree of lesion generalisation: 51.8% (14/27) in 2 organs and 100% (4/4) of the cases in 3 organs, respectively, whereas e. coli was more associated with localised infections in 42.8% of the cases (3/7). association of pathogens was observed to higher dissemination of the lesions. lastly, cpv, ccov, and cav variants were investigated. in 16 dogs (11.6%), a generalised cpv-vaccine strain was detected. in 15 of them, there was the copresence of other agents, and in 14 of them, there was also the cpv field strain. within the 38.4% of positive samples (53/107), cpv-2a was found to be the prevalent antigenic variant, followed by cpv-2b, which was found in 28.2% of the cases (39/107). in contrast, no cpv-2c alone was detected. an association of 2 variants was observed in 15/107 cases (10.8%). more specifically, we found 8 cases of cpv-2b and 2c, 5 cases of cpv-2a and 2b, and 2 cases of cpv-2a and 2c. for canine coronaviruses, type ii was the most prevalent, with 72.1% cases (31/43); of these, the ccov-iia variant was most common (28/31; 90.3%). finally, cav-2 had a higher prevalence than cav-1, with 87.8% (29/33) and 12.1% veterinary medicine international (4/33) of the cav infections, respectively; 1 case (3%) showed copresence of both strains. furthermore, in 16 cdv-positive samples, genetic characterisation was conducted on the hemagglutinin (h) glycoprotein, which showed a close match to arctic-like lineages, both in owned and ownerless dogs. breed, age, habitat, and stress are some of the risk factors that are known to predispose dogs to infections [3, 5, 8] . a compromised immune system, due to stress from overpopulation, long travels, or high levels of environmental contamination, can create conditions for viral infections and bacterial superinfections [9, 10] . defining the causative agent of death on the basis of clinical and/or necropsy data without ancillary tests is quite difficult because of the similarity of symptoms and lesions between pathogens [43] , and the presence of superinfectants can modify the original pathological findings of the diseases [44] . to assess infectious causes of death in dogs aged under 1 year of age, the circulation of agents in southern italy and whether age, lifestyle, gender, or breed can influence infections and relative lesion generalisation, a survey was conducted on 138 deceased pups. a strong association between generalisation of lesions and microbiological infections was observed in this study (p < 0.0001). necropsy examination showed that multipleveterinary medicine international organ lesions were more frequently detected (84%) than single-organ lesions (11.6%), mostly due to viral-bacterial coinfections. our results showed that multiple agents, with 2 or more pathogens detected, represented 62.31% of the cases. e presence of multiple agents creates conditions suitable for generalisation, because polymicrobism is reported to enhance other agent symptoms [18, 46] and diffusion of localised pathogens, as for coliform septicemia during parvoviral infection in puppies [17] . enteritis was the most prevalent lesion (83.3%) due to the high detection of enteric agents. in 6 necropsies (4.3%), no macroscopic sign was observed. it is likely that sudden death occurred (unpublished data), and histologic alterations could have been present, given that in 5 of these 6 cases (83.3%) one or more pathogens were detected, but this aspect was not investigated in our study. our study highlights the importance of lifestyle [1] ; indeed, in dogs of 0-1 year of age, a higher rate of coinfection is observed than in any other age group [9] . ownerless dogs showed a higher trend in lesion diffusion than dogs with owners (p � 0.013). environmental microbial contamination, lack of vaccination, overpopulation, and habitat are some of the risk factors reported to cause stress and predisposition to infections and coinfections [7, 8] . ese conditions are frequently identified in shelters. inappropriately constructed areas, dietary changes, and transport [47, 48] , together with continuous introduction of new animals, make kennels a place where exposure, transmission, and susceptibility to infections are more evident [8, 49] . e same stressful conditions are identified in other contexts too, such as with the illegal trading of puppies or in stray dogs. ownerless pups, in fact, showed a 3-fold higher risk for mixed viral infections (p � 0.0117), but they had lower susceptibility to bacterial infections (or � 0.2; p � 0.0120). e breed was also identified as a significant risk factor for the generalisation of the lesions (p � 0.0026). indeed, purebreds showed a higher prevalence for multiple-organ lesions than crossbreeds, in which lesions were found with 56.7% of double-organ afflictions but a lower rate of pure viral infections (or � 0.22; p � 0.0012). our survey showed high prevalence of cpv (77.5%) and canine coronaviruses (31.1%) that are described to be the main causative agent of enteritis worldwide [50] . eleni and colleagues in central-south italy demonstrated that cpv was the main causative agent of death in dogs under 1 year of age [27] . other studies in diarrheic dogs showed lower prevalence of this virus, with a range from the 16% up to 54.3% [10, 51, 52] . cpv is characterised by high morbidity (100%) and mortality (up to 91%) [53] ; thus, it is likely that the inclusion criteria of living dogs could be the main cause of difference with the other studies [10] , making it challenging to compare the results. it was also observed that all the three parvoviral antigenic variants are circulating in southern italy; cpv-2a was the most prominent genotype, followed by cpv-2b and 2c [54] . cpv was also the most common virus involved in coinfections and detected in 100% of mixed viral infections and in 94.8% of viral-bacterial ones. e second most prevalent agent was canine coronavirus (31.15%), which is consistent with earlier studies [10, 50] . as described earlier, a strong association of ccovs and cpv was observed [10] in 79% of the cases of coronavirus infection. when associated with other agents, coronavirus can cause either diarrhea by itself or exacerbate the symptoms of other viruses [18, 19, 55] . cav was found in 23.9% of cases, and the cav-2 strain was involved in 87.8% of the positive samples. lower prevalence of cdv (13.7%), chv-1 (3.6%), and rotavirus (1.4%) was observed. molecular typing of cdvs was conducted on the h glycoprotein, which is considered the best target for the determination of the lineages due to its high variability [56] . in italy, the europe-1/south africa-1 lineage is historically connected to cdv outbreaks; however, in the last 2 decades, the arctic-like lineage is spreading throughout the country among both domestic and wild animals [57] [58] [59] [60] . our study corroborates these data, since the arctic-like lineage was detected in cdv samples in both owned and ownerless dogs. is spread is speculated to be caused by dogs trading from east europe, where the lineage was first detected [57, 59] . lifestyle has been found to be an important risk factor for viral infections. ownerless dogs showed a higher predisposition to cpv than dogs with owners (or: 2.68; p � 0.0331), cav (or: 4.16; p � 0.0019), and ccov, which represented the highest hazard for this variable (or: 14.96; p < 0.0001), as assessed in other studies [8, 61, 62] . for the age variable, we did not observe the typical age trend of cpv infection, because these viruses are described to have the highest range of susceptibility from 3 to 6 months of age and are related to the decline of mda [56, 63] . in our study, younger dogs were less involved in parvoviral infections (p � 0.0444), as for cdv (p � 0.0007) [4] . another difference was observed for the breed and gender variable, which were, in general, associated with a higher risk for cpv and ccov infections [4, 10] . we thus speculated about the role of lifestyle, which could be a more important risk factor than other factors, and the low immune system defense caused by [63] [64] [65] . bacteria, mainly represented by enteropathogens, were rarely detected in pure infections (10.8%), and they were mostly associated with viruses in mixed infections (42%). above all, e. coli (23.9%), cpa (18.1%), and enterococcus spp. (8.7%) were the most common bacteria, followed by low percentages of other opportunistic bacteria. since many of them belong to normal enteric microflora, they were considered only when toxigenic or in cases of localisation other than the gut. dismicrobism, immunosuppression, and drug resistance are the predisposing causes for the passage of the bacteria from the gut to bile ducts and via the lymph-hematic route [14] [15] [16] that can lead to bacteremia [66] , as for overpopulation, high environmental contamination, and viral copresence. is causes young dogs to have more exposure to environmental bacteria; moreover, especially when vaccination is not provided, they are predisposed to infection and superinfection, given a lack of a completely competent immune system [9] . to the authors' knowledge, this is the first comprehensive survey on the circulation of several infectious agents in southern italy related to necropsy examination. our results indicate that lifestyle is an important risk factor for several viral pathogens and their generalisation, mostly identified in ownerless dogs that live in overcrowded habitat or are in contact with a stressful environment and intense conditions due to long travels. high circulation of enteric pathogens has been detected, with relative enteritis lesions in necropsy examination, mostly identified in cpv and ccov and frequently involved in coinfections. cpv has been found as the most common pathogen involved in viral and viralbacterial coinfections, highlighting its role in the suppression of the immune system. in conclusion, the application of a broad microbiologic diagnostic panels for the identification of infectious agents that are responsible for the death of young dogs is an important tool for understanding the presence of infectious agents in a determined area and to clarify their epidemiological role in the genesis of diseases that lead to death. in addition, it can also be used as support for intra vitam diagnosis and in the therapeutic approach of veterinarian practitioners. e data used to support the findings of this study are included within the supplementary information file. e authors declare that there are no conflicts of interest regarding the publication of this paper. immunodeficiencies caused by infectious diseases e pathological newborn in small animals: the neonate is not a small adult a comparative study on canine parvovirus infection of dog in bangladesh and india risk factors associated with parvovirus enteritis in dogs: 283 cases (1982-1991 canine parvovirus 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differentiation of cav-1 and cav-2 by polymerase chain reaction a duplex real-time pcr assay based on taqman technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2 phylogenetic characterization of canine distemper virus isolates from naturally infected dogs and a marten in korea determination of toxinotypes of environmental clostridium perfringens by polymerase chain reaction occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnostic dilemma canine influenza virus coinfection with staphylococcus pseudintermedius enhances bacterial colonization, virus load and clinical presentation in mice streptococcal infection in dogs: a retrospective study of 393 cases canine respiratory viruses behavioural and physiological responses of dogs entering re-homing kennels behavioural and glucocorticoid responses of dogs (canis familiaris) to kennelling: investigating mitigation of stress by prior habituation enteropathogens identified in dogs entering a florida animal shelter with veterinary medicine international 9 normal feces or diarrhea western european epidemiological survey for parvovirus and coronavirus infections in dogs comparison of the prevalence of enteric viruses in healthy dogs and those with acute hemorrhagic diarrhea by electron microscopy identification of enteric viruses circulating in a dog population with low vaccine coverage status-report-canine viral-enteritis molecular epidemiology of canine parvovirus type 2 in italy from 1994 to 2017: recurrence of the cpv-2b variant infectious canine hepatitis: an "old" disease remerging in italy canine distemper virus heterogeneity within the hemagglutinin genes of canine distemper virus (cdv) strains detected in italy a distinct cdv genotype causing a major endemic in alpine wildlife update on canine distemper virus (cdv) strains of arctic-like lineage detected in dogs in italy occurrence of different canine distemper virus lineages in italian dogs canine parvovirus: current perspective risk factors for delays between intake and veterinary approval for adoption on medical grounds in shelter puppies and kittens canine parvoviral enteritis; an update on clinical diagnosis, treatment and prevention effect of canine parvovirus strain variations on diagnostic test results and clinical management of enteritis in dogs factors affecting the occurrence, duration of hospitalization and final outcome in canine parvovirus infection role of the gut in multiple organ failure acknowledgments e authors would like to thank mr. domenico giudice for informatics support. e supplementary material contains raw data on age, breed, gender, and lifestyle of the dogs and the results of microbiological and necropsy examination. (supplementary materials) key: cord-300187-fr6tme32 authors: kearns, shawn title: infectious hepatopathies in dogs and cats date: 2009-11-26 journal: top companion anim med doi: 10.1053/j.tcam.2009.06.004 sha: doc_id: 300187 cord_uid: fr6tme32 this article serves to review the various infectious diseases that affect the liver primarily or as a part of systemic infection. although bacterial infections are probably the most common cause of infectious hepatitis, the clinician should be aware of other potential organisms and other commonly involved systems. therefore, this article includes a description of common bacterial, mycobacterial, viral, fungal, protozoal, parasitic, and rickettsial diseases in dogs and cats. alimentary flora circulates to the liver under various clinical conditions. these bacteria are extracted by kupffer cells, killed by neutrophils, or excreted in bile in healthy clinical states. a low-flow, low-pressure perfusion of hepatic sinusoids may allow superior removal of bacteria by phagocytes, and pressure differentials in the biliary system may limit retrograde access of enteric organisms. 2, 4, 5 changes in this sinusoidal flow may decrease the effectiveness of phagocytosis when portal flow is compromised. bowel disease, cholestasis, immunosuppression, and altered gut motility result in altered portal circulation, and the subsequently unchecked bacterial access to the liver may result in bacterial hepatitis or cholangiohepatitis. common isolates implicated in bacterial hepatitis and cholecystitis include escherichia coli, enterococcus spp, bacteroides spp, streptococcus spp, and clostridium spp. 6 cultures can be obtained by liver aspirate, liver biopsy, and cholecystocentesis. a combination of liver and gall bladder samples (fig 1) may increase the likelihood of identification of the offending organism(s). surgical or laparoscopic biopsies may be more rewarding for culture growth compared with aspirates. 6 in suspected cases, broad-spectrum antibiotics for common enteric isolates should be initiated pending specific culture results. focal micro-and macro-abscesses have also been documented in dogs and cats. 7, 8 predisposing causes include alterations in blood flow, trauma, ascending biliary infections, liver lobe torsions, 9 immunocompromised clinical states, 10 and neoplasia. 7, 11 microabscesses are often identified in association with extrahepatic infection and sepsis. 7, 12 ultrasound has greatly enhanced the early diagnosis of hepatic abscesses. 8 greater than 50% of solitary abscesses are polymicrobial. antimicrobial treatment should be directed at both anaerobes and aerobes regardless of whether anaerobic cultures are negative if a polymicrobial hepatic infection is documented. 2 bacterial isolates in hepatic abscesses are similar to those identified in diffuse bacterial hepatic disease. however, clinically rare isolates including klebsiella, listeria, salmonella, brucella, yersinia pseudotuberculosis, actinomyces, nocardia, and pasturella have also been documented. 13 focal abscesses may require surgical drainage and antibiotic therapy. treatment in all cases must be implemented for a minimum of 6 to 8 weeks. leptopirosis is an extremely common nonenteric bacterial infection in the canine liver. leptospires are thin, filamentous, spiral-shaped motile bacteria with a lipopolysaccharide outer envelope. direct transmission occurs via contact with infected urine, venereal and placental tissues, or fluids. indirect transmission can occur through contaminated water sources, soil, food, or bedding. the organism can stay stable for several months with the right environmental conditions. the organism initially penetrates the mucous membranes and rapidly multiplies after entry into the vascular space. dissemination and replication occur in many tissues, including the liver. however, the organism tends to persist in the kidney and can be shed for weeks to months after infection. certain serovars are more frequently associated with hepatic involvement and include leptospira icterohaemorrhagiae and l. pomona. young dogs (ͻ6 months of age) seem to develop signs of hepatic dysfunction more frequently in disease outbreaks. 14 profound hepatic dysfunction may occur without significant histologic changes because of subcellular damage produced by bacterial toxins. the endothelial damage, subsequent thrombosis, and possible disseminated intravascular coagulation seen in acute disease may contribute to hepatic damage. chronic hepatitis has been reported as a sequelae to leptospiral infection. 15, 16 diagnosis is usually made based on clinical signs and serologic titers. however, leptospirosis polymerase chain reaction (pcr) performed be-fore treatment may increase testing sensitivity given vaccinal interference and delayed seroconversion in the acute phase. 17 penicillins are the treatment of choice in the acute phase and must be followed by appropriate antibiotics to eliminate the carrier state. alternatively, doxycycline may be used for both the acute and carrier states. bartonella spp are gram-negative fastidious bacteria and are well adapted for the intracellular environment. a recent case report documented b. henselae and b. clarridgeiae dna in the liver of 2 dogs with granulomatous inflammation. both had a positive clinical response to azithromycin and demonstrated biochemical reduction in hepatocellular enzymes. 18 another dog with peliosis hepatitis (a rare vascular condition characterized by multiple, randomly distributed blood-filled cavities throughout the liver) had b. henselae dna amplified from multiple hepatic specimens by pcr. 19 helicobacter canis has been isolated from the liver of a single dog with hepatitis. 20 helicobacter spp have also been amplified from hepatic tissue in cats with cholangiohepatitis. further studies are required to determine whether these organisms are associated with inflammatory liver disease. these organisms are difficult to culture, and this failure may reflect the fastidious nature of these bacteria. pcr positivity may reflect the presence of intestinal helicobacter from the enterohepatic circulation or transient colonization rather than a true disease association. 21 francisella tularensis (tularemia) is a pleomorphic, gramnegative, nonspore-forming bacillus. this disease frequently occurs as a result of exposure to ticks or wildlife. macrophages are the primary host cells, and bacteremia with multiorgan involvement is common. lungs, spleen, liver, and skin are common sites for embolic spread, resulting in microabscesses and granulomatous disease. puppies and young cats appear more susceptible to infection, and dogs are generally more resistant to infection. clinical findings include depression, oral/lingual ulceration, regional or generalized lymphadenomegaly, hepatosplenomegaly, panleukopenia with severe toxic neutrophil changes, hyperbilirubinemia, and bilirubinuria. [22] [23] [24] examination for evidence of microscopic agglutinating antibody is most frequently used for diagnosis, although indirect fluorescent antibody testing may be useful as well. 24 aminoglycosides are the primary treatment in humans. however, tetracyclines (doxycycline), chloramphenicol, and quinolones are commonly used in dogs and cats. unfortunately, clinical relapse is common with these antibiotics. tyzzer's disease (clostridium piliforme) is caused by a flagellated, spore-forming, gram-negative intracellular parasite. although spores have been identified in rodent species, interspecies transmission via ingested feces has not been documented. however, spontaneous disease has been documented in dogs and cats. [25] [26] [27] [28] colonization of the liver results in multifocal, periportal hepatic necrosis and may result from a currently unidentified toxin. 29 minimal inflammation may be present despite extensive necrosis. 30 death usually occurs within 24 to 48 hours once the organism is in the liver. [31] [32] [33] [34] [35] [36] [37] rhodococcus is a soil-borne pleomorphic, gram-positive bacteria normally associated with suppurative infections in figure 1 . fine-needle aspirate and cytology from the gallbladder of a cat with cholangiohepatitis. the aspirate consists predominantly of bacteria of mixed type. the bacteria are frequently present in chains (black arrow). also, note dark brown-staining amorphous material (bile pigment: yellow arrow). the finding of bacteria in cytologic specimens of bile is considered abnormal. the following organisms were cultured from the bile: escherichia coli, streptococcus pneumoniae, an anaerobic bacterial rod, prevotella oralis, and a gram-positive rod that could not be classified. courtesy of the pathology department, angell animal medical center, boston, massachusetts. domestic livestock. inhalation from soil or wound inoculation are the suspected routes of transmission. disseminated infection and death have been reported in a single dog. 38 clinical reports are rare in cats. mycobacterium spp are aerobic, nonspore-forming, nonmotile bacteria with a wide host affinity and pathogenic potential. they are typically classified based on growth in culture and by the pathologic production of tubercles or granulomatous disease. mycobacterium tuberculosis and m. bovis are the most pathogenic, and humans are reservoirs for these species. aerosolized organisms in sputum are considered the primary mode of transmission. however, m. bovis can be acquired via uncooked meats and wildlife reservoirs. mycobacterial disease is often subclinical in dogs and cats, but signs may be associated with granuloma formation in various organs. 39, 40 nontuberculous mycobacterium, including those in the mycobacterium avium complex, are saprophytic opportunistic organisms primarily implicated in disseminated disease in cats [41] [42] [43] [44] [45] and occasionally in dogs. 46 -53 no clear associations have been identified with retroviral diseases. canine and feline breeds with potentially increased susceptibility include basset hounds, 51 miniature schnauzers, 53 siamese, 45 and abyssinians. 42 dogs with m. avium complex-induced disease will often demonstrate extensive granulomatous disease of the intestine, spleen, liver, and mesenteric lymph nodes. animals undergoing immunosuppressive drug therapy with inhibition of cell-mediated immunity may be at risk for disseminated disease, including renal transplant patients. 43 acidfast cytology can demonstrate bacilli, although false negatives can occur. negative bacterial images may be identified on routine stains (fig 2) . pcr may provide greater sensitivity and safety than culture. 52 combination therapies are often required, because organisms build resistance quickly, particularly with disseminated disease. although not a risk for immunocompetent individuals, dogs and cats infected with saprophytic mycobacterium pose a risk for immunodeficient people. mycobacterium lepraemurium was considered the main causative agent for feline leprosy until recently. however, m. visibilis has been associated with feline multisystemic granulomatous mycobacteriosis, resulting in diffuse cutaneous disease and widespread dissemination to multiple internal organs. 54 organisms responsible for disseminated fungal infections include histoplasma capsulatum, coccidioides immitis, blastomyces dermatitides, aspergillosis sp, cryptococcussp, and sporothrix schenckii. most are dimorphic, saprophytic, opportunistic fungi that exist in the mycelial stage in the environment. spores are produced in the mycelial stage and be-come pathogenic on inhalation, ingestion, or inoculation. dissemination occurs via the hemolymphatic system. specific environmental conditions are required for the individual organisms, and this dictates their geographic range. histoplasma capsulatum is located primarily in the temperate and subtropical areas of the world. organisms are phagocytized by mononuclear cells and replicate intracellularly once they are inhaled and converted to the yeast phase. the primary clinical signs in dogs are associated with the gastrointestinal system (diarrhea, tenesmus, mucous, fresh bloods in stools). clinical signs in cats are vague. dissemination to other visceral organs (including the liver) has been documented in both species. [55] [56] [57] clinically affected animals are usually young (1-4 years of age). diagnosis is usually achieved with fine-needle aspirate or exfoliative cytology of affected organs. aspergillosis is primarily associated with rhinitis. however, several reports have documented systemic infections in german shepherds and in non-shepherd breeds. aspergillus terreus 58 -62 and a. deflectus 63, 64 have been most frequently implicated in systemic infection. predisposing factors include optimal climatic conditions, access to a partial strain, or subtle defects in mucosal immunity. 65 disseminated aspergillosis has also been documented in cats. 66, 67 neurologic deficits, spinal column pain, urinary system disorders, and respiratory pathology are the primary presenting clinical signs. prototheca is a saprophytic, achlorophyllous alga found in the southeastern united states. three species of prototheca have been identified, but p. zopfii is the only one associated with disseminated disease. the organism is associated with sewage, slime flux of trees, and animal waste. transmission generally occurs through ingestion or penetration of injured skin or mucosa. disease can develop with diminished host resistance or concurrent diseases. 68 concomitant large intestinal diarrhea and ocular signs should prompt clinical consideration of prototheca infection. dissemination via blood or lymph to other organs including the liver is common. various stages of development of the organism may be identified on cytology or histopathology. urine culture and sediment are also useful in organism identification. 69 this disease is invariably fatal, although disease progression may be delayed with various antifungal and antibacterial agents. 70 -73 coccidioides immitus is a dimorphic fungus with preference for the alkaline sandy soil environment found in the lower sonoran life zone in the southwestern united states, western mexico, and central and south america. mycelia are produced during rainfall, but arthroconidia develop with soil drying and become airborne under dry and windy conditions. inhalation is the primary mode of infection in dogs and cats. the spherule (tissue parasitic form) undergoes division with eventual rupture. the severity and extent of clinical disease depend on immunocompetence and range from a mild, asymptomatic, pulmonic form to severe, life-threatening disseminated disease. dissemination most commonly involves the axial and appendicular skeleton and overlying skin. tissues from abdominal viscera, the central nervous system (cns), pericardium, myocardium, and prostate can also be involved. 74, 75 cytology or histology may reveal spherules, although diagnosis is often made based on history, clinical signs, and positive serology. antigens for sero-testing commonly use tube precipitin and complement fixation with agar gel immunodiffusion. sporothrix schenckii causes a chronic granulomatous disease of worldwide distribution. infection is usually the result of trauma and inoculation with infective conidiophores. the skin is the primary target organ. however, disseminated disease has been reported, particularly in the cat. no clear dissemination pattern has been identified because of low case numbers, but affected organs include the internal lymph nodes, liver, lungs, eyes, bone, muscles, and cns. 76, 77 diagnosis is frequently made by cytology. blastomyces dermatitidis is found primarily in mississippi, missouri, the ohio river valley, the mid-atlantic states, and some canadian provinces. growth of the organism requires sandy, acidic soil with some proximity to water. preferred sites for dissemination include the skin, eyes, bones, and lymph nodes, although dissemination to the liver has been reported. 78, 79 cryptococcus neoformans has a worldwide distribution. inhalation may be the primary mode of infection, and sites of infection tend to be areas of the body with cooler temperatures, including the respiratory passages and subcutaneous tissues. the fungus is occasionally disseminated to the kidneys and rarely to the liver. 80 treatment of most disseminated fungal infections involves the use of triazoles, including itraconazole and fluconazole, as well as amphoterocin b. 59,81-85 clinical signs may resolve in many cases, but relapses occur and patients with severe clinical illness generally have a poor prognosis. leishmania, transmitted by the sandfly (lutzomyia in the new world, phlebotymus in the old world), frequently causes cutaneous and visceral lesions in the dog. promastigotes transmitted by the female sandflies become amastigotes in the vertebrate and are phagocytized by mononuclear cells. the organism travels through hemolymph organs to remote dermal sites and other organs. clinical signs will not develop in all exposed animals, and the immune response at the time of infection appears important in determining development of disease. leishmania infection should be considered in dogs from endemic areas with marked hyperglobulinemia or in those with a travel history to endemic areas. mild increases in liver enzymes are often noted. however, unlike the kidneys, the liver is not a primary target organ. infection can be associated with chronic hepatitis. 86 definitive diagnosis is made by demonstration of organisms on cytology or histopathology, or by serology, culture, or pcr. amphotericin b in a soybean oil lipid emulsion has been intravenously administered for higher clinical cure success rates and greater numbers of negative posttreatment parasitologic tests compared with other treatments. other less successful treatment options include allopurinol and the pentavalent antimonials. 87 hepatozoon canis is a worldwide protozoal disease reported in domestic dogs and is most prevalent in subtropical and temperate climates. the primary vector is the rhipicephalus sanguineous tick, which is primarily located in warm and temperate regions. transmission occurs through ingestion of ticks containing mature protozoal oocyts. sporozoites are released in the intestine on tick ingestion and penetrate the gut wall, invade mononuclear cells, and disseminate. target organs include the bone marrow, spleen, and lymph nodes but can involve other internal organs such as the liver, kidney, and lungs. 88, 89 the most striking clinicopathologic abnormality is leukocytosis with evidence of parasitemia of the white cells on peripheral blood smears. clinical findings can range from incidental hematologic findings to severe life-threatening illness. hepatitis, glomerulonephritis, and pneumonitis have all resulted from h. canis infection. 90 coinfections with other protozoal diseases (toxoplasma, leishmania, and babesia spp) or other tick-borne diseases (ehrlichia spp) and immunosuppressive states can predispose animals to clinical illness. the hepatitis is associated with developing meronts within the liver and their associated neutrophilic and mononuclear inflammation. hepatozoon has also been documented in felines. [91] [92] [93] microscopic detection of gamonts in peripheral blood smears is the most frequently used diagnostic test. imidocarb is the treatment of choice in dogs. subcutaneous or intramuscular injections are administered every 14 days until gamonts are no longer visualized in the leukocytes. a new species, hepatozoon americanum, was identified in 1997, with the amblyomma maculatum tick as its definitive host. 94 this emerging disease has spread to the north and the east since its initial identification in the gulf coast region. clinical signs are often severe, even in the absence of other diseases or in the presence of immunosuppression. waxing and waning clinical signs are attributed to repeated cycles of asexual reproduction and pyogranulomatous inflammation. the primary site of infection for the merozoites is the cardiac and skeletal muscle. however, single zoites can enter circulation and reproduce asexually at distant locations. 95 diagnosis is most often made with muscle biopsy, although a recent study has identified promise in the use of pcr testing. 96 an enzyme-linked immunosorbent assay has been developed with sporozoites as the antigen. 97 no treatment effectively eliminates the tissue stages of h. americanum. however, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and an excellent quality of life. 98 the microsporidial parasite encephalitozoon cuniculi is an obligate intracellular protozoan. infection likely occurs by inhalation or ingestion of spores from contaminated urine or feces shed by infected hosts. the organism undergoes asexual reproduction or binary fission after infecting host cells and ruptures, leading to infection of new cells or shedding of resistant spores into the environment. typical organs of localized infection include the kidney, liver, lungs, and brain with resultant granulomatous inflammation. 99, 100 cats and older dogs are not commonly affected, and renal disease predominates in young dogs. cytological examination of fluids (particularly urine) is important in making a diagnosis in animals with disseminated disease as other tests are commercially unavailable. cytauxzoon felis is a tick-borne protozoal disease of domestic and wild cats. the bobcat is the natural reservoir in north america and is usually asymptomatic despite persistent erythroparasitemia. the tissue phase of infection consists of the development of large schizonts in mononuclear phagocytes. the schizonts line the lumens of vessels in most organs, eventually leading to vessel occlusion. merozoites are released into blood or tissue fluid once the host cells rupture and infect red blood cells. late-stage parasitemia can often be detected on blood films at about 1 to 3 days before death. most clinical signs, including those associated with liver abnormalities, are due to schizont-associated mechanical obstruction. however, parasite by-products may also be toxic, pyogenic, and vasoactive. the anemia is regenerative but mild in comparison with clinical icterus. this may be useful in differentiating this infection from hemotropic mycoplasmas. demonstration of piroplasms in wright's-stained or giemsa-stained blood films most frequently provides a definitive diagnosis. histopathology reveals schizont-laden mononuclear phagocytes in the veins of the lungs, liver, and spleen. the prognosis is generally considered poor, but different geographic strains may have varying virulence. 101 treatment with diminazene or imidocarb has been somewhat successful. 102 toxoplasma gondii is an obligate intracellular coccidian parasite that infects almost all warm-blooded animals. do-mestic cats are the definitive hosts and excrete the infective oocyts. three stages of the life cycle are considered infectious, including oocyst sporozoites, tissue cyst tachyzoites, and tissue cyst bradyzoites. transmission can occur through ingestion of oocysts or tissue cysts and via congenital transmission. other reported modes include lactation, transfusions, and transplantation. 103 a higher frequency of disease is reported in dogs and cats fed raw meat or those in a rural/ feral environment. the extra-intestinal life cycle is the same in all hosts, and sporozoites encyst in the intestinal lumen, penetrate cells, and divide into tachyzoites. the tachyzoites can form cysts in the cns, muscle, and visceral organs, and may persist for the life of the host. clinical signs were diverse in 100 cats with histologically confirmed toxoplasmosis, and more than 90% had pulmonary, cns, and liver manifestations. 104, 105 in dogs, disseminated infection is most often associated with canine distemper, other infections including ehrlichiosis and immunosuppression, or vaccination with live attenuated vaccines. 106 clinical cases in cats have been seen with steroids, cyclosporine use, hemotropic mycoplasms, and viral disease. [107] [108] [109] liver and lung involvement is associated with quicker mortality than other organ involvement. tachyzoites may be detected on cytology of various organs and body fluids. however, diagnosis is most frequently based on clinical signs, serology (immunoglobulin g, immunoglobulin m), and response to treatment. clindamycin is the treatment of choice. neospora caninum is a protozoan similar to toxoplasma. dogs and coyotes are considered definitive hosts, and deer and cattle are intermediate hosts. the predominant mode of transmission is transplacental in the dog, and clinical signs are usually secondary to exacerbation of a congenital infection. acute phases of infection include widespread dissemination to many organs, including the liver, whereas chronically infected animals are restricted to muscular and neuronal sites. 103 serology and muscle biopsy often provide a diagnosis, although tachyzoites may be detected in other parasitized tissue or body fluid. 110 sarcocystis canis is an apicomplexan protozoan with no particular geographic distribution. infection results in disseminated disease, including protozoal hepatitis. 111, 112 many reports involve puppies, suggesting the presence of congenital infection. however, the life cycle is still unknown. sarcocystis canis is the only sarcocystis species known to form schizonts in canine tissue. infectious canine hepatitis (ich) is caused by adenovirus type 1. this is the only virus with primary tropism for the liver. 113 infection leads to severe hepatic necrosis and can also cause ocular and renal changes. the virus localizes in the tonsils after oronasal exposure, spreads to regional lymph nodes, and disseminates via the thoracic duct. hepatic parenchymal cells and vascular endothelial cells are the prime targets of viral localization, and injury leading to centrilobular to panlobular hepatic necrosis ranges from self-limiting to fatal. most affected dogs are less than 1 year of age and unvaccinated. severely affected dogs can become moribund and die within hours of disease onset and with few predictive clinical signs. if patients survive the acute phase, they may develop clinical signs including vomiting, diarrhea, and abdominal pain. 114, 115 those that survive may go on to develop chronic hepatitis and fibrosis, likely secondary to self-perpetuating liver inflammation rather than chronic infection. 116 diagnosis is frequently made based on clinical signs and serology, although the virus can be isolated in cell cultures. this disease is rarely encountered because of the high efficacy of vaccination. canine acidophil hepatitis is believed to be caused by a viral agent. however, the specific agent is not yet identified. disease has been reproduced via injections of sterile liver homongenates from spontaneously affected animals. acute infections can lead to acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. diagnosis is made on histology because acidophils are scattered throughout lesions. this disease has only been reported in great britain. 117, 118 canine herpesvirus causes tissue necrosis and localized mucosal or generalized systemic infections in young or immunocompromised animals. the virus only infects dogs because of specific cell-surface receptors. replication occurs via viral dna synthesis within the host nucleus. transmission occurs through direct contact with mucosal secretions from the respiratory or genital tract of animals. factors predisposing to infection in puppies include hypothermia and a poorly developed immune system. newborns can acquire disease in utero, during passage through the birth canal, during contact with infected littermates, from oronasal secretions of the dam, and from fomites. puppies less than 1 week of age are more susceptible to generalized fatal infections. dissemination leads to hemorrhagic necrosis in several organs including the adrenal glands, kidney, liver, lungs, and spleen. clinical signs include loss of interest in nursing, loss of body weight, soft yellow-green feces, abdominal discomfort, and dullness. a marked increase in alanine aminotransferase is often noted on biochemistry profile. definitive diagnosis is by viral isolation. 119 feline leukemia virus is a single-stranded retrovirus that replicates in many tissues. clinical illness is generally related to the hematopoietic system and the immune system. feline leukemia virus has also been associated with icterus and various inflammatory and degenerative liver diseases including focal liver necrosis. 120 feline infectious peritonitis (fip) is a feline coronavirus that has undergone frequent rna mutations, resulting in an ability to enter and replicate in macrophages. an immunemediated vasculitis occurs if the virus is not eliminated. affected cats develop signs related to target organ lesions (kidney, liver, cns, intestine) or due to fluid redistribution. abnormal liver enzymes can occur because of hepatitis, hepatic lipidosis, or prehepatic sequalae of vasculitis, erythrocyte destruction, and hypoxia. hyperbilirubinemia is common and usually secondary to vasculitis in the liver. 121 histopathology is required for definitive diagnosis but is sup-ported by history, physical examination, and laboratory findings. a new pcr test may also prove useful in the diagnosis of fip. 122 treatment is generally unrewarding. conflicting information exists on the usefulness of feline recombinant interferon, although it may be beneficial for a subpopulation of fip-infected cats. 123, 124 rickettsial diseases the most common agents encountered in dogs with clinical evidence of liver involvement include the ehrlichia sp, rickettsia rickettsii, and borrelia burgdorferi. these organisms can infect either hepatocytes or endothelial cells. hepatic involvement in erhlichia infections occurs in more than 80% of human patients, leading to mild transient increases in transaminases. 125 liver injury may be related to organism proliferation in hepatocytes and stimulation of immunologic and nonspecific inflammatory mechanisms. rocky mountain spotted fever is vasculotropic in nature and can cause moderate increases in transaminases. experimental evidence with borrelia suggests direct hepatic invasion by the spirochetes in conjunction with cellular and humoral immunologic mechanisms. 126 an association with borrelia was observed and confirmed with liver biopsy in 2 dogs. lesions were consistent with lobular dissecting hepatitis and mixed multifocal inflammation leading to focal pyogranulomas in the other. 2 chronic cholangitis associated with liver fluke infestation in endemic areas is primarily observed in cats and less frequently in dogs. most infections are due to opistorchus and metorchis, which require 2 intermediate hosts. the first hosts are water snails, and the second hosts include a wide variety of fish with encysted metacercariae. the final host acquires infection by ingestion of fish, and the young liver flukes migrate to the liver through the bile ducts. this results in bile duct thickening and dilation. rarely, cysts may be formed as well. 127 a slight to moderate inflammation may be seen both within the ducts and in the portal areas. although eosinophils may be present, they are usually limited in numbers. the number of liver flukes and eggs within the dilated bile ducts varies markedly, and often only limited evidence of liver flukes or eggs is identified. platynosomum concinnum is a trematode of the feline biliary system. terrestrial snails, lizards, toads, and terrestrial isopods act as intermediate hosts based on geographic location. disease is most prevalent in the tropical and subtropical climates. clinical cases involve adult indoor or indoor-outdoor cats. the severity of clinical signs is proportional to the number of adult flukes as well as to the duration of parasitemia. early diagnosis can be difficult. however, diagnosis is easier when eggs have been identified in the bile. 128 treatment of p. concinnum and liver fluke infections is best accomplished with praziquantel. there are many infectious diseases that ultimately affect the liver. few, however, have primary tropism for hepatic tissue. testing should be directed based on signalment, geographic locale, and primary presenting complaint. cytology and/or histopathology of the liver will most frequently provide a definitive diagnosis in clinical situations with liver involvement. the prognosis is guarded with many disseminated infections. toxic, metabolic, infectious, and neoplastic liver diseases infectious diseases of the dog and cat detection of portal and systemic bacteremia in dogs with severe induced hepatic disease and multiple portosystemic shunts diseases of the gallbladder and biliary tree cholangitis/cholangiohepatitis complex in the cat bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease: 1998-2003 hepatic abscesses in cats: 14 cases hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options liver lobe torsion and liver abscess in a dog hepatic abscesses associated with diabetes mellitus in two dogs hepatocellular carcinoma with secondary abscessation in a cat hepatic abscesses in dogs hepatic abscesses in dogs: 14 cases (1982-1994) infectious diseases of the dog and cat chronic active hepatitis in dogs associated with leptospires chronic hepatitis associated with leptospiral infection in vaccinated beagles clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease peliosis hepatis in a dog infected with bartonella henselae helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis association of helicobacter with cholangiohepatitis in cats feline tularemia on tularemia in a cat acute tularemia in three domestic cats bacillus piliformis infection in an adult dog tyzzer's disease in kittens with familial primary hyperlipoproteinaemia tyzzer's disease in a puppy tyzzer's disease in puppies infectious diseases of the dog and cat the liver in systemic disease. an innocent bystander naturally occurring tyzzer's disease as a complication of distemper and mycotic pneumonia in a dog tyzzer's disease in a dog tyzzer's disease in an adult cat naturally occurring tyzzer's disease in a cat naturally occurring tyzzer's disease in a puppy tyzzer's disease complicated with distemper in a puppy tyzzer's disease in puppies vapa-negative rhodococcus equi in a dog with necrotizing pyogranulomatous hepatitis, osteomyelitis, and myositis mycobacterium tuberculosis complex infection in a dog putative transmission of mycobacterium tuberculosis infection from a human to a dog disseminated mycobacterium avium infection in a cat disseminated mycobacterium avium infection in young cats: overrepresentation of abyssinian cats disseminated mycobacterium avium complex infection following renal transplantation in a cat tuberculosis in cats disseminated mycobacterium avium complex infection in three siamese cats fatal mycobacteriosis with hepatosplenomegaly in a young dog due to mycobacterium avium a case of disseminated tuberculosis in a dog caused by mycobacterium avium-intracellulare systemic mycobacterium smegmatis infection in a dog disseminated mycobacterium avium infection in a dog disseminated mycobacterium avium complex infection in a dog tuberculosis in five basset hounds systemic mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat disseminated mycobacterium avium infection in three miniature schnauzer litter mates histologic and genotypic characterization of a novel mycobacterium species found in three cats atypical histoplasma capsulatum infection in a dog disseminated histoplasmosis in dogs: 12 cases (1981-1986) disseminated histoplasmosis in cats: 12 cases (1981-1986) disseminated aspergillosis in two dogs in israel long-term survival of four dogs with disseminated aspergillus terreus infection treated with itraconazole disseminated aspergillus terreus infection in a dog disseminated aspergillosis in a dog with diskospondylitis and neurologic deficits disseminated aspergillosis in a dog systemic mycosis due to aspergillus deflectus in a dog disseminated aspergillosis attributable to aspergillus deflectus in a springer spaniel canine disseminated aspergillosis systemic aspergillosis and mucormycosis in 23 cats feline disseminated aspergillosis altered immune function in a dog with disseminated protothecosis urinary tract manifestations of protothecosis in dogs more than meets the eye: subretinal aspirate from an acutely blind dog disseminated protothecosis causing acute blindness and deafness in a dog disseminated protothecosis in a dog infectious diseases of the dog and cat deep mycotic infections in cats disseminated coccidioidomycosis in a dog pathology of sporotrichosis in 10 cats in rio de janeiro disseminated sporotrichosis in a cat ocular changes in a cat with disseminated blastomycosis legendre am: blastomycosis, in greene ce fatal disseminated cryptococcosis and concurrent ehrlichiosis in a dog disseminated opportunistic fungal disease in dogs: 10 cases infectious diseases of the dog and cat cryptococcosis coccidioidomycosis and paracoccidioidomycosis infectious diseases of the dog and cat chronic hepatitis associated with canine leishmaniosis (leishmania infantum): a clinicopathological study of 26 cases initial and long term efficacy of a lipid emulsion of amphotericin b desoxycholate in the management of canine leishmaniasis hepatozoon canis infection in two dogs canine hepatozoonosis in oklahoma hepatozoon canis infection feline hepatozoonosis granulomatous cholangiohepatitis in a cat due to a protozoan parasite resembling hepatozoon canis hepatozoon species infection in domestic cats: a retrospective study a new hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in north america characterization of stages of hepatozoon americanum and of parasitized canine host cells diagnosis of canine hepatozoon spp. infection by quantitative pcr an indirect enzymelinked immunosorbent assay for diagnosis of american canine hepatozoonosis treatment of dogs infected with hepatozoon americanum: 53 cases (1989-1998) mammalian microsporidiosis experimental encephalitozoonosis in neonatal dogs cats surviving natural infection with cytauxzoon felis: 18 cases (1997-1998) administration of diminazene aceturate or imidocarb dipropionate for treatment of cytauxzoonosis in cats toxoplasmosis and neosporosis infectious diseases of the dog and cat acute primary toxoplasmic hepatitis in an adult cat shedding toxoplasma gondii oocysts fatal toxoplasmosis in five cats acute toxoplasmosis following renal transplantation in three cats and a dog feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis neonatal toxoplasmosis in littermate cats histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952-1990) neospora caninum associated with septic peritonitis in an adult dog fatal cutaneous and visceral infection in a rottweiler dog associated with a sarcocystis-like protozoan fatal hepatic sarcocystosis in a puppy with eosinophilia and eosinophilic peritoneal effusion canine viral diseases viral hepatitis of dogs (rubarth's disease) infectious canine hepatitis (hepatitis contagiosa canis use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis a new transmissible agent causing acute hepatitis, chronic hepatitis, and cirrhosis in dogs persistent hepatitis and chronic fibrosis induced by canine acidophil cell hepatitis virus canine herpesvirus frequency and significance of feline leukemia virus infection in necropsied cats feline infectious peritonitis and feline enteric coronavirus feline infectious peritonitis: typical findings and a new pcr test use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis effect of feline interferonomega on the survival time and quality of life of cats with feline infectious peritonitis ehrlichial diseases of humans: emerging tickborne infections host-pathogen interactions in the immunopathogenesis of lyme disease severe cholestatic liver disease secondary to liver fluke key: cord-279551-py2awuav authors: willi, barbara; spiri, andrea m.; meli, marina l.; grimm, felix; beatrice, laura; riond, barbara; bley, tim; jordi, rolf; dennler, matthias; hofmann-lehmann, regina title: clinical and molecular investigation of a canine distemper outbreak and vector-borne infections in a group of rescue dogs imported from hungary to switzerland date: 2015-07-16 journal: bmc vet res doi: 10.1186/s12917-015-0471-0 sha: doc_id: 279551 cord_uid: py2awuav background: canine distemper virus (cdv) is a major pathogen of dogs and wild carnivores worldwide. in switzerland, distemper in domestic dogs is rarely reported. in recent years, the import of dogs from eastern europe to switzerland has steadily increased. in the present study, we describe a distemper outbreak in 15 rescue dogs that were imported from hungary to switzerland by an animal welfare organisation. the data on vaccination and medical history were recorded (14 dogs), and the samples were collected to investigate cdv and vector-borne infections (13 dogs) and canine parvovirus infection (12 dogs). the dogs were monitored for six months. results: one dog was euthanised directly after import. thirteen dogs showed clinical signs after arrival, i.e., diarrhoea (57 %), coughing (43 %) and nasal and/or ocular discharge (21 %); radiographic findings that were compatible with bronchopneumonia were present in four dogs. cdv infection was diagnosed in 11 dogs (85 %); 10 dogs (91 %) tested pcr-positive in conjunctival swabs. vector-borne infections (babesia spp., leishmania infantum, dirofilaria immitis) were found in 4 dogs (31 %). three dogs were hospitalized, and six dogs received ambulatory therapy for up to two months until recovery. none of the dogs developed neurological disease. cdv shedding was detected for a period of up to four months. because dogs were put under strict quarantine until cdv shedding ceased, cdv did not spread to any other dogs. the cdv isolates showed 99 % sequence identity in the ha gene among each other and belonged to the arctic-like lineage of cdv. conclusions: the present study highlights the imminent risks of spreading contagious viral and vector-borne infections through the non-selective import of sick dogs and dogs with incomplete vaccination from eastern europe. cdv shedding was detected for several months after the cessation of clinical signs, which emphasised the roles of asymptomatic carriers in cdv epidemiology. a long-term follow-up using sensitive pcr and strict quarantine measures is of upmost importance in preventing the spread of infection. dog owners and animal welfare organisations should be educated regarding the importance of complete vaccinations and the impact of dog imports on the spread of viral and vector-borne pathogens. canine distemper virus (cdv) is one of the most important viral pathogens in domestic dogs and causes high morbidity and mortality worldwide, particularly in unvaccinated dogs or dogs with incomplete vaccination [1] . cdv is a small, enveloped rna virus that belongs to the family paramyxoviridae and the genus morbillivirus [2] . cdv has a wide natural host range that includes a variety of terrestrial carnivores [2] . dogs are thought to be the major reservoir host for cdv [3, 4] . infection occurs by direct contact with oronasal secretions of infected animals [5] ; indirect transmission plays only a minor role in cdv epidemics because the virus is quickly inactivated in the environment [6] . the course of the cdv infection is strongly dependent on the immune response in infected animals [7] . in this context, vaccination is critically important. dogs that develop an adequate immune response can clear the virus from most tissues, whereas in dogs that show an intermediate immune response, cdv infects the epithelial tissues and induces clinical signs. in dogs that have a weak immune response, cdv disseminates to various tissues, and the clinical signs are usually severe with the persistence of the virus until death [8] . invasion of the central nervous system occurs when viraemia is sufficiently high [9, 10] . more than 50 % of all cdv infections are perceived to be subclinical [11] . in clinically affected dogs, the disease usually starts with fever and a serous-to-mucopurulent conjunctivitis, followed by a dry to productive cough, depression, anorexia, vomiting and diarrhoea [1] . neurological signs usually develop within one to three weeks after recovery from systemic illness, but can occur weeks to months later [12] . since the introduction of highly protective cdv modified live virus (mlv) vaccines more than 60 years ago [13] , the incidence of cdv infection in completely vaccinated dogs has decreased [8] . however, in regions with a low proportion of vaccinated dogs, in stray dogs and in shelter environments, the incidence of cdv epidemics is high. in switzerland, the last cdv epidemic in domestic dogs occurred in 1984-1985; this outbreak was suspected to be attributed to an inadequate vaccination rate in the swiss dog population at that time [14] . furthermore, a cdv epidemic associated with high morbidity and mortality commenced in the spring of 2009 in wild carnivores in switzerland [15] . the latter was perceived to be part of a large transnational outbreak that spread from eastern to western europe. only one domestic dog was affected in switzerland during this outbreak [15] . remarkably, the 2-year-old mixed breed dog died of a cdv-associated neurological disease, although it had received the standard anti-cdv vaccination protocol. according to the animal identity service (anis) in switzerland, the import of dogs increased by 23 % within one year (2011-2012) [16] . the imported dogs comprised primarily stray dogs that were adopted by animal welfare organizations or pure breed dogs to meet the increasing demand of miniature breeds in western europe. in the present study, we report on a distemper outbreak in rescue dogs that had been imported from hungary to switzerland. the study provides data on vaccination, medical history, clinical examinations and diagnostic imaging of the dogs and cdv testing, testing for canine parvovirus (cpv) and vector-borne infections. additionally, the study gives prospectively collected follow-up data on the treatment, clinical course and outcome of the infections and the period of cdv shedding. finally, a molecular characterization of the cdv isolates was performed. a group of 15 rescue dogs that derived from a shelter in kecskemét, hungary, was imported to switzerland in october 2013. one dog was euthanised within several days of import because of clinical deterioration; no data regarding this dog were available. the other 14 dogs comprised nine female (5 spayed) and five male (4 castrated) mixed breed dogs, aged 6 months to 8 years old, and weighing 5 kg to 30 kg ( table 1 ). all of the dogs had received rabies vaccination (rabisin®, biokema sa, crissier, switzerland) six to 33 weeks before import and deworming (containing praziquantel, pyrantel and fenbendazol, uniwerm®, provet, beograd, serbia) seven to nine days before their arrival in switzerland. additionally, the dogs had been vaccinated with one shot of a combined mlv vaccine containing cdv, canine adenovirus-2, cpv, leptospira spp. and canine parainfluenzavirus (biocan® dhppi & l, table 1 ), either seven to eight days (dogs 1 to 6 and 8 to 14) or one month prior to arrival in switzerland (dog 7); dog 12 had been revaccinated in switzerland one week prior to sample collection for cdv pcr (table 1) . after arrival on october 22, 2013, the rescue dogs were directly distributed to 14 private households throughout switzerland (table 1 , fig. 1 ). seven dogs (dogs 3, 6, 7, 8, 9, 13 and 14) were placed in multidog households. after arrival, the new owners observed clinical signs in 13 of the 14 dogs (table 1) , i.e., diarrhoea (57 %), coughing (43 %), nasal and/or ocular discharge (21 %), vomiting (14 %), gagging (14 %), lameness (14 %), apathy and sneezing (each 7 %). because of these symptoms, five dogs (dogs 1, 2, 3, 4 and 8) were presented to private veterinarians within one week of arrival. three of these dogs (dogs 1, 2 and 3) were subsequently referred to small animal clinics for additional investigations. haematology and blood biochemistry results were available for 13 dogs (tables 2 and 3 ), 11 of which were cdv-pcr positive (dogs 1 to 11, see below). at initial presentation, anaemia (9/13, 69 %), leucocytosis (8/13, 62 %), eosinophilia (8/11, 73 %), neutrophilia (6/11, 55 %) and monocytosis (5/12, 42 %) were common ( table 2) . dogs 3 and 4 showed severe pancytopenia and moderate bicytopenia, respectively; both were cdv pcr-positive, co-infected with babesia spp. (dog 3) or positive for anti-leishmania infantum antibodies (dog 4, see below), and they exhibited fever, increased inspiratory lung sounds, purulent ocular and nasal discharge and radiographic signs that were compatible with bronchopneumonia (tables 1 to 5 ). dog 8 showed slight anaemia and leucopenia ( table 2) ; this animal was co-infected with cdv and babesia spp. (tables 4 and 5) . dog 13 showed a pronounced eosinophilia ( table 2) ; this animal was cdv-pcr negative but dirofilaria immitis positive (tables 4 and 5 ). blood biochemistry results revealed only unspecific changes in the dogs (table 3) . the radiographic examinations of the thorax revealed moderate-to-severe interstitial lung changes with variable bronchial thickening in four dogs (dogs 1 -4). the changes were generalised and most pronounced in the dorsal (dog 2), perihilar (dogs 2 and 4) and caudal lung areas (dog 3, fig. 2 ). the radiographic findings were compatible with bronchopneumonia in all four dogs, and all of the dogs tested cdv pcr-positive. the thoracic radiographs of dog 13 revealed mild right-sided cardiomegaly and mild generalised bronchointerstitial lung changes; echocardiography showed mild tricuspid and aortic regurgitation but no signs of pulmonary hypertension or right ventricular pressure overload. dog 13 tested d. immitis-positive but was negative for cdv. eleven of the 13 dogs tested cdv pcr-positive during the initial examination ( table 4 ). the positive pcr results were most commonly obtained from conjunctival swabs (10 of the 11 cdv-positive dogs, table 4 ). the vaccine-specific real-time reverse transcription (rt)quantitative (q)pcr was negative for all ten dogs that were tested, which supports the finding of infection with a wild-type cdv strain. all three vaccines that were tested (biocan® dhppi & l, bioveta, ivanovice na hané, czech republic; nobivac® dhhpi, msd animal health, luzern, switzerland; canigen® sha2ppi, vibac, glattbrugg, switzerland) exhibited a positive pcr result. in gel electrophoresis of the pcr products, a appropriate-sized band was detected for all ten dogs, as were the three vaccines, as expected [17] . the sequence of the amplification product of the biocan® dhppi & l vaccine used in the dogs of the present study was clearly distinct from the sequence alb albumin; 7 ap alkaline phosphatase; 8 alat alanine aminotransferase; 9 na sodium; 10 k potassium; 11 p phosphorus of the cdv isolates of the ten rescue dogs (fig. 3 ) and most closely related to the cdv vaccine strain onderstepoort (99 % nucleotide identity to ab250738). sequencing of the ha gene of cdv isolates of five of the infected dogs revealed that the dogs were infected with a similar cdv strain (99.9-100 % nucleotide identity among the ha gene of cdv isolates of dogs 1, 5, 6 and 10); the ha gene of the cdv isolate of dog 9 differed in only one nucleotide position from the ha gene sequences of the other four cdv isolates. the phylogenetic analysis revealed that the isolates from the five import dogs belonged to the arctic-like lineage of cdv (fig. 4) . the ha gene sequences of the cdv isolates were most similar to a published ha gene sequence of a cdv strain from a domestic dog from italy (kf914669, 99 % nucleotide identity, fig. 4 ) [18] . they were only distantly related to cdv strains isolated during a cdv epidemic in wild carnivores in switzerland (jf810109 and jf810111, 92.6 % nucleotide identity, fig. 4 ) overall, two dogs (dogs 5 and 11) exhibited cdv pcrnegative results one month after the initial examination (table 4 ). two months after the initial examination, another three dogs were found to be cdv pcr-negative (dogs 7 to 9); three months after the initial examination, dog 3 was cdv-negative and the remaining three dogs tested cdv-negative four months (dog 6) and five months (dogs 1 and 2) after the initial examination (table 4) . all twelve dogs that were tested for cpv at the initial presentation were pcr-negative (table 5) . vector-borne infections were detected in 4 dogs (31 %, table 5 ): infection with babesia spp. was detected in dogs 3 and 8; infection with l. infantum was diagnosed in dog 4 and infection with dirofilaria immitis was found in dog 13. dog 13, which tested positive in d. immitis antigen and knott tests, had received a certificate from a laboratory in budapest, hungary, that stated a negative result in the knott test in august 2013. none of the rescue dogs tested positive for ehrlichia canis (table 5) . the cdv-infected dogs 1, 3 and 4 were hospitalized for two to three days and received intravenous infusions of crystalloids, intravenous antibiotic therapy and inhalation (table 1) . dog 3, which was co-infected with babesia spp., was treated with two imidocarb injections two weeks apart. dog 4, which was co-infected with l. infantum was treated using allopurinol. all three dogs showed rapid clinical improvement with treatment and were discharged with oral antibiotic therapy. repeated haematological examination in dog 3 two weeks later revealed that the pancytopenia had resolved and had returned to moderate neutrophilia, eosinophilia and slight monocytosis. mild anaemia was still present in dog 3 at that time (data not shown). repeated haematology in dog 4 two weeks after the initial presentation showed normal platelets counts and nearly normal pcv values (data not shown). all three dogs were clinically asymptomatic in the 6-month follow-up period. dog 2 was ambulatory and was treated with oral antibiotics and antibiotic eye drops ( table 1 ). the dog showed several relapses with purulent nasal and ocular discharge after antibiotic therapy ceased and was repeatedly treated with antibiotics for two months. thereafter, there was no relapse, and the dog was clinically asymptomatic in the remaining 4-month follow-up period. five cdv pcr-positive dogs (dogs 5 to 8 and 10) received oral antibiotic therapy (amoxicillin clavulanic acid or doxycycline) for seven to ten days after their arrival in switzerland. dog 5 developed watery diarrhoea two weeks after arrival and was additionally treated with metronidazole, deworming and a highly digestible diet. dog 8, which was co-infected with babesia spp., received two injections of imidocarb diproprionate two weeks apart and antibiotic ear drops because of otitis externa. at the end of the 6-month follow-up period, all of the cdv pcr-positive dogs had recovered and none had developed neurological signs. all of the owners of the cdv pcr-positive dogs were instructed by the first author (bw) to quarantine the dogs until they tested cdv pcr-negative. the owners of the cdv-positive dogs in multidog households (dogs 3, 6, 7, 8 and 9) were instructed to separate the infected dog from the other dogs in the household. however, several dogs (dogs 6, 7, 8 and 9) had already had contact with adult dogs within the household at the time when the cdv diagnosis was made. all of the contact dogs had been vaccinated against cdv, although several dogs had only received the initial vaccination series as puppies and had received no booster vaccinations (data not shown). two dogs that were in close contact with dogs 7 and 9 were tested for the cdv infection with pcr one month and two months after the initial cdv diagnosis in dogs 7 and 9. one contact dog exhibited a single, very weak cdv-positive result in the conjunctival swab in the first sampling but was negative in all of the swabs collected one month later (data not shown). the other contact dog tested pcr-negative in all of the collected samples (data not shown). in all of the other multidog households, no samples were collected for cdv pcr, but no clinical signs of the disease were noted in the 6month follow-up period. the present study describes a distemper outbreak in rescue dogs in switzerland that had been imported by an animal welfare organization. one dog had to be and of ten wild-type cdv isolates of the rescue dogs (dogs 1 to 3 and 5 to 11) is shown. grey-shaded letters: identical nucleotides between vaccine strains and wild-type isolates. black letters: nucleotides that differ between vaccine strains and wild-type isolates. grey bars: schematic drawing depicting the positions of the primers and the probe used in the cdv vaccine specific real-time rt-qpcr assay [17] . consensus: consensus sequence of 100 % identical bases matching all of the sequences (most of the ambiguities) euthanized directly after import for humane reasons, whereas nine dogs required therapy for up to two months; three of these dogs were hospitalized at veterinary clinics. the imported animals shed cdv for up to four months, which necessitated long-term quarantine measures. moreover, four of the dogs were infected with vector-borne pathogens. the present study underscores the risk of introducing contagious or vector-borne pathogens to central european countries by importing rescue dogs with incomplete vaccination. based on the data of the anis in switzerland, 43.9 % of the newly registered dogs in 2012 were imported [16] . the imported dogs primarily comprised small and miniature purebred dogs to meet the increasing demand for these types of breeds in switzerland, and mixed breed dogs that are rescued by animal welfare organisations [16] . the dogs of both groups are at risk for carrying infectious diseases because of the inadequate vaccination policies and quarantine measures in many breeding kennels and animal shelters in eastern europe. in the animal shelter in hungary where the dogs originate, no quarantine measures or reliable vaccination policies had been implemented. the dogs had only received a single cdv vaccination either one week or one month before importation to switzerland. at this time point, several dogs had likely been infected with cdv. after the outbreak, the rescue organisation was instructed to introduce vaccination policies and quarantine measures within the animal shelter. in switzerland, the vaccination rate in dogs is insufficiently high to provide population immunity against the cdv infection. the proportion of vaccinated dogs in a population must be > 70 % to provide protection for the dog population, in contrast to protection of only a single vaccinated dog [19] . a vaccination rate of 60-70 % has been estimated for swiss dogs based on the numbers of sold vaccine doses in 2009 [20] . mandatory courses for new dog owners have been introduced in switzerland in which freshly adopted dogs come in close contact with each other. together with the decreasing vaccination rate and the increasing number of imported dogs, this situation has clearly increased the risk for canine distemper outbreaks. dog owners and animal welfare organisations should be informed concerning the critical importance of complete vaccination schedules in domestic dogs. in the present outbreak, the infection could be prevented from spreading to other dogs by extensive followup examinations of the dogs and a thorough education of the dog owners concerning the critical importance of strict quarantine measures. luckily, no cdv-positive dogs of this study had already had contact with young unvaccinated dogs at the time of diagnosis. the cdv vaccines are known to induce a strong and long-lasting immunity when no interference with maternally derived antibodies occurs [21, 22] . cdv was reported to be shed by infected animals for up to three months [11, 23] . we demonstrated cdv shedding in some dogs in the present study even for up to four months. shedding was detected for several months after the cessation of clinical disease. our results underscore the role of asymptomatic carriers in cdv epidemiology and the importance of a long-term followup of cdv-positive dogs for preventing the spread of infection. several studies have reported the excretion of vaccine strains after cdv vaccination [11, 24] . this was not observed in the present study: the dogs that tested cdv pcr-positive were shown to be infected with wildtype cdv, although the majority of the dogs had received mlv cdv vaccination within one to seven weeks before pcr testing. consistent with the published data [25] , pcr from conjunctival swabs was found to be most reliable for detecting cdv in acutely infected animals and during follow-up examinations. however, in one animal, only the nasal but not the conjunctival swab tested positive for cdv. nine of the dogs in the present study were hospitalized or required ambulatory therapy for up to two months. another animal was euthanised directly after import because of clinical deterioration. in all of the affected dogs, the respiratory and gastrointestinal symptoms predominated. in four dogs, signs of bronchopneumonia were evident. remarkably, none of the dogs developed a neurological disease. whether the latter was due to the intrinsic properties of the cdv strain or to the age and immune status of the dogs is unknown. the sequence analyses of the cdv strains detected in the rescue dogs indicated that all of the dogs were infected with the same wild-type cdv strain, which belongs to the arctic-like lineage of cdv [18, 26] . the initial description of arctic-like lineage of cdv dates back to the late 1980s, when epizootics were observed in seals in northern europe and siberia [27] [28] [29] . (see figure on previous page.) fig. 4 phylogenetic relationship between selected cdv strains based on the complete haemagglutinin (ha) gene sequence. the cdv isolates analysed in this study appear in bold. nine cdv lineages are shown: asia-1, europe, america-2, europe-wildlife, africa, arctic-like, asia-2, asia-3 and america-1. phocine distemper virus (pdv-1) was used as the outgroup. genbank accession numbers, host species and geographical origin are indicated, if known. the numbers at the nodes were generated from 1000 bootstrap resamplings; only values > 70 are shown. the bar represents the mean number of differences per 200 sites. strain af178038 (giant panda isolate) is the resultant of a genetic recombination between "asia-1" and a "europe-wildlife" strain [44] the strains of the arctic-like lineage are closely related to the cdv strains in north america, china and greenland and were recently isolated from domestic dogs in hungary [30] and domestic dogs and wolves in southern italy [31, 32] . the present study shows how fast these strains can spread to central european countries by import of infected domestic dogs. the present study indicates that rescue dogs may also play an important role in the spread of vector-borne infections to central european countries. babesia spp., l. infantum or d. immitis infections were detected in four of the 13 rescue dogs. several dogs were reported to be free of these pathogens based on the laboratory certificates provided by the animal welfare organisation. in the case of dog 13, d. immitis infection was diagnosed in this study using the antigen enzyme immunoassay and the knott test in november 2013. the dog was found to be negative in the knott test in august 2013. the d. immitis antigen and knott tests are known to turn positive not before five to eight months after infection [33] ; therefore, the infection could have been missed in the first testing. l. infantum serology specimen, which was positive in dog 4, has similar limitations in that negative serological results cannot exclude infection and seroconversion can occur many months after infection [34] . future dog owners should be properly informed concerning the limitations of these tests and the costs of treatment for vector-borne infections. the present study highlights the risks of spreading contagious viral and vector-borne infections by a nonselective import of sick and unvaccinated dogs or dogs with incomplete vaccination from eastern european countries. the animal welfare organisations should be thoroughly informed concerning the critical importance of complete vaccination schedules and quarantine measures in animal shelters to combat the outbreaks and the spread of viral pathogens to other countries. dog owners in switzerland should be educated regarding the risk of and the potential costs of adopting sick dogs or dogs with incomplete vaccination and the critical importance of sufficiently high vaccination rates in domestic dogs in switzerland. the present study describes a distemper outbreak in fifteen dogs originating from an animal shelter in kecskemét, hungary, and the prospective follow-up of the infected animals. the dogs were imported to switzerland by an animal welfare organisation on october 22, 2013. one dog had to be euthanised within a several days of arrival. no data on that dog were available. of the remaining fourteen dogs, the signalment, vaccination and medical history and clinical signs were recorded (table 1) and dogs were monitored for six months. sample and data collection and sample processing nasal and/or conjunctival swabs were collected from 13 dogs for cdv-specific real-time rt-qpcr and rectal swabs from 12 dogs for cpv real-time qpcr as indicated in tables 4 and 5 . edta blood and serum samples were collected to obtain haematology and blood biochemistry results, and for cdv real-time rt-qpcr and testing for vector-borne infections (babesia spp., e. canis, l. infantum and d. immitis) as indicated in tables 2 through 5. all of the samples were processed within 12 h of collection. in 10 of the 11 cdv-infected dogs (dogs 1 to 3 and 5 to 11), monthly follow-up examinations for cdv were performed from conjunctival, nasal and oropharyngeal swabs ( table 4 ). the swabs were collected by the owner based on written and image instructions regarding how to collect and ship the samples. all of the swabs were sent by priority mail to the clinical laboratory, vetsuisse faculty, university of zurich, within one day of collection. follow-up was continued in each dog until the dog tested cdv pcrnegative, except for dog 10, in which the owner declined further testing despite a pcr-positive result at three months of follow-up. haematology and blood biochemistry tests were performed at the clinical laboratory, vetsuisse faculty, university of zurich (dogs 1 and 2, 5 to 11 and 13), at the clinical diagnostic laboratory, vetsuisse faculty, university of bern (dog 3), at a private laboratory (dog 4: alomed, radolfzell-böhringen, germany) or by a private veterinarian (dog 8, tables 2 and 3 ). the laboratory's own device-specific reference intervals were applied; for dogs aged 6 to 8 months (dogs 2, 5 to 7), published reference intervals for phosphorous, alkaline phosphatase, urea and creatinine were used [35] . at the time of the initial examinations, conjunctival, nasal or rectal swabs were incubated for 10 min in 300 μl of phosphate buffered saline (pbs) at 40°c; the swabs were subsequently turned upside down and centrifuged for 1 min at 6440 × g and the supernatant was used for tna extraction (see below). for the follow-up examinations, the two conjunctival and the two nasal swabs, respectively, were pooled in a total of 400 μl of pbs before incubation at 40°c for 10 min and tna extraction. the tna extraction was performed from 100 μl of edta blood, 200 μl of swab supernatant or vaccine material that was resuspended in 500 μl of pbs (see below) using the magna pure lc (roche diagnostics ag, rotkreuz, switzerland) and the magna pure lc tna isolation kit (roche diagnostics) following the manufacturer's instructions. with each batch of extraction, a negative control consisting of 200 μl of pbs was used to monitor for cross-contamination. the tna was stored at −80°c until pcr analysis was performed. for cdv testing, a published real-time rt-qpcr assay was used as previously described [36] . for the detection of cpv, a published real-time qpcr assay developed for the detection of feline parvovirus (fpv) was applied [37] . the assay amplifies a 107 bp sequence of the highly conserved vp1/vp2 gene region using the following primers and probe: pv3294f: 5′-actgcatcattgat ggttgca-3′; pv3400r: 5′-ggtatggttggtttc catgga-3′ pv3375p: 5′-fam-cccaatgtctcaga tctcatagctgctgg-6-tamra-3′. sequence comparison revealed that the primer and probe-binding sites in the vp1/vp2 gene region of fpv and cpv showed > 99 % sequence identity (genbank accession numbers m38246, m38245, m19296, m74849, m74852, m24000, m24003, km457142, jq268284). during cdv follow-up, a published canine (c)gapdh pcr assay was applied to ensure that the quality of the swabs collected by the dog owners was adequate [38] . during follow-up, dogs were stated cdv-negative if they tested pcr-negative for cdv in conjunctival, nasal and oropharyngeal swabs and the swabs showed a cgapdh threshold cycle below 32. all of the real-time pcr reactions were run using an abi 7500fast real-time pcr system (applied biosystems, rotkreuz, switzerland). negative and positive controls were included in each pcr run. to ensure that the cdv real-time rt-qpcr signal was due to infection and was not due to a recent vaccination, a published real-time rt-qpcr system based on the cdv m gene and m-f intergenic region was used [17] . the primers of the pcr assay amplify the mlv vaccine and wild type cdv strains, whereas the probe of the assay only binds to the mlv vaccine strains (fig. 3) . because no sequence data have been published on the m gene and the m-f intergenic region of cdv u39 strain contained in the vaccine used in the rescue dogs (biocan® dhppi & l, bioveta), the latter vaccine, together with two other cdv vaccines that are commercially available in switzerland (nobivac® dhhpi, msd animal health; canigen® sha2ppi, vibac) were tested to confirm that the cdv strains contained in these vaccines are detected by the assay. the tna from the three vaccines and from the conjunctival or nasal swabs from 10 of 11 cdv pcr-positive dogs (dogs 1 to 3 and 5 to 11) were subjected to real-time rt-qpcr. the pcr products were separated using 2.5 % agarose gel and appropriate-sized bands (294 bp) cut out, extracted using the minelute® gel extraction kit (qiagen, hombrechtikon, switzerland) and sequenced (microsynth, balgach, switzerland). the complete haemagglutinin (ha) genes of the cdv isolates from five dogs were sequenced (dogs 1, 5, 6, 9 and 10). the tna extracted from the conjunctival swabs was used as a template. for amplification, five previously published primer pairs were used [39] ; single nucleotides in one primer pair (472f and 1172r) had to be changed because of mismatches within the primer binding sites for the five cdv isolates (472f_new: 5'-ctgtacat caccaagtcata-3' and 1172r_new: 5'-tagaatac catcttgtgaat-3'). reverse transcription was performed using the high capacity cdna reverse transcription kit (applied biosystems) according to the manufacturer's instructions. the pcr amplification was conducted using 5 μl of 5 x hf pcr buffer (finnzymes, bioconcept, allschwil, switzerland), 500 nm each primer, 200 μm each dntp (sigma-aldrich, buchs, switzerland), 1 u phusion high-fidelity dna polymerase (finnzymes), and 2.5 μl template cdna made up to 25 μl with water. the thermal cycling conditions comprised 98°c for 2 min, 35 cycles at 98°c for 30 s, 50°c for 30 s, 65°c for 1 min, and a final elongation at 72°c for 3 min. after the pcr run, the amplification products were separated using 2 % agarose gel; appropriately sized products were excised and purified using the minelute gel extraction kit (qiagen). direct sequencing of the purified amplicons was performed using the amplification primers in a commercial laboratory (microsynth, balgach, switzerland) under standard conditions. vector-borne infections were tested in 13 dogs, as shown in table 5 . the analyses were performed at the clinical laboratory (for e. canis) and the institute of parasitology (for babesia spp., l. infantum and d. immitis) of the vetsuisse faculty, university of zurich, and at private laboratories (dog 8: labor am zugersee, hünenberg, switzerland; dog 4: alomed; dog 12: idexx diavet ag, bäch, switzerland) ( table 5 ). the laboratories' own reference values were used for defining the positive, negative and intermediate results. the microscopic blood smear evaluation for the presence of babesia spp. organisms was conducted in dog 3 by the private veterinarian and in dog 4 by a commercial laboratory (alomed) ( table 5) . the obtained sequences were edited and aligned with a consensus sequence using geneious version 7.1.8 [40] . only the nucleotides available for all of the included sequences (2005 nucleotides of the ha gene) were used to calculate the percent nucleotide identities and perform the phylogenetic analyses. for the phylogenetic analyses, the sequences were aligned with known distemper sequences from genbank (see fig. 4 ) using geneious version 7.1.8. a bootstrap phylogenetic tree demonstrating the relationship between the isolates was created using the maximum-likelihood method [41] and a distance matrix corrected for nucleotide substitutions based on the kimura 2-parameter model [42] . the dataset was resampled 1000 times to generate bootstrap values. the phylogenetic and molecular evolutionary analyses were conducted using the mega version 6 [43] . nucleotide sequences obtained in this study have been submitted to 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leishmania infections in dogs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors thank the animal rescue organisation and the owners of the dogs for providing the data and supporting diagnostic work-up. we thank t. meili, e. goenczi, s. childers and the technicians of the clinical laboratory for their excellent laboratory assistance. the laboratory work was performed using the logistics of the center for clinical studies, vetsuisse faculty, university of zurich. as was supported by a research grant (forschungskredit, fk-53210-01-01) from the university of zurich. the authors declare that they have no potential conflicts of interest to disclose.authors' contributions rhl and bw conceived the study. bw and ams were responsible for the study coordination and the data and sample collections. lb, tb and rj were responsible for the clinical work-up and medical care of the dogs. mlm was responsible for the molecular laboratory aspects, fg was responsible for the analyses of vector-borne infections, br was responsible for the haematology and blood biochemistry tests and md was responsible for all aspects of diagnostic imaging. rhl and bw drafted the manuscript. all of the authors read and approved the final manuscript. key: cord-253525-r6ocr18h authors: fontbonne, alain title: small animal reproduction: scientific facts versus dogmas or unverified beliefs date: 2020-03-11 journal: theriogenology doi: 10.1016/j.theriogenology.2020.03.014 sha: doc_id: 253525 cord_uid: r6ocr18h small animal reproduction is a rather recent topic in the field of animal reproduction. although some continuing educational societies aim to improve the level of general knowledge, published scientific facts are still limited, and there are many hypotheses or affirmations that remain unverified or even sometimes dogmatic or empirical. through examples, this article reviews the main causes that may sometimes challenge a veterinary practitioner faced with a dog or a cat presenting reproductive problems. it could be difficult to orientate the diagnosis or the therapeutic approach, not knowing if what is found in the literature is a scientific proof or just an unverified affirmation. this problem may result from the fact that there are still physiological processes that remain unclear, but also that some common beliefs are based on old studies that have not been repeated or verifiedin addition, to make it more challenging to seek for the information, there are unverifiedaffirmations,unclear nomenclature, clinical conditions which have not been standardized in the literature, clinical conditions mostly described through case reports, lack of studies on specific topics, areas of small animal reproduction that have been neglected by researchers, contradictory data or even studies that lack objectivity. the growing interest for research in small animal reproduction will probably reduce the gap between unverified beliefs and scientific facts. small animal reproduction (sar) is a topic that has developed recently. until the eighties, within most veterinary faculties the teaching content in the field of animal reproduction was mainly e and sometimes only e based on reproduction in horses and farm animals, that were considered having an economic value. but progressively, following the growing interest in companion animals in modern societies, the scientific and academic interest for sar expanded. some precursors such as professor patrick w. concannon in the usa who began publishing some scientific data about canine endocrinology in 1975 [1] , found the basic knowledge essential for further research. the first international symposium on canine and feline reproduction (iscfr) was organized in dublin (ireland) in 1988, bringing together for the first time around 100 specialized academics or practitioners. it was the beginning of an exponential interest for dog and cat reproduction, including both domestic and wild canids and felids, with an international symposium occurring every 4 years since then. in europe, a species-oriented continuing education society e the european veterinary society for small animal reproduction (evssar) -was created in the nineties and held its first annual meeting in 1998 in barcelona (spain), and every year since then. in 2001, the first academic book entirely devoted into sar was written by shirley johnston, margaret root-kustritz and patricia olson [2] . it extensively reviewed all the published data until that date and is still nowadays considered as a reference book for most students and academics. however, it probably represented a tremendous amount of work to prepare and since 2001, although some dedicated manuals on sar have been published in different countries, no similar extensive review on published scientific data has been made and the book of johnston et al. [2] has not been updated. it means that some data found in this book may have changed in some articles published later, but may not be accessible to veterinary students or general practitioners. this probably contributes to the difficulty of differentiating scientific facts and unverified beliefs in the field of sar. in 2004, a study based on an open questionnaire was published. the survey was performed in 86 institutions of veterinary education belonging to 32 european countries, 15 within the european union (eu) and 17 outside the eu, in central and eastern europe and the european free trade association (efta). it was aimed to provide a view of the general status of education and research in sar in europe [3] . it showed that, although more than 60% institutions were providing a well-balanced comparative teaching in animal reproduction, in some places the teaching was more species orientated. the author concluded that, in the absence of comparative data and research used in education and in the development of new technologies in sar, there was a risk that sound sciencebased academic education might be constrained. to counteract these negative trends, sar should be recognized as a very important branch of the department of animal reproduction. this should lead to the development of a high quality research rejecting empiricism and dogmas. in the present article we aim e using some relevant examplesto demonstrate that even nowadays, our knowledge in sar is often based on unverified beliefs more than on scientific facts, and that an effort has to be made to catch up the level of excellence that exists in the field of large animal reproduction or even in human gynaecology, andrology or obstetrics. furthermore, it appears important to develop clinical approaches based on evidence based medicine (ebm). there are still some unclear physiological pathways that have not been elucidated in sar. one of the best examples of a common claim that should be more closely looked at is ovulation in the queen. in most books and academic reviews, including the reference book from johnston et al. [2] , the queen is presented as an induced ovulator, and luteinizing hormone (lh) release from the pituitary gland, and subsequent ovulation, is said to be induced by copulation. however, since the early nineties some authors have reported cases of spontaneous ovulation in groups of queens housed together [4, 5] . in wild felids, some species such as lions, clouded leopards, leopards, pallas' cats, fishing cats or margays may show alternatively induced or spontaneous ovulations [6] . moreover, it is well known that some non-mated queens may develop pyometra during their life, and that this disease e apart from treatment with progestins -is often linked to progesterone impregnation that follows ovulation [7] . although a study conducted on demand of an insurance company in sweden [8] showed that the overall incidence rate (ir) of this disease appeared low (17 cats per 10,000), a significant breed effect was observed. the breed with the highest ir (433 cats per 10,000) was the sphynx, and other breeds with ir over 60 cats per 10,000 were siberian cat, ocicat, korat, siamese, ragdoll, maine coon, and bengal. pyometra was more commonly diagnosed in older animals, with a marked increase in cats over 7 years [8] . an objection to the claim that the queen is a strictly induced ovulator is that a so called "spontaneous ovulation", which appears without any coitus, seems especially frequent in breeding catteries. in domestic shorthair queens living in groups of females, the level of spontaneous ovulations may reach up to 87% [5] . in a preliminary study conducted in our experimental cattery [9] , we aimed to determine the occurrence and frequency of spontaneous ovulations in a group of 11 queens composed of domestic shorthair cats (2/11), thai cats (6/11), and cross-bred cats (3/ 11) . no male cats were housed in the same building. during the study 24 oestruses were detected in a 7 months period and in 6/24 cases (four queens only), a significant increase of progesterone after oestrus was detected (26.26 ± 9.99 ng/ml). among these four queens that presented this phenomenon, three were thai cats and one was a cross-bred queen between a thai and a domestic shorthair. of course, this is not a scientific demonstration but it raises the question of whether the cat should be considered as an alternatively induced or spontaneous ovulatory species, depending on the breed, on the age, and on the housing. another point that remained unclear for a long time in cats concerns the steroidogenic capacity of the placenta as a supplemental source of progesterone during pregnancy, and the possibility for a pregnant queen to maintain her pregnancy even after ovariectomy. in 2001, johnston et al. [2] wrote that the feline corpora lutea are the main source of progesterone in the pregnant queen, and that placental progesterone is of minor or nonexistent importance. although in 1993, verstegen et al. [10] had observed a pregnant queen which, after being treated with the dopamine agonist cabergoline, was able to maintain her pregnancy with a plasma progesterone concentration below 1 ng/ml for more than 7 days. these authors suggested e among other possibilities -that placental paracrine progesterone may allow pregnancy to continue. surprisingly, this very important issue for feline reproduction was not confirmed until 2012, when siemienuch et al. [11] demonstrated that placental progesterone concentration was low in early pregnant queens, but increased with gestational age. their results finally confirmed that the feline placenta is an additional source of progesterone in pregnant queens and could be considered as an endocrine organ helping to maintain pregnancy in this species. of course, further studies have to be conducted to see when, for how long and in which conditions placental progesterone alone could help to maintain pregnancy in an ovariectomised queen, but at least this discovery has helped to solve an unverified belief. another frequent statement is that bitches -to the contrary of queens -are non-seasonal breeders, because they may exhibit oestrus at any time of the year and litters are born during each month of the year [2, 12] . a study published in 2011 even stated that the bitch is "typically non-seasonal" [13] . most publications mention that primitive breeds such as the australian dingo, the wolf hybrids (saarlos, czeck) or the african basenji may display oestrus once a year [2] . however, some studies have shown that there may be an influence of the season on reproductive activity in bitches. in a study, greyhound bitches were mostly in heat in winter, with a highest incidence in february, while the lowest frequency of their cycles was in november [14] . in another study, the data collected during a four year period showed that a seasonal pattern was observed when the cumulative distribution over years was analyzed, with a higher frequency of oestrous cycles observed during winter and summer [15] . an analysis of 319 interoestrous intervals of 36 beagles, 36 german shepherd and 20 labrador retrievers claimed that bitches kept under artificial temperature and light conditions are not as influenced by season as are bitches kept outdoors and thus exposed to seasonal variations in climate and day length [16] . an influence of the breed upon the seasonal aspect of cyclicity was also mentioned in these two studies [15, 16] . these data are often neglected but could be interesting to develop new approaches in the control of reproduction in the bitch. for example, the prevention of oestrus may be considered differently depending on the breed, or the use of compounds mimicking a change in the photoperiod, such as melatonin, may be considered for the control of oestrus in bitches, as it is the case in the domestic cat. some dogmas exist in sar that may not be completely proven by scientific studies. in this respect, the meta-analysis studies made by the group of beauvais et al. on the effect of neutering on the risk of developing mammary tumours in dogs [17] and also on the risk of developing urinary incontinence in bitches [18] are perfect examples. concerning the belief that an early neutering lowers significantly the risk for a bitch to develop mammary tumours later in her life, it is based on a study published in 1969 which has not been repeated since then [19] . this may raise some skepticism when we suppose that the way of evaluating the quality of scientific articles may have changed considerably since the sixties. indeed in the first study by beauvais et al. [17] , among 13 selected reports in englishlanguage peer-reviewed journals addressing the association between neutering, age at neutering and mammary tumours, nine were judged to have a high risk of bias. the remaining four were classified as having a moderate risk of bias. one study found an association between neutering and a reduced risk of mammary tumours. two studies found no evidence of association. one reported "some protective effect" of neutering on the risk of mammary tumours, but no numbers were presented. due to the limited evidence available and the risk of bias in the published results, the authors concluded that evidence that neutering reduces the risk of mammary neoplasia, and the evidence that age at neutering has an effect, are judged to be weak and are not a sound basis for firm recommendations. even if this belief appears to be probably true for most veterinary practitioners following bitches throughout their lives (very few bitches spayed at an early age develop mammary tumours), it is wrong to present this as a scientific fact. the association between spaying and the subsequent development of urinary incontinence in bitches is another example. another study from beauvais et al. [18] based on a systematic review of seven selected peer-reviewed original english analytic journal articles treating the effect of neutering or age at neutering on the risk of urinary incontinence concluded that four articles were judged to be at high risk of bias. of the remaining three studies, which were at moderate risk of bias, there was some weak evidence that neutering, particularly before the age of three months, increases the risk of urinary incontinence. for these authors, the evidence overall was neither consistent nor strong enough to allow for strict recommendations on the effect of neutering or age at neutering on the risk of urinary incontinence. these two studies based on the cochrane guidelines for systematic reviews of interventions [20] give a good approach on how we should re-analyze, under the light of updated reviewing standards, some old articles or common veterinary protocols that are considered as "true", just because they have been published in peer-reviewed journals. some statements considered as true beliefs because that have been put forward by highly considered scientists may have to be reconsidered. let us take the example of the endocrinology of pregnant cycles in the bitch. concannon and his colleagues, in their preliminary studies in the eighties, described an increase of blood testosterone and oestrogens during pregnancy, suggesting heightened luteal secretion of these steroids [13, 21] . however, in a study in 1994, hoffmann et al. [22] , investigating the hormonal changes around parturition in the dog and the occurrence of pregnancyspecific non conjugated oestrogens, found that no pregnancy specific increase of oestradiol-17 beta could be observed. oestradiol-17 beta levels decreased prior to parturition concomitant with the decrease of progesterone, suggesting a likewise luteal origin of oestradiol-17 beta in the pregnant and non pregnant dog. no hints in respect to a specific placental oestrogen production were obtained when examining placental tissue. in a review published in 2012, kowalewski [23] stated that there is no pregnancy-and/or parturition-specific increase in oestrogens in the bitch. one example may be taken on the use of preputial smears to detect an abnormal oestrogenic impregnation in male dogs, often linked with a feminizing syndrome due to a sertoli-cell testicular tumour. elevated concentrations of oestrogen in serum are supposed to cause cornification of preputial epithelial cells, which may be evaluated as for a bitch in oestrus [24] . this technique is commonly used in daily practice avoiding measurement of blood oestrogens, which is often complicated to perform (it requires a specific laboratory able to assay oestradiol) and to interpret. to the best of our knowledge, the first study evaluating the sensitivity and specificity of this technique was published only in 2012 [25] , on 45 dogs with palpable testicular masses and 30 healthy control dogs. the authors concluded that the preputial cytology has a high sensitivity and specificity for the diagnosis of oestrogen producing testicular tumours in dogs. it means that for many years e preputial smears being an old and common technique e veterinarians were using "empirically" this cytological examination without a real scientific confirmation. to the contrary of preputial smears whose interest has been finally confirmed, there is probably still a huge number of beliefs and practices in the field of sar that haven't yet had the chance to be confirmed e or infirmed e by scientific works, and are supposed to be useful, although this may not be the case. another example is the common belief that a superfoetation (i.e. simultaneous presence in the uterus of foetuses of different gestational ages) may occur in the queen as a consequence of anecdotal oestrous activity during pregnancy and subsequent matings occurring in a pregnant queen [2] . in carnivores, this phenomenon, apart from the cat, has been suspected to occur in the badger (meles meles), the geoffroy's cat (leopardus geoffroyi), the lion (panthera leo); the american mink (mustela neovison) or the leopard (panthera pardus) [26] . personal communication with many cat breeders tend to indicate that most of them have observed queens delivering viable kittens up to several weeks apart. surprisingly, although this phenomenon seems to exist, there are very few e and only old e case reports that have been published on this topic and still no confirmation of its reality in domestic cats with experimental procedures in which the paternity of the born kittens could for example be tested. another factor that may disrupt the establishment of a consensus in the field of sar is the nomenclature. in other terms, different specialists may use similar terms to designate different problems or diseases. a good example is to be found when speaking about uterine diseases in bitches or queens. a more complete classification of the uterine diseases would be very helpful to ensure that everybody is using the same terminology. for example, there is often a confusion when using the term "metritis". some authors make a difference between metritis (usually occurring after an oestrus accompanied by mating) and pyometra (occurring during dioestrus) [27] . in 2006, fieni [28] makes a difference between metritis (putrid vaginal discharge with no enlargement of the uterine lumen), open pyometra (putrid vaginal discharge with enlargement of the uterine lumen) and closed pyometra (enlargement of the uterine lumen without vaginal discharge). two years later, verstegen et al., in 2008 [29] only used the term "pyometra". a similar confusion may exist when using the term "endometritis", as it may designate different pathological conditions, which may be acute or chronic, and may be associated or not with ceh [35] . for some authors [30] , it defines a sub-clinical inflammation of the endometrium that does not extend beyond the stratum spongiosum, corresponding histologically to a localized breakthrough into the endometrial epithelium, infiltration of inflammatory cells, vascular congestion, stromal oedema and accumulation of lymphocytes and plasma cells in the upper layers of the endometrium. this "endometritis" corresponds more or less to the second stage described by dow in 1959 [31] from pathological and histological observations. this author described 4 stages of degradation of the uterine endometrium: stage 1 (increase of the number and irregularity of endometrial glands), stage 2 (plasmocytic infiltration of the endometrium), stage 3 (cystic endometrial hyperplasia (ceh) with an acute inflammation and even zones of haemorrhage), stage 4 (ceh with chronic endometritis, lympho-plasmocytic infiltration of the endometrium and modifications of the myometrium). this heterogeneity of the nomenclature complicates our understanding of the role of sub-clinical endometritis as creating infertility in the bitch. for example, in a recent study [32] using in vitro explants of endometrium taken from bitches suffering from ceh, the authors postulated that these bitches were accurate models of a matinginduced endometritis because the bitches with endometrial hyperplasia had a mild histological inflammatory reaction similar to that noted by de bosschere et al., in 2001 [33] . however, in 2013 in the study of mir et al. [34] who analyzed the histological features of surgical biopsies taken on infertile bitches or bitches who suffered from pregnancy arrest, there were several cases of lymphoplasmocytic endometritis or neutrophilic endometritis that were not associated with endometrial hyperplasia.this need to clarify the nomenclature has been put forward by schlafer and gifford [36] , who defined histologically some different degrees of cystic conditions of the canine and feline uterus, and even introduced the definition of a new condition called "pseudo-placentational endometrial hyperplasia". another unclear statement concerns mammary fibroadenomatosis in the queen, a disease characterized by enlarged mammary glands, often under the influence of luteal progesterone or the use of exogenous progestins [2] . histologically, two types of conditions have been identified: a lobular hyperplasia e also called intraductal papillar hyperplasia e which appears to be a result of proliferation of the mammary gland duct epithelium, and a fibroepithelial hyperplasia e also called diffuse fibroepithelial hyperplasia which is a general enlargement of the mammary glands [37] . however, in practice two clinical conditions may be found: one is a diffuse mammary hypertrophy mostly diagnosed in young queens with solid e fibrous -content, that is commonly treated with success using the progesterone receptor blocker aglepristone [38] , another form is a cystic aspect of the mammary glands, which appear full of liquid with often a blue aspect of the skin. this second form develops often in older queens and doesn't respond very well to the use of aglepristone. if it is the same disease or if its pathogenicity is exactly the same is not really known. in order to avoid any confusion, the term of "mastosis" (by analogy with a human disease, in which the role of estrogens predominates) has been suggested by some others to describe the cystic clinical condition [39] . but specific veterinary research is still lacking to better characterize this disease in the cat. there are also some diseases or abnormalities that are commonly found but do not have a well-described monography. one of them is a perineal fold found in adult bitches and which is supposed to promote the development of vaginitis due to lack of drainage and maceration within the vaginal lumen [40] . this existing condition lacks a consensual description and denomination. meanwhile the term of "perineal fold" or "occluded vulva" has been proposed [40, 41] . episioplasty, which consists of the surgical ablation of the excess of perineal skin and associated subcutaneous tissue, is of great interest in this case [41] . in the field of sar, many diseases or clinical findings give rise to the publication of case reports, which may just be anecdotic and not at all representative of the most frequent clinical forms of a disease. in an analysis of all abstracts presented during the successive annual congresses of the evssar and which remained unpublished in peer-reviewed journals afterwards, robin and fontbonne [43] discovered that 92/375 abstracts (24.5%) were case reports, which may indicate an overrepresentation of rare or unusual cases in the field of sar. this is the case, for example, concerning ovarian diseases. in a recent analysis of the literature, arlt and haimerl [42] pointed out that only very little research has been performed on ovarian diseases in the bitch in the last decades, and that our knowledge is mostly based on published clinical reports. in addition, different definitions of diseases and different diagnostic procedures are applied. according to these authors, systematic research results on the clinical and reproductive features of ovarian diseases are not available. many publications are based on case reports that, in general, provide little evidence. also, studies with small sample sizes may provide biased impressions about the nature or presentation of a disease, if extreme or rare cases are frequently reported. regarding cystic ovarian diseases, mostly cases with follicular cysts have been published [42, 43] . this might be due to the fact that various amounts of hormones are secreted, either causing overt clinical signs and/or oestrous irregularities. most published case reports documented large cysts. this suggests that factors such as size, hormonal activities or other rare patterns may cause a publication bias meaning that weird cases are more likely to be published than ordinary ones [43] . this has to be considered when reviewing the literature on ovarian diseases and other rare conditions. the role of infectious diseases in reproduction is a good example. in dogs, with the notable exception of brucella canis, exogenous bacterial pathogens are sporadic causes of reproductive diseases [44] . most commonly, bacterial infections of the reproductive tract are endogenous in origin; many of the bacteria etiologically involved in reproductive disease are part of the urogenital microflora [44] . bacterial reproductive diseases are therefore frequently opportunistic, and predisposing factors must be present for disease to develop [44] . the role of mycoplasma and ureaplasma spp. on reproductive problems is still unclear [45] . certain viruses, such as the minute virus of canines (mvc) or canine herpesvirus (cahv), may also play a detrimental role, although the conclusions of different studies are not always clear and are often based on experimental conditions [46] . faced with pregnancy arrest or abortion in a breeding bitch, veterinarians often do not know what to search for, as the number of proven pathogens towards reproduction is low. indeed, there is a lack of data on the exact role of many diseases including e but not only -leptospirosis, neosporosis, q fever (coxiella burnetii) or the blue tongue virus. in cats, there are even fewer recent reviews about the role of infectious agents towards feline infertility [45, 46] . among viral diseases, only retroviruses (felv, fiv) and the parvovirus have been confirmed as detrimental agents for fertility or pregnancy. the role of feline herpesvirus (fhv) or calicivirus remains unclear and not fully demonstrated in the field, and more studies should be made on this aspect, as these two diseases are of major concern in most catteries. feline coronavirus is considered as an uncommon cause of reproductive problems [47] . but all these affirmations are based on very few studies and remain hypothetical. among bacterial diseases, the main concern is about chlamydophila felis. it has been suggested as a cause of infertility [48] , although this has not been fully demonstrated in the field. other publications report a potential negative role to fertility of coxiella burnetii, leptospira sp. or bartonella henselae. a report indicated also that cats may be sensitive to brucella sp. [49] . there is a special concern about toxoplama gondii. in experimental conditions, trans-placental contamination and even abortion may occur [50] , but in the field the situation remains unclear. another annoying lack of studies concerns the potential promoting effect of recurrent episodes of overt pseudopregnancy with lactation (psp) in the development of mammary tumours in bitches. according to some authors [51, 52] bitches presenting psp seem to have an increased risk of mammary tumours in comparison with bitches without psp. they claim that, in these animals, tumours are detected earlier and are more often malignant. the risk seems to be higher if the bitches have developed many successive psp and when they get older. there seems to be a significant risk after a bitch has undergone three clinical psp [52] . due to the very high frequency of psp diagnosed in veterinary clinics, such studies would be interesting to pursue and of high clinical interest. unfortunately very few retrospective studies have been presented and they were not conclusive [53] . sometimes the use of a drug or a procedure is too recent and the results are based on an insufficient number of animals to be able to claim that it brings strong scientific data. for example, the gnrh agonist deslorelin has been commercialized in europe for about 10 years, and has been used in experimental protocols in dogs and in cats, in-label and off-label. to have data on the reversibility after implanting male cats with the most common 4.7 mg sc implant, there is only one published study based on only 10 male cats housed in experimental conditions [54] , although there are some published studies based on the 9.4 mg sc implant. veterinarians should not consider this study as giving a definitive scientific conclusion. recent unpublished work in our group tend to show that there are considerable variations depending on the breed and on the environment [55] . thereforeit is recommended to remain prudent in drawing any conclusion until there is enough datacollected. another example may be the use of a mechanical drainage in the bitch for the treatment of pyometra. some authors have described successful treatments using an intra-uterine drainage, followed by subsequent pregnancies [56] . surgical drainage and intrauterine lavage resulted in further fertility in 100% of eight treated bitches [57] . but there is without any doubt too few studies to draw any conclusion. in some topics, there may be contradictory data between different authors, which may complicate some decisions in daily veterinary practice. contradictory results are common in fields of research in which just a few number of patients or cases can be enrolled or in research protocols that do not take into account bias in the proper way. contradictory results in different researches on the same topic means that no definitive conclusion can be drawn, and therefore there is a need to produce more research in a specific field. for example, spaying bitches before the first oestrus e with the reserves already mentioned [18] -is often sought by owners to reduce the risk of further mammary tumours. but does it promote the risk of urinary incontinence? a study done in switzerland on 206 bitches that had been spayed before their first oestrus and for which the owners were questioned on the occurrence of urinary incontinence as a result of spaying in at least the 3 years that followed, the incidence of urinary incontinence was approximately half that of spaying after the first oestrus [58] . a completely different conclusion was drawn by another study producing evidence that neutering before first oestrus increases the risk in comparison to after puberty [59, 60] . and as already said, a metaanalysis of all published papers does not permit one to draw conclusions because of the many biases [18] . many examples of contradictory conclusions exist in the field of sar. for example, veterinarians often wonder if it is necessary to spay a bitch that has been surgically operated for a mammary carcinoma, and if it will increase her chance of survival. some studies claim that it is indeed an effective adjunct therapy to tumour removal and that, depending on the timing of spaying, it may influence the survival [61] . other studies claim the contrary and declare that spaying when mammary tumours are removed does not have a significant effect on the progression of malignant disease and that about one in four bitches with a benign mammary tumour is likely to develop a further tumour in another gland [62] . in the topic of artificial insemination (ai) there are also a lot of contradictory data. in the dog, some authors claim that the addition of autologous prostatic fluid, when using frozen-thawed semen, increases the pregnancy rate and the litter size [63] . other authors claim that the pregnancy rate, whelping rate and litter size are reduced when frozen-thawed, prostatic fluid-supplemented semen is vaginally deposited [64] . also when inseminating a bitch with frozen-thawed semen it is a common belief that intra-uterine inseminations give better results than intra-vaginal deposition of semen. linde-forsberg et al. [65] obtained a whelping rate of 84.4% with intra-uterine deposition versus 58.4% for intra-vaginal inseminations. surprisingly, rota et al., in 2010 [66] claimed that 10/ 10 pregnant bitches were pregnant after intra-vaginal deposition of semen 4 and 5 days after the estimated lh peak, using 200 millions of sperm per ai. othaki et al. [67] published successful results of intra-vaginal ais with frozen semen that were above 85%. one hypothesis may be that nowadays veterinarians detect more carefully the time of ovulation than 10 or 20 years ago and that it may not be necessary anymore to deposit the semen inside the uterine lumen. it could also depend on the fertility of the bitch or the sire, and a distinction has to be made between experimental and clinical trials. in cats also, there are contradictory data. one of them is the potential detrimental effect that anaesthesia e mandatory in felids due to the difficulty of contention e has on ovulation and on the subsequent pregnancy rate when inseminating with frozenthawed semen. howard et al. [68] reported that queens inseminated (laparoscopic intrauterine) after ovulation produced more corpora lutea and embryos and had a higher pregnancy rate than those inseminated before ovulation (50% vs 14.3% pregnancy rate). because of the concern that anaesthesia may inhibit ovulation, many researchers elect to inseminate after ovulation has occurred (28e40 h). however, others have not found an effect of anaesthesia on ovulation rate and claimed even better pregnancy rate when anaesthetized queens were inseminated before ovulation than after (10/18 (56%) vs 5/24 (21%)) [69] . when freezing cat semen, the addition of the detergent equex stm-paste is controversial, with some authors claiming that the addition of equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing, but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of equex than in the controls at 6 h after thawing [70] . other authors wrote that even if sperm motility and membrane integrity decreased more rapidly in presence of equex than those in controls, total motility and sperm viability were similar at 3 h and 6 h after thawing [71] . some topics are discussed more than others in congresses and appear fashionable at some time. but it is dangerous to claim scientific facts before enough data has been collected and enough studies published. one recent example is the potential increased risk of developing cancer after spaying in the dog. increased discussion on the influence of neutering on cancer development has been recently prompted with several studies that seem to indicate that incidence of some cancers may be increased with castration or spaying in the canine populations. indeed these studies tend to show an increased risk of prostatic carcinomas, osteosarcomas, transitional cell carcinomas [72] , lymphomas, haemangiosarcomas [73] or mastocytomas [74] in spayed animals, often with a sexpredisposition. but most of them concern only specific breeds and for now it is impossible to say that neutering a dog promotes the development of cancer. although these data are thoughtprovoking, we should not extrapolate findings in single dog breeds to the entire species [75] . another topic that has to be considered with caution is early neutering in dogs and cats. for the past 10e15 years, it has developed following several publications and because it is encouraged by animal welfare societies to limit the number of feral dogs and cats and abandoned animals, but also by dog and especially cat breeders to protect their genetics and avoid others to breed animals bearing their genetics. sometimes it may appear that publications promoting this approach lack some objectivity. if such a practice seems indeed interesting to limit uncontrolled reproduction of non-pedigree pets and feral animals, it should not be forgotten that it may also promote potential health problems such as e among others -urinary incontinence [18] , diseases of the lower urinary tract, the development of a recessed or infantile vulva [76] or the risk of epiphyseal fractures due to the delay of closure of growth cartilages in long bones [77] . at the end of this review based on chosen examples, it appears important to develop clinical approaches using evidence based medicine (ebm), and to build further research on strict and statistically well managed protocols. following the example of the group of beauvais et al. [17, 18] it would be very valuable to make critical reviews of founding articles that have been published a long time ago. due to the growing interest in sar, a high quality of published data should be mandatory, which also implies finding accurate and meticulous reviewers. the growing interest for getting knowledge and learning techniques in the field of sar will probably reduce the gap between unverified beliefs and scientific facts. the authors have no conflict of interest to declare. the ovarian cycle of the bitch: plasma estrogen, lh and progesterone canine and feline theriogenology reflections upon the trends of education and research in small animal reproduction in europe ovulation without cervical stimulation in domestic cats incidence of spontaneous ovulation in young, group-housed cats based on serum and faecal concentrations of progesterone female reproductive cycles of wild female felids pyometra in small animals incidence of pyometra in swedish insured cats ovulation in 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tomcats contraceptive implants used by cat breeders in france, a study on 140 purebred cats uterine drainage in the bitch for treatment of pyometra refractory to prostaglandin f2a surgical uterine drainage and lavage as treatment for caninepyometra the relationship of urinary incontinence to early spaying in bitches acquired urinary incontinence in bitches: its incidence and relationship to neutering practices associations between neutering and early-onset urinary incontinence in uk bitches under primary veterinary care effect of spaying and timing of spaying on survival of dogs with mammary carcinoma effect of ovariohysterectomy in bitches with mammary neoplasms stimulation of mating-induced uterine contractions in the bitch and their modification and enhancement of fertility by prostatic fluid intravaginal insemination of bitches with fresh and frozenthawed semen with addition of prostatic fluid: use of an infusion pipette and the osiris catheter comparison of fertility data from 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development results of vulvoplasty for treatment of recessed vulva in dogs clinical use of deslorelin implants for the long-term contraception in prepubertal bitches: effects on epiphyseal closure, body development, and time to puberty the author wishes to thank mrs. felicity leith-ross for her great help in improving the english language, and all his colleagues of the cerca (centre d'etude en reproduction des carnivores), especially dr natalia santos and dr.cindy maenhoudt. key: cord-285096-g9y3au1a authors: mitchell, judy a.; brooks, harriet w.; szladovits, balázs; erles, kerstin; gibbons, rachel; shields, shelly; brownlie, joe title: tropism and pathological findings associated with canine respiratory coronavirus (crcov) date: 2013-03-23 journal: vet microbiol doi: 10.1016/j.vetmic.2012.11.025 sha: doc_id: 285096 cord_uid: g9y3au1a canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day 14 dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. tropism and pathological findings associated with canine respiratory coronavirus (crcov) a b s t r a c t canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day 14 dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. ß 2012 elsevier b.v. all rights reserved. since its initial discovery in 2003 (erles et al., 2003) ; canine respiratory coronavirus (crcov) is now considered to be a significant cird pathogen, most frequently detected in dogs with mild respiratory clinical signs during the early stages of cird onset (erles et al., 2003) . although crcov has been found worldwide (decaro et al., 2007; erles and brownlie, 2008; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 yachi and mochizuki, 2006; knesl et al., 2009) ; little is known regarding its pathogenesis, tissue tropism or virulence differences among global isolates in the canine host. it is postulated that crcov plays an important role during the early stages of cird by predisposing the dog to more severe clinical disease from secondary infections. through the use of an in vitro tracheal explant culture system, a moderate reduction in ciliary function and a down-regulation of pro-inflammatory cytokine mrna levels (tnf-a, il-6 and the chemokine il-8) was observed in response to crcov exposure (priestnall et al., 2009) . such alterations in the mucociliary and innate immune systems could be linked to increased susceptibility to secondary infection and is consistent with the proposed role for crcov in cird. however, the limitation of this in vitro model precludes the understanding of the clinical relevance and pathogenesis of a crcov infection in the dog. furthermore, given the global presence of this virus, insight into crcov pathogenesis among isolates originating from geographically distinct locations would be valuable to determine the need for a global vaccine. recently our group has collected findings from a preliminary in vivo challenge study of crcov. in that study we demonstrated that young dogs were susceptible to experimental infection with both crcov isolates, which gave rise to clinical signs of respiratory disease consistent with naturally occurring infection. crcov was detected in the oropharynx of infected dogs and spread rapidly to sentinel dogs which also displayed clinical signs of disease (mitchell et al., unpublished data) . here we extend this study to gain a better understanding of crcov pathogenesis in vivo. analyses specifically focused on the histopathological changes in the canine upper and lower respiratory tissues, virulence differences among crcov isolates derived from cird cases representing wide geographical locations; uk and usa [mo, ne, ut and mi] , and the demonstration of koch's postulates. the information obtained from this study vastly enhances our understanding of crcov pathogenicity and its involvement in the cird complex. five crcov isolates originating from geographically distinct regions of the uk and usa were used in this study (table 1) . crcov isolates uk 4182, np631, np787, and np742 were propagated in culture on human rectal tumour cells (hrt-18g; american type culture collection cell line, manassas, va, usa), and used at 10 6 tcid 50 /ml for intranasal challenge. crcov lu298 was not was not propagated or expanded in vitro, instead 0.2 mm filtered viral fluids obtained from the tissues of a crcov infected dog in the usa were used to challenge animals in to1. prior to challenge each of the crcov virus and control hrt-18g cell culture fluids were satisfactorily tested for sterility and canine extraneous agents (including canine distemper virus, measles, canine adenovirus-2, canine parainfluenza virus, canine rotavirus, rabies, canine parvovirus and canine enteric coronavirus). thirty-six, 12-16-week old specific pathogen free (spf) purpose bred beagle dogs were used in this study. all dogs were demonstrated as crcov negative and seronegative for crcov and b. bronchiseptica prior to the study. dogs were housed in temperature controlled isolation rooms with dedicated shower in and out procedures, disinfection and sterilisation of all items prior to entry and a pasteurised diet were used to maintain bio-security. colony dogs are screened quarterly to determine the spf status. throughout the study dogs from the same treatment group were housed in pairs to minimise stress. dogs were randomly divided into six treatment groups (t1-t6) (table 1) , each consisting of six dogs. dogs in groups t1-t5 inclusive were challenged with the crcov isolates as described in table 1 . dogs in t6 were mock challenged with uninfected hrt-18g cell culture supernatant to serve as negative controls. intranasal inoculations took place over two consecutive days (study day zero and one). on each challenge day dogs were sedated prior to intranasal administration with 1.0 ml of virus or control material (0.5 ml/nostril). dogs were monitored throughout the trial for clinical signs of disease. on study days 3, 6, and 14, as detailed in table 1 , dogs were humanely euthanatized within each treatment group as determined by randomisation completed prior to the start of the trial, and necropsies were performed immediately. gross pathological changes were documented throughout each necropsy and the lungs of each animal were photographed. all experiments involving animals were carried out at a contract research organisation, in compliance with national legislation, and subject to local ethical review. general health observations were performed on each dog, twice daily, and scored for general appearance, breathing, sneezing, coughing, and ocular and nasal discharge as detailed in table 2 . body temperatures were recorded twice daily via implanted microchips, and dogs were weighed on study days à7, à1 and on the day of euthanasia. appetite was recorded according to the quantity of food eaten per room. oropharyngeal viral swabs for rt-pcr analysis (sterilin, uk) and virus isolation (dacron swabs, puritan, in 3 ml virus transport medium (pah)) were collected on study days à1 (pre challenge) 2, 3, 4, 5, 6, 8, 10, 12, and 14 from all dogs on the study. rectal swabs were collected from each dog at necropsy. after sampling, viral swabs were frozen and stored at à70 8c until processed. swab tips for rt-pcr analyses were immersed in 1 ml of rpmi medium (sigma, dorset, uk) and mixed prior to analysis. samples from the following tissues were harvested at necropsy and stored at either à70 8c or fixed in 10% buffered formalin: nasal cavity (included ciliated area), nasal tonsil, palatine tonsil, trachea, apical lung lobe, diaphragmatic lung lobe, and bronchial lymph node. each tissue sample was taken using a new set of sterile instruments. lung lavage fluid samples were collected in dmem cell culture medium and stored at à70 8c. formalin-fixed tissues were processed and stained for histology and were examined by a veterinary pathologist as described in section 2.9. on study days à1, 3, 6, and 14, serum and edta whole blood samples were collected for serological and haematological analysis as described below. swabs and tissue samples were tested for the presence of crcov by rt-pcr and virus isolation. prior to inoculation on to hrt-18 g cells, swab and lung lavage samples were clarified by centrifugation, and filtered (0.2 mm filter). similarly, approximately 1 g of each tissue sample was homogenised in 3.0 ml of virus transport medium (vtm), clarified by centrifugation and filtered (0.2 mm filter). filtered samples were inoculated onto confluent t25 or t150 monolayers of hrt-18 g cells. inoculated cultures were maintained in dmem supplemented with final concentrations of 200 mm l-glutamine, 22.5 mg/ml gentamicin, and 1% fbs. inoculation media was also supplemented with trypsin. trypsin concentrations were determined for each batch (circa 1 mg/ml) based of cell toxicity. cell culture fluids were sampled weekly for 2 weeks to test for the presence of crcov by immunofluorescence assay (ifa) (section 2.5.3). rna was extracted using the rneasy mini kit (qiagen, crawley, uk) as recommended by the manufacturer from 200 ml of the swab fluid or a 0.5 cm 2 piece of tissue. rna was transcribed into cdna using random hexameres (ge healthcare, little chalfont, uk) and improm ii reverse transcriptase (promega, southampton, uk) according to the manufacturer's protocol. all samples were tested for the presence of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) by pcr as described previously (grone et al., 1996) . results were expressed as a positive or negative outcome to ensure successful nucleic acid extraction. samples were analysed for crcov using the nested spike gene pcr as described previously (erles et al., 2003) . formalin-fixed tissues were processed for histology and sections were stained using haematoxylin and eosin. the histological sections were examined by a veterinary pathologist who was blinded to the study groups. each tissue was ascribed a histological and cilia score as described in table 3 . a single overall histological ''weighted'' score was assigned to each dog, in which scores assigned to the lower respiratory tract (trachea, lungs, bronchial lymph nodes) were considered more noteworthy than those in the upper respiratory tract (nares, tonsils). this weighting of the overall score was used to take into account that histological changes (especially inflammatory reactions or lymphoid hyperplasia) in the upper respiratory tract are not uncommon, especially in young animals, and unless considerable, are considered part of the normal defences of the upper respiratory tract. conversely, such changes in the lower respiratory tract are more likely to be clinically significant. thus moderate to marked lymphoid hyperplasia or neutrophilic inflammation of the palatine tonsil (score 4) would have less effect on the overall histology score for that individual dog than would a similar grading of lymphoplasmacytic or neutrophilic aggregation in the tracheal mucosa or lungs. paraffin-embedded formalin-fixed tissues (4 mm) were mounted on superfrost plus microscope slides (menzel-glä ser, braunschweig, germany). slides were heated at 60 8c for 1 h, deparaffinised and dehydrated. endogenous peroxidase was blocked with 3% h 2 o 2 for 10 min then slides were washed in dh 2 o, then incubated in prewarmed (37 8c) protease xiv 0.05% (sigma) in tbs for 15 min. slides were rinsed in dh 2 o then incubated with blocking serum (2% normal goat serum [vector laboratories, peterborough] in tbs). staining for crcov was performed as described previously (priestnall et al., 2009) . positive cells were identified microscopically by the presence of staining. haematological analysis of edta blood samples was performed using a cell-dyn 3500 automated haematology machine. a 100 cell differential count of the white blood cells was performed on blood smears stained with a hematek 1 automated stainer using modified wright's stain, by a board certified veterinary clinical pathologist blinded to the treatment groupings. the results for day à1 were analysed for normality and reference intervals were estimated (n = 36). total neutrophil, band neutrophil, lymphocyte and monocyte concentrations were calculated using the automated total wbc concentration, and the observed percentages of each leucocyte type determined by the differential count. the results within each treatment group for days 3, 6 and 14 were compared with each other and with those at day à1 to evaluate trends or significant differences. the paired t-test or wilcoxon signed ranks test with repeat measures were used if distribution was normal or non-normal, respectively. statistical significance was set at p = 0.05. hrt-18g cells were cultured to confluency in 96 well plates. monolayers were inoculated with a usa crcov isolate, incubated for 5 days, and fixed with 80% acetone. canine serum samples were diluted 1:40 in pbs supplemented with 1% bovine serum albumin (w/v) (steris corporation, mentor, oh, usa) and 0.09% sodium azide (mallinkrodt chemicals, hazelwood, mo, usa) followed by two-fold serial dilutions to 1:1280. diluted serum was dispensed at 100 ml/well onto the fixed cells, incubated for 1 h, and then washed twice with water. bound antibody was detected with 50 ml/well of fluorescein isothiocyanate-labelled secondary antibody (rabbit anti-dog igg) (sigma-aldrich, jerusalem, israel) diluted 1:250 in pbs supplemented with 1% bovine serum albumin (w/v) and 0.09% sodium azide (w/v), incubated and washed as before. endpoint crcov titres were observed using fluorescence microscopy and defined as the inverse of the last dilution of serum exhibiting definite crcov fluorescence. in instances where no virus-specific fluorescence at the 1:40 dilution was observed, dogs were considered seronegative or non-exposed to crcov. dog sera collected on study day 14 were tested for serum neutralising antibody titres against 1 uk, and 10 usa crcov isolates. hrt-18g cells were grown to confluency in a 96 well plate. cells were rinsed once in serum-free dmem and then pre-treated for 1 hour with 100 ml/well of serum-free dmem containing c1 mg/ ml trypsin. heat inactivated serum samples were diluted 2-fold, 1:10 through 1:1280, in dmem containing 1% fbs. a further 1:2 dilution of the serum was made in 50-300 tcid 50 of virus per 100 ml, to obtain final serum dilutions of 1:20-1:2560. virus:serum mixtures were incubated for 1 h at room temperature. cells were inoculated in quadruplicate with 200 ml/well of the virus: serum mixture, incubated for 5-7 days, and fixed with 80% acetone. virus growth was detected by ifa as described previously (section 2.5.3). the fifty percent neutralisation endpoint for each serum sample was calculated by the statistical methods of spearman-karber. oropharyngeal amies swabs (sterilin, uk) and lung lavage fluid samples were collected from all dogs at necropsy in order to screen for the presence of bacterial pathogens. briefly each swab was plated onto 1â chocolate agar, 1â macconkey agar, and 2â blood agar (1â aerobic and 1â anaerobic) and submitted to the clinical services division at the royal veterinary college for analysis. samples were also plated onto mycoplasma experience agar (mycoplasma experience ltd. surrey, uk) and incubated at 37 8c with 5% co 2 for 3 days. dogs from all six treatment groups were healthy prior to challenge. dogs in t6 (negative control) remained healthy for the duration of the study, the only exception being one dog which had some serous ocular discharge prior to challenge, and throughout the study. all 36 dogs completed the study on the pre-assigned day. following challenge, a number of dogs from t1 to t5 inclusive displayed mild clinical signs of respiratory disease which included nasal discharge, sneezing and coughing. table 4 shows the total scores for each observation by treatment group. there were no significant changes in body temperature all dogs had normal or fair appetites and gained weight over the course of the study (data not shown). no consistent differences between dogs treated with different strains of the virus were observed, although respiratory signs were recorded most frequently and most severely in one dog from t3 and one from t5 (table 4 ). it is worth noting that in both instances these dogs were euthanized on study day 14 and displayed the clinical signs of respiratory disease recorded throughout the 14-day study period, although the scores were highest in these two dogs on study days 10-13. oropharyngeal swabs were analysed for crcov by both rt-pcr and virus isolation (fig. 1) . all dogs from all six treatment groups were negative for crcov on study day à1 (pre-challenge) using both techniques. all dogs in t6 (negative control) remained negative for the duration of the study. shedding of crcov in t1-t5 was consistently detected for up to 6 days post challenge by both rt-pcr and vi. all rectal swabs, collected at necropsy, were positive for the internal pcr control gapdh. all rectal swabs collected from dogs in t6 (negative control) were negative for crcov by both rt-pcr and vi for the duration of the study, as was the case for dogs in groups t3, t4 and t5. in t1, two dogs euthanized on study day 3, and one dog euthanized on study day 6 were positive for crcov, as was one t2 dog euthanized on study day 3. the remaining dogs in these groups were negative. all rectal swabs were negative for crcov by vi (data not shown). tissues collected at necropsy were tested for the presence of crcov by rt-pcr and vi (fig. 2) . crcov was detected in at least five of the eight tissues [diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, bronchial lymph node, nasal and palatine tonsil and lung lavage fluid] from groups t1-t4 by rt-pcr. in t5 crcov was detected in all the tissues tested. vi was successful in seven of eight tissue types from groups t1-t5 including the diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, nasal and palatine tonsil as well as the lung lavage fluid. only bronchial lymph nodes were negative for crcov by vi. overall, crcov was detected most frequently from the trachea, followed by the lung lavage fluid, nasal cavity and nasal tonsil, in all five treatment groups. histological examination was carried out on the palatine tonsil, external nares, nasal tonsil, trachea, apical and diaphragmatic lung lobes, and bronchial lymph node. the most significant findings were seen in the trachea and nares where inflammation, with notable changes in the length and distribution of the cilia were observed as described below and shown in figs. 3 and 4. in t6 the overall weighted histology score was consistently minimal (average 1.1), with minimal changes in all the tissues examined. in t1 the weighted score indicated minimal to modest abnormalities (average 1.7), including inflammatory aggregates in the nares and inflammation in the trachea which was associated with loss of cilia from the mucosal surface. one dog in particular on day 3 showed marked changes (scoring 3 or 4) in all of the tissues examined, with the exception of the diaphragmatic lung lobe and nasal tonsil where only minimal to modest changes were observed. in t2 and t3 the overall weighted score showed modest to marked histological abnormalities with average scores of 2 and 2.5, respectively. in the nares modest to marked inflammation was seen, which in t3 trailed off in the later stages of the trial period, whilst inflammation in the trachea of dogs from both groups was mild but with moderate shortening and irregularity of the cilia. in t4, with the exception of two dogs (one each on days 3 and 6), marked histological changes in the nares were observed throughout the trial period. in addition marked changes were also observed in the trachea and cilia. one dog on day 6 also had marked changes in the bronchial lymph node and diaphragmatic lung lobe (data not shown). the overall weighted scores indicated consistently marked abnormal histological changes in all dogs in this group with an average score of 3.3. in t5 modest to marked inflammation was observed in the trachea of dogs in t5 which was reflected in the condition of the cilia, whilst only modest changes were observed in the nares. on study day 14 marked changes were also seen in the bronchial lymph nodes. the overall weighted score showed consistently marked abnormal histological changes (average 2.8), with the exception of one of the dogs at the first time point which had a score of 1. in t1-t5 inclusive histological abnormalities in the lungs, although not marked, were variable and tended to be lymphoid aggregates adjacent to airways or blood vessels, an observation which was not seen in the broth control which yielded consistently mild scores. in all groups, including the broth control, the palatine tonsils had medium to high histological scores (data not shown). coronavirus antigen-positive cells were detected within the epithelium of the trachea and bronchioles of the infected dogs (fig. 5) . positive staining was present in the cytoplasm of ciliated columnar epithelial and goblet cells. positive cells were widespread but often found in focal clusters, and often associated with small aggregates on the luminal surface of the trachea. positive cells were surrounded by areas of vacuolation within the epithelium, possibly representing a cytopathic effect of the virus. seroconversion to crcov was measured by ifa. all dogs were seronegative (ifa < 40) for crcov on study days à1, 3 and 6, and all dogs in the t6 control group remained seronegative throughout the study whilst all dogs in the crcov challenge groups seroconverted by study day 14. crcov serum cross-neutralisation antibodies from serum collected on study day 14 were tested against 1 uk and 10 usa crcov isolates (table 5 ). all serum samples tested from dogs in crcov treatment groups were shown to be serum neutralisation positive against all the crcov isolates tested. serum collected from t6 control dogs showed no neutralising activity against any of the crcov strains. the mean concentrations of lymphocytes, neutrophils and monocytes, for each group on study days à1, 3, 6 and 14 were determined (data not shown). in t6, and t3 no statistical differences in the lymphocyte, neutrophil or monocyte concentrations were detected. in t1, t2, t4 and t5 increases in total white blood cell concentrations were observed as a result of lymphocytosis which was significant in t1 (p = 0.002) on day 6 and t5 (p = 0.027) on day 3. in t2 a marked lymphocytosis was seen on day 14. in t4 there was moderate lymphocytosis accompanied by significant changes in monocyte concentrations which peaked on day 6 [>day 3 (p = 0.003), >day 14 (p = 0.006)]. in contrast there was a significant decrease in neutrophil concentration on day 3 in t1, t4 and t5 (p = 0.004, p = 0.001 and p = 0.005, respectively) compared to day à1, which in t1 resulted in 3 of the dogs becoming neutropenic. across the groups seven dogs had rare to occasional toxic neutrophils exhibiting foamy cytoplasm, mild cytoplasmic basophilia or rare dohle bodies, and detectable levels of band neutrophils. in group t1, all dogs had detectable levels of band neutrophils on day 3, which was a significant increase when compared to day à1 (p = 0.031). on day 3 one dog in each of t2, t4, and t5 had band concentrations higher than the estimated upper reference limit; however this did not result in statistically significant changes at a group level and no other significant changes could be detected. oropharyngeal amies swabs collected from all dogs on day à1 and at euthanasia yielded growth of normal respiratory flora. a majority of dogs also yielded scanty growth of either streptococcus canis or staphylococcus intermedius. in the vast majority of dogs the lungs were sterile for bacterial growth, with the exception of the growth of mycoplasma spp. in four dogs, one from each treatment group t2-t5 inclusive (data not shown). previous publications have collectively demonstrated the global distribution of crcov and its association with respiratory disease in dogs under field conditions (erles et al., 2003; decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 yachi and mochizuki, 2006; knesl et al., 2009) . this is the first publication to report experimental infection of dogs using crcov, and the comparison of different crcov isolates from wide geographic origins. each crcov isolate was derived from dogs with respiratory disease in mo, mi, ut and ne in the usa, and one isolate from london, uk. this study builds upon a preliminary experimental challenge study (mitchell et al., unpublished data) . in that study we demonstrated rapid shed-spread of crcov from experimentally infected dogs to sentinel dogs within 4 days of exposure. peak viral loads were detected in oropharyngeal swabs at 4 and 6 days post infection in experimentally infected, and sentinel dogs, respectively; with shedding lasting for 8-10 days in both groups. both inoculated and sentinel dogs displayed clinical signs of mild respiratory disease, and seroconverted to the virus. in the current study prolific shedding of crcov from the oropharynx of dogs in all treatment groups (detected by rt-pcr and vi from oral swabs) was seen by day 2. in a majority of dogs viral shedding ceased after day 6, although crcov was detected up to 10 days post infection by rt-pcr in one dog. small differences in the duration of viral shedding between the two studies are most likely to be explained by differences in the age of the dogs used in each of the studies (preliminary study: 1-3 weeks old, current study: 12-16 weeks old). despite differences, both studies clearly illustrate that crcov is a quick-hit respiratory pathogen, and supports field data in which the rapid spread of crcov throughout a large rehoming centre was observed (erles et al., 2003) . consistent with observations made during naturally occurring infection, dogs in this study also displayed clinical signs of mild respiratory disease following viral challenge (nasal discharge, sneezing, and coughing); whilst the control group remained healthy. there appeared to be no profound difference in the clinical observations made between groups of dogs challenged with the different strains. two dogs however (one each in t3 and t5) showed more severe and prolonged clinical signs compared to the others. this included increased respiratory noise, and more frequent and prolonged coughing and sneezing. the reason for this was unclear. disease in these two dogs did not appear to be associated with any secondary bacterial infections in the lung, nor with notably different histopathological changes when table 5 study day 14 cross neutralisation titres. two dogs remained on the study at day 14 in each treatment group. samples with sn values of 14 were considered negative at the 1:20 starting dilution. serum samples collected at earlier time points were negative for crcov antibody. treatment group (isolate) lu131 lu172 lu189 lu295 lu317 np599 np604 np617 np631 np634 uk4182 t1 (lu295) 320 80 95 113 113 190 135 135 95 113 80 381 135 80 160 160 226 160 135 135 135 113 t2 (uk4182) 369 80 48 57 113 113 95 113 40 135 57 67 34 28 40 40 28 28 17 40 57 57 t3 (np631) 40 28 48 28 28 28 34 17 34 48 28 95 95 80 95 67 48 95 48 67 113 compared to other challenge dogs in the same group (data not shown). given its multifactorial aetiology; modelling cird under controlled conditions, to mimic disease as it appears in the field is difficult to achieve. when studied in isolation dogs infected with other viruses involved in the cird complex, such as cpiv and cav-2, also cause only mild respiratory disease such as a dry cough and nasal discharge (appel and binn, 1987a,b; castleman, 1985; buonavoglia and martella, 2007) . more severe clinical signs, representative of the disease complex, are likely to occur when other viruses or bacterial respiratory pathogens are also present, and when environmental stresses such as those encountered in rehoming facilities are experienced (appel and binn, 1987a,b; buonavoglia and martella, 2007) . overall there appeared to be little difference in tissue tropism, when comparing the different crcov isolates investigated. crcov was detected in at least five of the eight necropsy samples examined (lung lavage, diaphragmatic lung lobes, trachea, nasal tonsils and nasal cavity) in dogs from all five challenge groups. crucially, isolation of crcov was achieved from six of the seven tissues collected (the bronchial lymph node remained negative) as well as the lung lavage fluid. re-isolation of virus from experimentally infected dogs displaying clinical signs of disease, signifies a causal relationship between crcov and respiratory disease, which until now has been best demonstrated through epidemiological surveys (erles et al., 2004 (erles et al., , 2003 decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 knesl et al., 2009) . in all treatment groups crcov was detected most frequently in the trachea, nasal tonsil and lung lavage fluid, and these same tissues also exhibited the highest viral titres (data not shown). this is consistent with previous findings from a small cohort of naturally infected dogs in which the highest viral loads, detected by quantitative rt-pcr, were seen in the trachea and nasal tonsil . importantly, histological analyses were consistent with our molecular and virological findings. ihc revealed coronavirus positive staining in the cytoplasm of ciliated epithelial and goblet cells of the trachea and bronchioles of crcov infected dogs. this pattern of staining is consistent with previous work which identified coronavirus antigenpositive epithelial cells within the trachea of dogs naturally infected with crcov (ellis et al., 2005; priestnall, 2007) , and in crcov infected tracheal sections maintained in culture (priestnall et al., 2009; priestnall, 2007) . furthermore, histopathological analysis showed a clear association between exposure to crcov and inflammation in the nares and trachea, with loss or damage to cilia in the latter. these changes were particularly marked in dogs belonging to t4 and t5, which may indicate a higher degree of pathogenicity for those crcov strains; although, with the exception of one dog in t5 this was not apparent from the clinical signs. ciliary clearance is a key strategy for the removal of pathogens from the respiratory tract. reductions in the efficiency of ciliary clearance would potentiate infection with secondary respiratory pathogens, leading to increased severity and duration of disease. in addition to the trachea, the nasal tonsil may also represent an important site for crcov infection and pathogenicity; given the consistency and duration of crcov isolation and detection in this tissue. preliminary ihc analysis has shown that crcov infection in the nasal tonsil is associated with the epithelium, and with large mononuclear cells which have the appearance of macrophages (priestnall, 2007) . a clear association of crcov with macrophages is yet to be confirmed, but this could present another interesting area given the importance of macrophages in the pathogenesis of disease associated with other coronaviruses, such as feline infectious peritonitis virus (fipv), severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus 229e (hcov-229e) (desforges et al., 2007; rottier et al., 2005; shieh et al., 2005) . the detection and re-isolation of crcov from the lungs indicates the virus can also establish an infection in the lower airways. gross and histopathological analysis showed that both the control and challenged dogs had focal reddening of the lungs at necropsy, however no significant histological abnormalities were associated specifically with this reddening, and such reddening is assumed to be an artefact of barbiturate euthanasia (lopez, 2001) . nonetheless, in most cases there were microscopic differences between the lungs of these dogs, which could not be appreciated grossly, and therefore the gross appearance of the lungs was not considered an accurate predictor of histopathological changes in these experimental conditions. pulmonary inflammation in crcov challenge dogs was associated with lymphoid aggregates adjacent to the airways or blood vessels. the significance of this is uncertain, but notably these aggregates were not a striking feature of the control dogs. histological changes in the lungs and bronchial lymph nodes of dogs in t6 tended to be more consistent and mild; whilst scores for dogs in crcov challenge groups, although not marked, were more variable throughout the study. histological changes in the lymphoid tissues of most dogs in the study (t1-t6 inclusive) included hyperplasia. lymphoid hyperplasia is not uncommon in dogs at this age, particularly in the palatine tonsil. acute inflammatory responses were evident; however in some dogs this may have been associated with the presence of fragments of foreign material (e.g. hair) in the tonsillar crypts. where marked lymphoid hyperplasia was seen, this was reflected in the histological scores, which were more variable in the challenge dogs compared to those in the broth control animals throughout the trial period. the close genetic relationship between crcov and bovine coronavirus (bcov) (erles et al., 2003 , raised the question as to whether crcov may have an extended tropism which involves the gastrointestinal tract, as seen with some bcov strains (park et al., 2007) . the molecular detection of crcov in the rectal swabs of some dogs from t1 is interesting, and consistent with findings from a number of previous reports of dogs naturally infected with crcov (decaro et al., 2007; yachi and mochizuki, 2006; mitchell et al., 2009) . dogs in the t1 challenge group were unique in that they were the only dogs to be challenged with virus that had not been grown in vitro. the inability to detect crcov from dogs in other challenge groups may therefore be a result of cell culture adaptation; alternatively, this may be a strain specific phenomenon. at present there is no conclusive evidence that crcov displays a true dual tropism for respiratory and enteric tissues. the failure to isolate crcov from enteric samples collected during the peak oropharyngeal shedding period, suggests that crcov may pass through the canine gut as a bystander, without infection. nevertheless, enteric shedding could have implications for managing the spread of disease, and therefore further investigation is warranted. in addition to the histological changes observed in the tissues, significant changes in the leukogram can also be detected in association with crcov infection. this is summarised by a statistically and also clinically relevant lymphocytosis between days 6 and 14; most clearly seen in groups t1, t2, and t5. this observation is not unexpected due to the viral antigenic stimulation, and the lymphocytic reactions found in various tissues. what was somewhat less expected is the high number of dogs with decreased neutrophil concentrations, including a number which developed neutropenia, mild left shift and toxicity, best seen in groups t1, t2, t4, and t5. these changes suggest an acute inflammatory reaction with a high demand for neutrophils and accelerated production in the marrow. transient neutropenia is not uncommon during infections with some viruses; however, there is currently limited data relating to the leukogram profile following infections with other beta coronaviruses. in one report detailing the experimental infection of cows with bcov, significant reductions in neutrophil concentrations were observed at 2 days post infection, followed by neutrophilia at 14 days post infection (traven et al., 2001) . in sars coronavirus infected patients the picture is mixed. neutropenia has been reported in some cases, however, in most cases high blood neutrophil concentrations were observed, and this is often associated with a poor clinical outcome manocha et al., 2003; wong et al., 2003) . neutrophil infiltration of tissues infected with different coronaviruses such as sars, mhv and rat cov has also been described (bhatt and jacoby, 1977; iacono et al., 2006; ding et al., 2003) ; however, their role in viral clearance and possible immune pathology is largely unknown. considering the presence of mostly lymphoid aggregates in crcov infected tissues in this study, the observed changes are intriguing, and a direct effect of crcov on the myeloid series in the bone marrow cannot be ruled out. seroconversion to crcov and the acquisition of neutralising antibodies to heterologous strains from distinct geographical locations within the usa and uk occurred in all challenge dogs remaining on the study at day 14. this degree of serological cross reactivity is not unexpected given the high degree of amino acid similarity seen in the spike protein of different isolates published to date. whilst the correlation between neutralising titres and protection in vivo is yet to be determined, this finding supports published data which demonstrated that the presence of crcov specific antibodies in dogs significantly decreased the risk of developing respiratory disease upon entry to the kennel (erles et al., 2003) . these findings support a possible role for vaccinating against crcov as part of a preventative strategy for respiratory disease in kennelled dogs. moreover, based on the high degree of cross neutralisation and high degree of amino acid identity among the crcov spike proteins described to date, it is anticipated that a single crcov isolate as a vaccine antigen will be sufficient to provide protection against crcov induced respiratory disease. in summary, this is the first study to describe the development of a model for studying crcov pathogenesis; and to fully demonstrate koch's postulates through the successful re-isolation of crcov from experimentally infected dogs with clinical signs of respiratory disease. isolation of crcov was achieved from a wide variety of respiratory and mucosal associated lymphoid tissues, lung lavage fluids and swabs collected over the 2-week period, thus providing clear evidence of tropism for the canine respiratory tract, accompanied by respiratory shedding. moreover, we have shown crcov infection is associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia alongside inflammatory responses. significant effects on the leukogram, in the form of clinically relevant lymphocytosis and neutrophil changes were also documented. strong serological and cross neutralising reactivity between heterologous crcov isolates demonstrates antigenic homogeneity among crcov from the two continents. this study provides vital evidence supporting a role for crcov in the cird complex. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. canine infectious tracheobronchitis short review: kennel cough canine infectious tracheobronchitis short review: kennel cough sv-5-like parainfluenza virus in dogs bordetella and mycoplasma respiratory infections in dogs and cats pathogenesis of canine bordetellosis experimental infection of adult axenic rats with parker's rat coronavirus canine respiratory viruses bronchiolitis obliterans and pneumonia induced in young dogs by experimental adenovirus infection mycoplasmas associated with canine infectious respiratory disease serological and 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winter dysentery bovine coronavirus in calves the role of a novel coronavirus in canine infectious respiratory disease. royal veterinary college serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus quantification of mrna encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (crcov) acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis survey of dogs in japan for group 2 canine coronavirus infection this study was funded by pfizer animal health. the authors would like to thank m. ikeh for technical assistance. rvc manuscript id number: pid_00399. key: cord-258783-ev0h95b9 authors: kapil, sanjay; yeary, teresa j. title: canine distemper spillover in domestic dogs from urban wildlife date: 2011-11-30 journal: veterinary clinics of north america: small animal practice doi: 10.1016/j.cvsm.2011.08.005 sha: doc_id: 258783 cord_uid: ev0h95b9 canine distemper virus (cdv) causes a major disease of domestic dogs that develops as a serious systemic infection in unvaccinated or improperly vaccinated dogs. domesticated dogs are the main reservoir of cdv, a multihost pathogen. this virus of the genus morbillivirus in the family paramyxoviridae occurs in other carnivorous species including all members of the canidae and mustelidae families and in some members of the procyonidae, hyaenidae, ursidae, and viverridae families. canine distemper also has been reported in the felidae family and marine mammals. the spread and incidences of cdv epidemics in dogs and wildlife here and worldwide are increasing. attachment factor, protein h, controls the host specificity and cell tropism and induces the majority of cvd-neutralizing antibodies. [13] [14] [15] humoral immunity due to the presence of neutralizing antibodies to cdv, elicited by either immunization or natural infection, is detectable within 10 to 14 days, providing protection against infection or reinfection. viral infection of a susceptible host cell begins when the h protein of cdv binds to the signaling lymphocyte activation molecule (slam; cd150) receptor site of the cell. 16 a conformational change of the h protein occurs on binding, which signals the f protein-mediated fusion of the cdv envelope with the host cell membrane. binding between slam and the h protein is a high-affinity, host-virus specific interaction. 14, 17 the h and f glycoproteins may mediate fusion activity between neighboring cells leading to syncytium formation and, ultimately, to cell lysis. 16 host cell surface sites cd46 and a heparin-like receptor have been suggested as putative h protein receptors in slam-negative cells, but strong supporting evidence is lacking at this time. 18, 19 disease distemper is a highly contagious disease that poses a threat mainly to concentrated populations of previously unexposed or unvaccinated, susceptible species. in these populations, distemper is almost always fatal. the disease is complex in that it presents varying clinical symptoms and may run varying clinical courses. outcomes of cdv infection range from complete recovery to persistent disease to death depending on the age and immune status of the animal infected. 12 robustness of the humoral immune response correlates with the disease outcome. canine distemper virus replicates initially in the lymphoid tissues of the upper respiratory tract followed by immune-mediated progression of the disease over a period of 1 to 2 weeks. a diphasic fever is a characteristic feature of the disease, occurring 7 or 8 days after infection, that drops rapidly and again climbs by day 11 or 12. clinical signs of distemper are often unapparent or initially mild during this time, and disease is characterized by mucopurulent oculonasal discharges, conjunctivitis, respiratory distress, anorexia, vomiting, diarrhea and dehydration, and cutaneous rash. anti-cdv antibody titers that develop 10 to 14 days postinfection contribute to viral elimination and recovery when a vigorous humoral response occurs characterized by highly specific anti-h protein antibodies. cell-mediated immunity also plays a role in recovery from cdv infection, and a strong t-cell-mediated cdv-specific immune response causes viral elimination in convalescing dogs. 20 weak humoral and cell-mediated responses lead to systemic intracellular spread of virus to the epithelial cells of the gastrointestinal and urinary tracts, skin, and the endocrine and central nervous systems causing direct virus-mediated damage. additional clinical signs that may occur are localized twitching, ascending paresis/ paralysis, and/or convulsions. hyperkeratosis of the foot pads and nose may be seen. the infection may either prove fatal or persist resulting in subacute or chronic central nervous system (cns) signs. delayed lymphocytolysis correlates with persistence of cdv in the cns. 21 within 1 to 3 weeks after recovery from gastrointestinal and respiratory signs, depression and neurologic signs indicating cns involvement are often evident, although sometimes neurologic impairment does not occur until months later, even without a history of systemic signs. 12 dogs that recover from acute disease with persistent infection may shed virus in urine and through the skin on the foot pads. these animals should be isolated from contact with unvaccinated animals, especially puppies. canine distemper infection can be challenging to diagnose because many diseases can cause symptoms resembling canine distemper. the respiratory symptoms of canine distemper may be mistaken as canine respiratory disease complex. canine parvovirus, coronavirus, bacterial, and internal parasite infections should be ruled out as causes of vomiting and diarrhea. often, cdv-infected animals that exhibit neurologic signs are mistaken as having rabies. neurologic symptoms must be differentiated from other infections, trauma, and ingestion of toxins. vaccination history of the affected animal, clinical symptoms, and laboratory testing support a probable diagnosis of cdv infection. state and commercial veterinary diagnostic laboratories offer testing for canine distemper and advice practitioners on appropriate specimens to submit, tests to order and the limitations of test results given the circumstances of each individual case submitted. the following 5 diagnostic methods are commonly offered: 15 use of monoclonal antibodies to differentiate recent field isolates from older field isolates and vaccine strains of cdv has met with limited success and the reagents developed are not widely available. 24, 25 currently, 2 in-clinic serologic test kits are licensed for sale in the united states, the titerchek cdv/cpv elisa-based assay (synbiotics, san diego, ca, usa) and the immunocomb canine vaccicheck (modern veterinary products, coral gables, fl, usa). 26 both kits evaluate an immune response to cdv from vaccination or infection but neither differentiates between titers to the vaccine or infection with wild-type cdv strains. licensed rt-pcr kits for detection of cdv are not available in the united states. among the commercial and state veterinary diagnostic laboratories that perform rt-pcr testing of their own design to detect cdv, one of the challenges is differentiating between vaccine strains and wild-type isolates that may be present concurrently in samples. the rt-pcr assays are typically designed to amplify a portion of the h, f, m, or n gene to verify the presence of cdv rna in specimens. absolute identification of strains and differentiation between vaccine and wild-type cdv may be performed by sequence analysis of the cloned rt-pcr amplified h gene region. 10 rapid methods have been designed to differentiate cdv strains as either wild-type or vaccine derived without the need to perform time-consuming gene sequencing. two popular methods are based on rt-pcr of a specific cdv structural protein genes followed either by a restriction fragment length polymorphism (rflp) analysis of the amplified nucleic acid or by a second round of nested pcr with analyses by electrophoresis. 10, [27] [28] [29] other unique approaches that have been developed are multiplex rt-nested pcr (rt-npcr) of the m protein and amplification refractory mutation system (arms)-pcr of the cdv m-f intergenic and untranslated, prepeptide regions of the f gene followed by rflp. 30, 31 most cdv vaccines in the united states, canada, and europe are of the american-1 (onderstepoort) lineage with the exception of the vanguard vaccine (pfizer animal health, madison, nj, usa), which is of the america-2 genotype. 27, 32 the major vaccine strains were isolated in the 1930s and it is not known if they continue to circulate in nature as they have not been detected for many years. 33, 34 although cdv vaccine strains have not changed in the past 60 years, there is potential for newer antigenic variants of cdv to emerge around the world. 23 however, the current vaccines have largely provided adequate protection against clinical disease when properly administered to healthy domesticated dogs in this country. core vaccination guidelines, including canine distemper mlv and recombinant canarypox vectored canine distemper virus (rcdv) vaccines, recommended by the american animal hospital association canine vaccine guidelines, were revised in 2006. 35 recommendations for administering the rcdv and mlv vaccines are similar. advantages of the rcdv vaccine is that it does not contain live virus that replicates and spreads from vaccinees and it is more likely to produce immunity in puppies that have passively acquired maternal antibodies. vaccination failures can occur when mlv vaccines are used to immunize puppies that have not cleared maternal antibodies. 36 maternal antibodies are adsorbed in the intestine from colostrum during the first 2 days of life and are cleared 6 to 12 weeks later. it is recommended that puppies receive a series of 3 vaccinations beginning at 6 to 16 weeks of age to achieve complete immunity to cdv followed by a booster at 1 year of age. canine distemper virus vaccines impart long-term immunity in dogs. 37 duration of immunity of 3 years has been reported for both mlv and rcdv vaccines. 38, 39 in animal shelters and high-risk environments, one dose of mlv or rcdv vaccine has been reported to be protective in puppies already exposed to cdv. 40 ferrets are also highly susceptible to cdv and the disease is virtually 100% fatal. the american ferret association recommends vaccinating ferrets with purevax ferret distemper vaccine (merial inc, athens, ga, usa), the only usda-licensed vaccine product labeled for use in ferrets, following the product label for kits or adults. 41 reasons that a vaccine may fail, in addition to the presence of maternal antibodies in puppies, are incomplete immunity due to failure to complete the puppy booster vaccination series, stressors in the physical environment, the animal's immune competence and specific responsiveness to cdv antigen or intercurrent exposure to other virulent viruses such as canine parvovirus or coronavirus or even parasites, and improper storage and handling of vaccine. 36, [42] [43] [44] a concern voiced by scientists is that new genetic cdv variants may be associated with pathogenesis changes or immune evasion in dogs vaccinated with current vaccines. 45 in infected dogs with a history of recent vaccination with mlv vaccine, exposure to wild-type cdv prior to vaccination is usually assumed to be the source of the cdv infection. 10 however, cdv infections reported in previously vaccinated dogs in japan, mexico, and the united states were caused by novel cdv lineages distantly related to the america-1 vaccine group. 10,44,46 -49 variation of key amino acid residues and the addition or loss of n-glycosylation sites on the h and f proteins may alter interaction between the h and f proteins during binding and fusion with susceptible cells, leading to changes in antigenicity, virulence, and tissues targeted by cdv variants. 50, 51 continued surveillance, study of genetic and antigenic drift in circulating cdv strains, and molecular analysis of emerging cdv variants are warranted to ensure that vaccines for prevention of distemper continue to be potent and efficacious in preventing infection in domestic dogs. in addition to immunization of domestic dog populations, hygienic measures are necessary. unvaccinated puppies should be isolated from dogs other than their bitches. strict isolation of dogs infected with cdv is the most important step in controlling the disease. virus is shed in all body secretions and excretions during the acute systemic disease. direct dog-to-dog contact and indirect aerosol transmission are the main routes of viral spread, but cdv can be transmitted from fomites at room temperature or lower for several hours. disinfection of cdv in the environment, particularly in shelters and kennels, is important. inactivation of canine distemper virus with benzalkonium chloride (0.05%), a quaternary ammonium compound, occurs in 10 minutes at room temperature. 52 similarly, 70% ethanol is effective against cdv. 53 nucleic acid sequence analysis of the h gene is the gold standard for phylogenetic analysis, classification, and genotyping of cdv because it has the greatest heterogeneity (about 10% amino acid variation) of the 6 structural proteins of cdv. 54 studies of complete h gene sequences have identified 12 distinct geographically separated clusters of cdv genotypes: american-1 (including most vaccine strains), american-2 (north america), arctic (arctic region and europe), asia-1, asia-2, asia-3, europe, european wildlife, south africa, argentina, rockborn-like, and a new genotype of primarily mexican strains. 28, 48, [55] [56] [57] serengeti isolates are distinctive from cdv isolates from other parts of the world. 5 in the united states, genotypes that have been identified in dogs and wildlife in addition to the american-1 and america-2 strains are the european wildlife, edomex, and arctic strains in domestic dogs. 10, 49, 55 amino acid sequence variation between the genotypes is greater than 4% and strains within each genotype have less than 2% amino acid variation. 12 characterization of cdv strains from south america may be of special interest. scientific archivists point to documentation of distemper-like epizootics occurring in peruvian dogs in the mid-1700s that may have spread to europe circa 1760 with the importation of diseased dogs by spanish colonials. 58 sequence analysis of cdv strains from different geographical locations and animal species indicates that the h protein gene undergoes genetic drift. 59 viral recombination in cdv has been documented in an isolate recovered from a giant panda. 60 recently, a cdv genotype designated "wildlife europe 2006 -2009 (we/06 -09)" found exclusively in wild carnivores was described that evolved and spread over a wide geographical area in northern italy in 10 months following its initial detection in 2006. 61 bavarian wildlife isolates collected during the 2008 distemper outbreak in the southern alps were 99.7% to 100% similar to the italian isolates. 62 the evolutionary origin of the group was estimated to have diverged from its most recent ancestor 5 months prior to identification of the first virus cdv. 63 the mean nucleic acid substitution rate in the new cdv genotype was estimated to be 10.53 ϫ 10 -4 subs per site per year, which was within the range typically observed for cdv. 63 phylogenetic analysis of 73 cdv h gene and h protein sequences from dog and non-dog hosts indicated that amino acid residues 530 and 549 are under positive selection, and these residues are located in the regions of the h protein that are important in binding to the host cell slam receptor and triggering activation of the f protein cellular entry. 17, 59, 64 this provides compelling evidence that repeated evolution at known functional sites of emerging strains of cdv is associated with multiple independent occurrences of disease emergence in a range of novel host species. facilitation of large-scale diagnostic and molecular epidemiologic studies of cdv requires rapid molecular-based methods that accurately differentiate among the genotypes and between vaccine and wild-type strains of cvd without the need to perform either full-length or partial sequencing of the h gene for each isolate. a hemi-nested pcr system was developed that can genotype 5 of the 12 cdv lineages (america-1, europe, asia-1, asia-2, and arctic) using specific primers targeted to the h gene. 32 the arms-pcr method followed by rflp also differentiates a broad variety of lineages. 31 further development of rapid protocols for distinguishing among all cdv genotypes is needed to advance epidemiologic studies of this important pathogen. genotyping is important for tracing the relatedness of cdv isolates and cross-transmission between and within species of carnivores. distemper outbreaks in rhesus monkeys (macaca culatta) have occurred since 2006 at the largest monkey breeding farm in mainland china that supplies breeding stock for biomedical research facilities and zoos. 65 over 10,000 monkeys contracted the disease and more than 4,250 died at the farm and at the facilities it serves. the entire genome of the isolated virus was sequenced. phylogenetic analysis of the h gene places it within the larger clade of asian genotypes yet it is unique in the number of amino acid changes to its structural proteins. although monkeys and monkey-derived cell cultures have been experimentally infected with cdv, only one other natural cdv outbreak of monkeys (macaca fuscata) occurring in japan was reported in 1989. 66 canine distemper is not a clinically recognized entity in domestic cats; however, large felids are susceptible to infection with cdv. most of the large cats are threatened or endangered species; thus surveillance of pathogens that have the potential to cause their extinctions is critical. where cdv has caused widespread distemper outbreaks in nondomestic cats, domestic dogs, raccoons, or wild canids have been implicated as reservoirs of the disease. cdv outbreaks with multiple mortalities were reported in lions, tigers, jaguars, and leopards in zoos and wildlife safari parks in the 1980s. 67 raccoons living in the area surrounding one suburban zoo had increased numbers of fatal distemper cases and may have transmitted the disease to the large cats. cdv isolated from large felids in the zoo was of the america-2 genotype circulating in the local feral raccoons. 64 a retrospective immunohistochemistry study of paraffin tissues from 42 necropsy cases of lions and tigers from swiss zoo and circus cats collected from 1972 through 1992 indicated that 19 were cdv positive. 4 of 56 asiatic lions from 6 captive breeding centers in western india tested in 2007 for antibodies against cdv, 88% were positive. 68 in addition to domestic dogs, urban wildlife in the united states such as raccoons, foxes, and skunks may play a role in direct transmission of distemper to large felids and other carnivores in zoos, wildlife parks, circuses, and captive breeding facilities. many studies of canine distemper in free-ranging large felids have been reported. 69 -75 african lions of the serengeti are the most intensively studied of the large felids with regard to the prevalence of cdv. in 1994, a cdv epidemic in serengeti lions caused fatalities in 30% of the population with only an estimated 2,000 lions remaining in 1996. 69 prior to 1994, disease-related mortality due to cdv infection of lions had not been documented, although retrospective serology tests indicated that 29% of lions that were living in the area from 1984 to 1989 had titers to cdv. a single cdv genotype was common among the susceptible animal species living in the serengeti during the 1994 cdv outbreak that included lions, hyenas, bat-eared foxes, domestic dogs, and jackals. 5 unowned, feral domestic dogs living in or near the serengeti are not vaccinated, experience periodic distemper outbreaks, and likely serve as a primary reservoir of cdv. jackals and hyenas may be amplifying species that spread cdv throughout the park to lions and other felids. 6,76,77 a brazilian study was performed in 2 state parks with the goal of determining the prevalence of cdv titers in wild felid populations (jaguars, pumas, and ocelots) and correlating it with the prevalence of cdv titers in, and density of, domestic dogs in the areas adjacent the parks. 72 dog owners in small rural settlements surrounding the parks were questioned about the cdv vaccination status of their dogs. unvaccinated dogs were tested for cdv titers. jaguars (60%) and pumas (11%) from one park had titers to cdv and 100% of the dogs living adjacent to the park were seropositive for cdv. none of the large felids tested at the second park had cdv titers and only 35% of the local unvaccinated dog population was seropositive for cdv. the occurrence of cdv in wild felids appears to be related with home range and close association with unvaccinated, infected domestic dogs living nearby. vaccine coverage of 95% of domesticated dogs is needed to control canine distemper in these pets. 78 currently the best means for breaking the circulation of cdv between susceptible wildlife populations and domestic dogs is through regular vaccination of pet dogs and preventing them from roaming freely and interacting with unvaccinated dogs and wildlife that may harbor the virus. free roaming wildlife are not vaccinated in the united states unless federal and state authorities determine that an endangered species may benefit from vaccination in captive breeding programs designed to stabilize and increase existing populations for release back into the wild. one study reported the vaccination of wild raccoons with mlv canine distemper vaccine prior to 1997 in a forest preserve near a chicago area zoo. 64 in the 1960s through the 1980s, primarily killed vaccines (kv) were used to vaccinate endangered wildlife and zoo animals against cdv. 79, 80 virus-neutralizing titers developed postvaccination to the kv were generally quite low, and several exotic species that had been vaccinated died from outbreaks of cdv infection. use of mlv cdv vaccines is often fatal to many wildlife and zoo animals; thus they have only been used in rare situations in the united states to control disease in endangered species and display animals in zoologic parks. 1,79 -82 after the univalent canarypox vectored recombinant distemper vaccine, purevax ferret (merial inc), was licensed and marketed in 2001, many north american zoological institutions began using the rcdv vaccine to vaccinate numerous at-risk species. 83 currently, the american association of zoo veterinarians' distemper vaccine subcommittee recommends the extralabel use of the rcdv purevax ferret distemper vaccine (merial, inc) in all susceptible zoological display animals where cdv is endemic in local wildlife. 84 vaccination of endangered species that are susceptible to cdv has been an important in the success of recovery programs. initially, commercial kv and mlv cdv vaccines were used to vaccinate the endangered black-footed ferret but these products proved to be nonprotective or fatal. 80, 85, 86 in 1988, an experimental canarypox vectored rcdv vaccine (merial inc) used to vaccinate ferrets in the captive breeding program successfully prevented distemper, one of several diseases that had threatened the species with extinction. 87 all wild-born black-footed ferrets are trapped and vaccinated. after the 1999 cdv outbreak on santa catalina island, california, the native island fox population plummeted from 1,300 to less than 100 individuals. infected domesticated dogs or stowaway raccoons from boats anchoring on the island mingling with the foxes may have caused the outbreak. 88 the federally endangered island fox was vaccinated with the rcdv vaccine to reestablish the population beginning in 1999 with permission from the california department of fish and game. 89, 90 wildlife rescue and research organizations also vaccinate cdvsusceptible animals in areas where distemper is endemic. the rcdv vaccine, purevax, is used prevent disease in captive southern sea otters at california institutions. 9 free-ranging sea otters are susceptible to cdv. immune-stimulating complexes (iscoms), a novel form of adjuvant that, combined with antigens, generally induces strong activation of both the cell-mediated and humoral immunity. african wild dogs (lycaon pictus), which are on the international union for conservation of nature red list of threatened species, cannot be vaccinated with mlv cdv vaccines, which are always fatal. 91 one study reported the use of iscoms incorporating the f and h proteins to vaccinate african wild dogs. 92 the dogs initially vaccinated at the beginning of the captive breeding program in 1995 developed protective immunity. however, in 2000, when the 49 of 52 dogs in the colony succumbed to distemper, neutralizing anti-cdv antibodies were not measurable despite a recent vaccination. although the use of iscoms appeared to be promising for control of cdv in a variety of wildlife, the successes have been limited. 82 oral bait vaccines to control zoonotic diseases like rabies and plague in wildlife are currently in use. oral vaccines to control wildlife distemper are not yet available. two major issues in developing an efficacious oral bait vaccine for distemper are achieving an adequate mucosal immune response in the gut and overcoming interference from maternal antibodies in infant animals. attempts at inducing mucosal immunity using vaccinia and canarypox vectored cdv vaccines have been reported using ferrets as model animals. 80, [93] [94] [95] highly attenuated vaccinia and canarypox virus strains expressing the h and f proteins of cdv were administered by parenteral, intranasal, and intradoudenal routes. juvenile ferrets receiving either vaccine intramuscularly or intranasally had 100% survival rates, but intradoudenal vaccination protected only 60%. 93 in studies of infant ferrets with and without maternal antibody, the vaccinia and canarypox vectored vaccines were administered parenterally or intranasally. all infant ferrets vaccinated parenterally with either vaccine in the absence of maternal antibody survived challenge. parenteral vaccination with either vaccine in the presence of maternal antibody did not protect against death from cdv challenge. intranasal vaccination with either vaccine, in ferrets with or without maternal antibody, was not protective against cdv. 94 other studies have shown low efficiency in producing a protective immune response with the nonparenteral delivery of cdv canarypox vectored vaccines. 80, 95 as with the raboral v-rg (merial, inc), the cdv vaccinia vectored vaccines stimulate a stronger protective mucosal immune response. 93 if an efficacious cdv oral bait vaccine can be developed for wildlife, vigorous domestic dog vaccination programs here and abroad will continue to be the primary means to control the disease. in the united states, several federal agencies are tasked with surveillance of animal diseases of wildlife. the u.s. department of agriculture-aphis wildlife services' administers the national wildlife disease program (nwdp), which participates in wildlife disease monitoring and surveillance in all regions of the united states. 96 additionally, nwdp assists state, federal, tribal and international agencies, and nongovernment organizations, with development of local wildlife disease monitoring programs and nationally coordinated wildlife surveillance systems. canine distemper is among diseases of interest to the surveillance program, although minor. 97 over the past 10 years, the nwdp has assisted in distemper surveillance monitoring and research activities with state agencies and veterinary colleges. 96, 98, 99 the usda national wildlife research center is currently assisting the zambian wildlife authority and the african wild dog conservation trust in the development of conservation management plans for several critically endangered species including african wild dogs, african lions, bat-eared foxes, and leopards. it has been postulated that diseased village dogs are the reservoirs of distemper, rabies, parvovirus, and a number of parasites that are infecting african wildlife. 100, 101 three programs within the u.s. department of the interior also monitor threats to wildlife and wildlife health in the united states: the fish and wildlife service (fws), national park service (nps), and u.s. geological survey (usgs). 102 the fws administers health monitoring programs for endangered and threatened terrestrial and freshwater species under the endangered species act of 1973. in 1988, in association with state and private organizations, the fws began a captive breeding and vaccination program of black-footed ferrets, which were nearly extinct due to outbreaks of canine distemper and sylvatic plague. 86, 103 the fws was involved in the captive breeding and vaccination program and continuing surveillance of the santa catalina island fox population after the 1999 canine distemper outbreak. by the end of 2010, the fox population rebounded from 100 foxes to 1,008 individuals. 88, 104, 105 grey wolves reintroduced by the fws to yellowstone national park are monitored for canine distemper, which caused population declines in 1999, 2005, and 2008. 106 the nps biological resource management division performs surveillance and disease management of wildlife health within the federal park system. the usgs national wildlife health center, which provides wildlife health and disease investigative, research, and training support to federal, state, local, and international conservation agencies, was designated as an oie collaborating centre for research and diagnosis of emerging and existing pathogens of wildlife, by the world organization for animal health (oie) in july of 2011. the epidemiology and transmission of cdv are complicated by the wide host range of animals susceptible to distemper. 2 canine distemper virus is present on all continents wherever there are carnivores. domestic dogs are considered to be the primary reservoir of cdv, which disseminates between free-ranging, unvaccinated or incompletely vaccinated dogs (pets and feral) and urban or rural wildlife. 1 raccoons, foxes, and skunks have adapted well to urban environments and, in the united states, raccoons, a secondary reservoir of cdv, are among the most common wildlife species found in cities and towns. cyclical outbreaks of distemper commonly occur in north america among raccoons associated with an increase in their populations. the periodic increase in distemper outbreaks in raccoons leads to spillback to domestic and feral dogs and spillover to other wildlife (skunks, foxes, badgers, coyotes, wolves, etc.). over the past decade, many outbreaks of canine distemper in urban wildlife have been reported in the united states and canada, prompting health officials to issue advisories to the public to avoid feeding or otherwise attracting wildlife to their property, keep dogs current on cdv vaccinations, and confine their pets in fenced enclosures or on a leash. 11, 107 infection with cdv also is an important conservation threat to many carnivore species in their natural habitats, especially for small, endangered populations that already face environmental insults. 108, 109 distemper has contributed to population declines in black-footed ferrets, catalina island foxes, native florida mink, gray wolves, coyotes, sea otters, pumas, and ocelots in the united states and many other wild carnivores worldwide. often, multiple competent hosts for cdv exist within a region, allowing localized persistence of disease. 110 susceptible captive animals that are held in high densities are especially vulnerable to infection; thus quarantine, vaccinations, and meticulous hygiene are important measures to take, as is reducing the potential for contact with free roaming wildlife that serve as reservoirs of disease. 4,64 -68 transmission of cdv between animals is via aerosol or respiratory secretions (coughing, sneezing, barking, licking) and bodily excretions (urine and feces) or through direct contact with shared, virus-contaminated food and water bowls, garbage, compost piles, and other organic materials. other disease-causing contacts include chasing, mating, fights, simultaneous and sequential feeding events at carcasses, and grooming. 76 wild animals with distemper have similar symptoms as infected dogs. they are often mistaken as rabid because they display unusual behavior, disorientation, aimless wandering, and/or aggression and walk with an unusual gait due to cns involvement. the majority of cases in wildlife are most often observed in spring and summer since juveniles are more susceptible to infection, but cases occur year round. studies of threatened, endangered, or reintroduced carnivore species in the greater yellowstone ecosystem and in the serengeti national park, tanzania, have supplied a wealth of information on the epidemiology of cdv in these expansive natural habitats over many decades. 69, 76, 77, 88, 110, 111 however, little is known of the overall health status and disease problems in free-ranging wildlife populations that have direct and regular contact with domestic dogs. the domestic dog is the most numerous of carnivores in the world with an estimated population of over 500 million worldwide. 112 domestic dogs have been sources of many zoonotic viruses, bacteria, helminths, arthropods, protozoa, and fungi and have served as a link for exchange of pathogens among livestock, wildlife, and humans. [113] [114] [115] an international expert meeting on dog population management was held in banna, italy, in march 2011 as a joint effort between the food and agricultural organization of the united nations and the world society for the protection of animals with technical support from the world health organization, to address the challenges of domestic and stray dog population management throughout the world. 116 regular domestic animal health care is not universally available in developing nations or even in remote areas of developed countries. this hinders development of effective disease detection and preventative veterinary medicine programs. 115 lack of vaccination to achieve herd immunity, uncontrolled reproduction of domestic dogs, and free-roaming dogs, they are whether owned, abandoned, or feral, are major roadblocks to preventing further spread of cdv to all susceptible species. 72, 73, 117, 118 studying the demographic characteristics of dog populations in urban and rural areas is critical for understanding the epidemiology of canine infectious diseases and to make decisions in planning and implementing dog population management schemes to control zoonotic diseases and diseases that are of conservation interest such as cdv. 72, 117, 118 three recent prospective studies of large felids in brazil, iberian lynx in andalusia, spain, and wolves in the remote north coastal mainland and islands of british columbia, canada, suggest that unvaccinated dogs in towns and small settlements do pose a significant risk; seroprevalence for cdv exposure in these animals is high. 72, 73, 119 additional prospective studies of disease in threatened and endangered species and dog populations that reside in transecting areas of urban populations, towns or settlements, and wilderness areas are needed to provide baseline health and serologic information. the heterogeneity of cdv genotypes that have been isolated in restricted geographical areas within the united states, europe, and elsewhere are postulated as being the result of intense, legal, or uncontrolled trade and travel of domestic dogs and uncontrolled movement receptive wild species. 10, 12, 120 recent reports of european wildlife and edomex genotypes isolated from north american dogs that have not traveled outside the united states underscore the need to gather additional sequence information to elucidate the epidemiologic patterns of cdv on a local and global scale. 10 characterization of circulating cvd genotypes in domestic dogs and wildlife within a discrete territory over a protracted timeline would also further our understanding of how the virus spreads and evolves within and between species. reliable information about transmission of cdv among domestic and wild carnivores should enable more effective management of the disease. 76 canine distemper is a highly contagious disease of domestic dogs that also infects multiple wildlife hosts, some that serve as secondary or amplifying reservoirs of the virus. transmission of cdv among dogs and other susceptible hosts continues to present many challenges in the united states and worldwide. control of distemper in dog populations requires a strong commitment by many constituencies. cdv is the most significant viral threat to the extinction of endangered carnivores, eclipsing rabies. effective vaccines for distemper are available to control cdv in domestic dogs, although the vaccine strains that are used in commercial vaccines have not changed in the past 60 years. client education about the serious consequences of cdv to both their pet dogs and to wildlife is the critical first step to curtail the spread of cdv, followed by reducing reproduction rates of dogs and abandonment of pets. it is important for veterinarians, dog owners, animal control officers, wildlife wardens, and quarantine officers to understand that canine distemper can cross continents during the transportation of dogs. a major challenge in diagnostic testing is differentiating infection due to attenuated vaccine virus from infection caused by wild-type virus so that recently cdv-vaccinated dogs are not unnecessarily euthanized where outbreaks of distemper occur, particularly in animal shelters. because canine distemper is an rna virus, a potential for emergence of antigenic variants exists, particularly in situations where wildlife that are infected with a strain of cdv that has adapted to that host spills back to domestic dogs. introduction of novel canine distemper viruses in improperly vaccinated dog populations with insufficient immunity can cause new outbreaks of cdv. increased surveillance of cdv in dog and wildlife populations to identify new genotypes and trace movement of strains within and between species will broaden our epidemiologic knowledge base and advise the veterinary profession and biologics industry as to the need for changes to vaccine strains to protect domestic dogs. canine vaccination-providing broader benefits for 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of parasitic zoonoses infectious diseases and extinction risk in wild mammals dogs as sources and sentinels of parasites in humans and wildlife, northern canada world society for the protection of animals. wspa and the fao call for global dog population body demography of domestic dogs in rural and urban areas of the coquimbo region of chile and implications for disease transmission urban domestic dog populations as a source of canine distemper virus for wild carnivores in the coquimbo region of chile exposure to infectious agents in dogs in remote coastal british columbia: possible sentinels of diseases in wildlife and humans canine distemper: still a major concern in central europe. lucrari stiintifice universitatea de stiinte agricole a banatului timisoara key: cord-303187-ny4qr2a2 authors: belo, vinícius silva; struchiner, claudio josé; werneck, guilherme loureiro; teixeira neto, rafael gonçalves; tonelli, gabriel barbosa; de carvalho júnior, clóvis gomes; ribeiro, renata aparecida nascimento; da silva, eduardo sérgio title: abundance, survival, recruitment and effectiveness of sterilization of free-roaming dogs: a capture and recapture study in brazil date: 2017-11-01 journal: plos one doi: 10.1371/journal.pone.0187233 sha: doc_id: 303187 cord_uid: ny4qr2a2 the existence of free-roaming dogs raises important issues in animal welfare and in public health. a proper understanding of these animals’ ecology is useful as a necessary input to plan strategies to control these populations. the present study addresses the population dynamics and the effectiveness of the sterilization of unrestricted dogs using capture and recapture procedures suitable for open animal populations. every two months, over a period of 14 months, we captured, tagged, released and recaptured dogs in two regions in a city in the southeast region of brazil. in one of these regions the animals were also sterilized. both regions had similar social, environmental and demographic features. we estimated the presence of 148 females and 227 males during the period of study. the average dog:man ratio was 1 dog for each 42 and 51 human beings, in the areas without and with sterilization, respectively. the animal population size increased in both regions, due mainly to the abandonment of domestic dogs. mortality rate decreased throughout the study period. survival probabilities did not differ between genders, but males entered the population in higher numbers. there were no differences in abundance, survival and recruitment between the regions, indicating that sterilization did not affect the population dynamics. our findings indicate that the observed animal dynamics were influenced by density-independent factors, and that sterilization might not be a viable and effective strategy in regions where availability of resources is low and animal abandonment rates are high. furthermore, the high demographic turnover rates observed render the canine free-roaming population younger, thus more susceptible to diseases, especially to rabies and leishmaniasis. we conclude by stressing the importance of implementing educational programs to promote responsible animal ownership and effective strategies against abandonment practices. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the relationship between dogs (canis familiaris) and men goes back to the beginning of civilization, about 13,000 years ago [1, 2] . it is generally accepted that dogs were domesticated from the wolf (canis lupus pallipes or c. lupus variabilis) in a process of symbiosis that evolved through selective breeding [3] . indeed, dogs are termed as 'domestic' or 'domesticated' animals due to its association with humans and to the role that humans exercised in the emergence of this lineage [4] . since domestication, this relationship became even more intense and dogs are ubiquitous in the cultural context of every society, constituting the most abundant carnivore animal on the planet [5] . dogs have been associated with their owners' welfare and well-being [6, 7] and have started to play different functions [2] due to their malleable personalities, docile behavior and utility as guardians and hunters [8] . dogs that are not under immediate human supervision and have unrestricted access to public property are named "free-roaming" or free-ranging [1] . these terms encompass both owned dogs (family and some neighborhood dogs) and ownerless dogs (stray or feral) [3] . the existence of these dogs that can circulate freely in the streets can be harmful to both the animals and to human beings [1] . the abandonment and breeding of dogs in unrestricted environments have been attributed to behavioral, religious, cultural, ecological and socioeconomic factors, constituting important issues in public health and animal welfare [9, 10] . unrestricted dogs, in general, have their psychological and physical health compromised, are more likely to acquire infectious diseases and have a lower life expectancy compared to pet dogs [11] [12] [13] . their presence can be detrimental to humans since they are associated with the occurrence of biting incidents, transmission of diseases, damage to wild animal populations, accidents and pollution [14] [15] [16] [17] [18] [19] . different strategies are used to control the population of unrestricted dogs [20] . elimination by killing is not considered effective, since the number of removed animals is compensated for the increased entry and survival of the remaining ones. in addition, this method is the subject of much criticism based on ethical issues [20] [21] [22] . as a result, actions towards promoting responsible animal ownership, the strengthening of legislation against abandonment, and surgical control have been established in different countries [20, 23] . annually, thousands of unrestricted dogs are sterilized in veterinary clinics and in campaigns run by governments and non-governmental organizations. nevertheless, the effectiveness of this measure, in the long term, has been poorly evaluated [24, 25] a proper evaluation of actions that aim at controlling the free-roaming canine population requires non-biased estimates of parameters driving the dynamics of the target population [26, 27] . even though several studies have yielded estimates of the population size of free-ranging dogs, most of them used inadequate analytical methods and were susceptible to biases, which casts doubt on the validity of these estimates, as evidenced in a recent systematic review [28] . additionally, there is no published data of capture and recapture procedures that consider the canine populations as open, that is, subjected to deaths, births and migrations [29] . despite the perceived need and usefulness of such parameter estimates and recommendations for the most appropriate approaches applicable under such study designs [30] , survival and recruitment estimates of free-ranging dogs had not been obtained using methods of capture and recapture. in this study, we present estimates of abundance, survival and recruitment rates, and the probabilities of capture of two free-roaming dog populations by means of analytical models for open populations, so far unexplored in previous studies. these dogs were followed for 14 months in a city located in the southeast region of brazil. we report temporal variations of the estimates during the study period regarding gender and the effectiveness of surgical sterilization. been approved by cepea-commission of ethics in research involving animals of the federal university of são joão del rey (protocol no. 24/2010). prior to the implementation of the activities described in the next section, we performed a pilot study to define the areas of study and to identify potential problems requiring further attention. the pilot study lasted for four days, each day spent in a different neighborhood of the city. each one of the four neighborhoods belonged to four different eligible areas defined with municipal health authorities based on previous information on the occurrence of freeroaming dogs and on the feasibility of carrying out the research. those with similar features and with the highest raw number of captured and released animals were selected. data acquired in this stage were not used in the analyses. we conducted seven capture and recapture procedures in a period of one year and two months, one every two months. dogs found wandering in the streets during the capture period were included in the study, provided an owner with a dog leash did not accompany them. adapted vehicles drove around all the streets of the study areas screening for free-roaming animals. the work team consisted of one driver, two municipal health agents, one veterinarian and two individuals responsible for collecting and recording data. in area a, activities took place in the first week of the collecting months, while in area b, they took place in the second week of the same month. screenings always followed the same route, so that they covered all the streets of each region at least once. the same team collected the data in both areas. captured dogs of areas a and b were taken to the health surveillance reference center of the city (crevisa) in adapted vehicles of public health service. in crevisa they underwent clinical tests conducted by veterinarians and were screened for canine leishmaniasis (canl). the diagnosis of canl was made in the parasitology laboratory of universidade federal de são joão del rei through the techniques of "enzyme-linked immunosorbent assay" and "indirect fluorescent antibody test". seropositive dogs were euthanized according to the recommendations of the brazilian ministry of health [31] . dogs that tested negative were microchipped for identification if recaptured. these animals were also de-wormed and received a vaccine against rabies and another against distemper, leptospirosis, hepatitis, parainfluenza, parvovirus, coronavirus and adenovirus. dogs captured in the area b (intervention) were sterilized (s1 appendix). healthy animals were returned to the same place where they had been captured, after screening for canl and total rehabilitation of the surgical procedure (area b dogs) (s1 appendix). recaptured animals were re-examined. animals screened and found negative for canl were released again. animals that tested positive were euthanized. all dogs, even those not captured, were photographed for posterior identification in the same sample interval and in recaptures. for identification, distinctive characteristics of the dogs were sought in their craniolateral and/or dorso-caudal portions. for analytical purposes, dogs not physically captured but photographed were considered captured. this information was added to the database serving as input for the estimation of the population dynamic parameters. we released informative materials to the local population with the purpose of increasing their awareness regarding responsible animal ownership and visceral leishmaniasis (s2 appendix). we entered the individual history of capture and recapture of each animal into a microsoft excel (2013) database formatted as "encounter history" for captured animals tagged alive [30] . euthanized animals carried a negative sign, indicating the occurrence of death during the capture procedure. there were no deaths attributed to other factors. general procedure. the jolly-seber model with popan parametrization served as the starting structure for model fitting [34] . we estimated the following three parameters using his approach: φ i (survival): probability of a marked or unmarked animal surviving (and not migrating) between the captures i and i+1. p i : (capture probability): the probability of finding or seeing a marked or unmarked animal in a given capture i, given the animal is alive and in the area of capture. b i : (probability of entrance): considering the existence of a "super-population", comprised of all animals that would ever be born to the population, this parameter constitutes the probability of an animal of this hypothetical "super-population" entering the population between the occasions i and i+1. the parameters above allow for the estimation of recruitment (b: number of animals that enter the population between two capture procedures) and population size (n). we used mark, version 6.2 for fitting the statistical models. goodness of fit of highly parameterized models. we evaluated the goodness of fit (gof) of the model with the largest number of parameters prior to fitting models that were more parsimonious [35] . this step was necessary to check the premises of the jolly-seber approach. we checked model's gof based on tests 2 and 3 of the release suite of mark software, the gof statistics obtained via the bootstrap, as well as the "median c-hat" statistics. among the procedures, we adopted the one that indicated the highest variance inflation factor (c-hat). we first considered the model with the variables "sex" (male or female), "area" (a or b), "time" (sampling period), and their respective interaction terms. a c-hat value of 2.52 suggested sparsity in different periods of capture. we changed our model search strategy accordingly and partitioned the previous model into two models: a model with "sex", "time" and interactions and a second model with "area", "time" and interactions. c-hat estimated in these cases was 1.17 and 1.25, respectively for each model, and there were few indications of sparse data. modeling procedures. the gof analysis reported above prompted us to investigate factors associated with survival estimates, probability of capture and probability of entry separately for the variables "sex" and "area". in both cases, we built models considering timedependent or time-independent parameters and the presence of interactions between the variables "sex" and "time" or "area" and "time". we also fitted additive models containing parameters expressed as a function of two or more factors, in this case, area and time or sex and time, without the presence of interactions. in total, we fitted 50 models in both groups (s3 appendix). all models supported temporal variations for the "probability of entry" (b i ). model selection followed the usual approach by searching for the most parsimonious structure that retained the best balance between explained variability and precision of estimates. we ranked all models based on akaike's information criterion corrected for finite sample sizes (aicc). this statistic provides a summary balance between the goodness of fit to the data of each model and the number of necessary parameters. "data cloning" was used to identify the correct number of estimated parameters [36] . the presence of overdispersion in the data indicated the need to further correct the aicc statistics by the c-hat values to obtain the quasi-aicc statistics (qaicc). lower values of these latter statistics point to models that were more parsimonious [37] . after ranking all models based on qaicc, we evaluated the force of evidence in favor of each model (aic weight-"w"). this statistic can be interpreted as the conditional probability of a given model being the best among the set analyzed. thus, higher values of "w" indicate higher force of evidence in favor of the model. models with values of "w" lower than 0.01 were disregarded. we further evaluated the importance of each variable in a context of a set of models by adding up the weight (w) of each model containing a given variable [37] . we repeated this procedure for all predictors considered. variables with higher weights are considered more important than those with lower weights in explaining the variance observed in the data. parameter estimation. estimates for the parameters survival probability, probability of capture, probability of entry in the population, abundance and recruitment rate relied on the technique known as "model averaging" [38] . under this approach, we calculated the weighted average of parameter estimates from all models fitted to data using as weights the relative support (w) of the respective model. therefore, this technique accounts for both sources of variance: the specific conditional variation present in each one of the models and the nonconditional variation present in the model selection process. in this way, parameter estimates express more faithfully the sources of uncertainty associated with the estimation process. in time-dependent models under popan not all parameters are identifiable [30] . this is the case of the probability of capture in the first and in the last captures (p 1 and p k ), the probability of entry between the first and the second captures (b 1 ) and between the penultimate and the last captures (b k-1 ), and the survival probability between the penultimate and the last captures (φ k-1 ). thus, only the remaining parameters whose estimation was possible are described here. the effectiveness of sterilization was analyzed by comparing the evolution of abundance and of the other parameters estimated in the areas: a (control) and b (intervention). we estimated dog:human ratio by the ratio of population size and the dog mean abundance in each of the areas. during the study period, 171 dogs were identified individually in region a (control) and 157 in region b (intervention). the proportion of males in areas a and b was 56% (96 dogs) and 62% (98 dogs) respectively. one hundred and thirty-three animals (77 males and 56 females) were captured in more than one effort of captures. one hundred and thirty-eight dogs (88%) were sterilized. twenty-four were euthanized for testing positive to canl. sixty-six different individual histories of captures were registered and 38 of them included animals not captured in the first effort. all recaptures and visualizations took place in the same area where the dogs were initially detected. most free-roaming dogs were neighborhood dogs, i.e. several human residents in the area provided the needed resources to them [3] . models including the variable "gender". we fitted 50 models containing the variable gender to the data (s3 appendix). five had w-statistics greater than 1% and are shown in table 1 , along with statistics qaicc, δqaicc (difference, in module, between qaicc values of the best model and the analyzed model). the relative support for each model is also expressed as the ratio of its w-statistics to the largest value of this statistic among the five models considered. the model in which survival, probability of capture and probability of entry varied with time, but not between male and female dogs, was considered the most parsimonious (w = 73.24%). the weight for this model was 6.48 times higher than the model in which survival varied additively with gender; and 8.37 times higher in relation to the model in which the probability of capture varied between genders. other models had weights lower than 5% and low support, when compared to the most parsimonious model. the sum of each variable's weight (w) considering all models (table 1 ) are presented in s5 appendix. time-dependent parameters displayed higher weights. survival and capture probabilities varied between genders but these variables' "w" conferred weak support to this statement. for entrance probability, there was no evidence for the existence of group variation. models with the variable "area". models containing the variable "area" behaved similarly to the previous set containing the variable "gender". table 2 presents the six models in this group with w-statistics greater than 1%. results for the remaining models are described in s3 appendix. the model containing time-dependent parameters, but constant between areas control (a) and intervention (b), was the most parsimonious in this group. its weight, however, was lower when compared to the models containing the variable "gender" (table 1 ). it had 3.11 times more support from the data than the model in which there was variation in survival between areas, and 3.99 more support than the model in which the probability of capture also varied between areas. differences were significantly higher when comparing the most parsimonious model to the remaining models since the latter models received even lower support from the data. we observed stronger weights associated with time-dependent variables (s5 appendix). however, the observed weights were lower than those associated to the variables in the set of models containing the variable "gender". we also observed stronger weights in variables describing differences in survival and probabilities of capture between areas. the remaining variables were associated with lower weights. in particular, the probabilities of entry did not vary between control and intervention areas. models with the variable "gender". we estimated a population abundance of 148 females and 227 males in the target population in the entire study period. table 3 depicts gender-specific parameter estimates and their respective confidence intervals (ci). they result from model-specific estimates weighted by the relative support (w) of the respective model. taken together, these results show that gender-specific differences regarding the estimated parameters were not relevant. in contrast, time-dependent differences were significant. survival probabilities increased steadily, going from 0.75 in the interval between the first and second captures, to 0.99 between the fifth and sixth. on the other hand, probability of entry in the population was close to zero between the fifth and the sixth captures, and varied between 0.12 and 0.15 in other intervals. probability of capture reached the highest value in the second capture (0.68), and decreased subsequently until the fifth capture when it reached its lowest value (0.39). estimates of abundance highlight the majority of males in its composition. additionally, there was a higher entry of male dogs in all intervals in which the number could be estimated. population increased in size during the study. we estimated the presence of approximately 59 females and population dynamics and effectiveness of sterilization of free-roaming dogs 92 males in the second capture, 71 and 69 females and 104 and 105 males, respectively, in the fifth and sixth captures. models with the variable "area". we estimated the presence of 199 dogs in area a (control) and 177 in area b (intervention) throughout the study period. estimates of additional parameters stratified by control and intervention areas are presented in table 4 . they reflect the weighting mechanism by the relative support of all models analyzed as explained in the methods section. analogously to the models containing gender, differences between the estimates of each area were small, even though they were slightly higher than those seen between genders. owing to the fact that stronger weights were attributed to models that did not show a difference between strata in either set of models, estimates of survival, capture probabilities and entry probabilities for the areas were similar to those described for gender. on the other hand, the recruitment was similar in both areas, contrasting with our findings comparing males and females. population size increased in both areas. abundances seen in the second capture were smaller, 82 animals in area a and 70 in area b, contrasting with abundances observed in the fifth capture, 96 dogs in area a and 83 in area b. dog:human ratio in area a was one dog to 42 human beings. in area b, this ratio was one dog to 51 humans. we estimated critical parameters (survival, recruitment and abundance) that describe the population dynamics of free-roaming dogs based on a capture and recapture study design and on models suitable for open populations. our study demonstrated the increase in population size in both areas, the predominance and greater recruitment of males, the temporal variability in recruitment and in survival probabilities, the lack of effect of sterilization on population dynamics, the influence of abandon and of density-independent factors and a high demographic turnover. such information on the dynamics of free-ranging dogs are useful for informing control interventions of unrestricted dog populations and against canine visceral leishmaniasis and rabies, both neglected tropical diseases endemic to various countries. the dog:man ratio observed in our study was smaller than that observed in counts performed in urban regions of nigeria (1 dog to 25 men) [39] and that among rural dog populations in india (1 dog to 35 men) analyzed by mean of beck's method [40] . it was larger, however, than the counts obtained by hossain et al. [41] in a rural area of bangladesh. demographic, socioeconomic, environmental and cultural factors able to explain differences in abundances between and within regions have been underexplored in the literature [28] . abundance of free-roaming dogs in general is lower in rural than urban areas [42, 43] . regions under poorer socioeconomic conditions and higher population densities tend to have a larger concentration of dogs [44] . in the present study, abundance possibly reflects the intermediary socioeconomic condition, the urban environment and low population density of the study areas as well as the different methodology applied. for most animal species, survival is the demographic parameter with highest impact on population size [45] . few studies, however, aimed at estimating the survival of free-ranging dogs in urban environment. reece et al. [46] used data from a sterilization program to estimate the survival of castrated females in jaipur, india. annual survival of females aged over one year old was 0.70 and of females in their first year of life was 0.25. the assumptions leading to these estimates were implausible and might have biased the results. pal [47] conducted four annual capture efforts, in bengal, india, and estimated the canine mortality from the number of dogs observed in the captures after the first one. annual survival for adult dogs was 0.91, and for dogs in their first year of life, 0.18. this study did not report capture probabilities and included in estimation only dogs found dead. this approach possibly contributed to an overestimation of the survival probability. survival probability reported by beck [1] , in a study conducted in baltimore, canada, with dogs of all age groups, was 0.70. this author relied only on existing information regarding the number of dead dogs, also possibly leading to an underestimation of mortality and consequently to an overestimation of survival probability. although limited, estimates obtained in the literature suggest that survival is lower in young free-roaming dogs [46, 47] , a pattern already seen in different animal species [48, 49] . once the proper identification of the dogs' ages was outside the scope of our project, estimates in the present study refer to the general survival probability of the population and not age-specific probabilities. annual survival in our study was higher than that estimated for dogs aged less than one year [46, 47] , and lower than the survival probability estimated for adult dogs [46, 47] and for beck's study population [1] . the low survival probability identified in the population results from the different sources of mortality experienced by free-roaming dogs in the study setting. residents often reported roadkill and poisoning episodes during the study period. the high prevalence of canl-seropositive dogs, especially in the first months of the study, is another relevant factor leading to the removal of many animals by euthanasia. additionally, government actions towards street dogs were restricted to rabies vaccination. the lack of additional prophylactic measures or treatment may have contributed to the increased susceptibility of dogs to infections and other conditions. females have lower survival rates [50, 51] in a large number of animal species due primarily to the effects of reproduction. given the predominance of males in different studies, it is hypothesized that this pattern also happens in the canine populations [52] . we observed no difference in survival probabilities between genders, although a higher abundance and recruitment of males occurred. most pet owners prefer male dogs since they do not get pregnant and are better guard dogs [28, 41] . therefore, the higher survival of male puppies of owned but free-ranging dogs or of pet dogs subsequently abandoned by their owners could probably explain the predominance of males in the free-roaming dog population. to our knowledge, we report for the first-time the temporal evolution of the survival probability of free-roaming dogs. annual point estimates of survival probability found in the literature do not bear a longitudinal structure. our results show that survival of unrestricted dogs displays variations, even in short temporal scales. among the models fitted to the data, those in which survival did not vary with time had significantly lower weights, indicating that a constant value is not appropriate to representing the entire period. estimates of survival probabilities in other mammal species also show a temporal dependence, especially in young individuals [53] [54] [55] . long-term studies are required to uncover the intrinsic and extrinsic determinants driving these temporal dependencies. this would be useful for understanding the population dynamics of free-ranging dogs and improving the validity and precision of predictive modelling procedures. such studies are difficult to perform, and thus are rare in the literature [56] . despite being a short-term study, survival, recruitment and population size displayed an increasing tendency. this pattern suggests that density-independent factors could be responsible for driving the variations observed in survival probabilities of dogs in both areas. density-dependent mechanisms are the subject of several studies focusing on different animal species [57] [58] [59] [60] . in epidemiological and ecological modeling, one assumes that survival and recruitment rates in free-ranging dogs are driven by the availability of resources in the environment, a density-dependent mechanism [61] . however, as pointed out by de little et al. [56] , extrinsic factors not regulated by density may determine fluctuations in population size when those populations have not yet reached their carrying capacity or when environmental conditions are favorable. according to morters [61] , human beings are the major agents responsible for providing care and adequate food for dogs. as a result, human related factors such as living together with free-ranging dogs, the low dog-human ratio and the availability of residents' resources to maintain these animals, may explain why the increase in density had no influence upon mortality and recruitment. reducing the availability of shelters and food is an ethically questionable measure for population control of free-roaming dogs. however, this alternative has been presented in a recent study [62] . it is not possible to affirm whether population growth, attributed to the large number of animals entering the population, would keep the reported increasing trend constant if the study had a longer duration. maximum survival and lack of recruitments between the fifth and the sixth captures suggest potential instabilities. in the presence of increasing abundance, density-dependent factors could start to play a stronger role in regulating the population [63, 64] and in the behavior of residents regarding their support to dogs. there is considerable uncertainty in assessing the role played by vital rates and intrinsic and extrinsic factors in driving the population size of free-ranging dogs and other mammals [54, 61, 65, 66] . estimates of recruitment obtained from capture and recapture models do not allow us to disentangle the sources of entry attributable to births and immigration. we observed no females with their brood along the study period. we might infer that breeding females were located in less visible areas or put to adoption by the city public service and returned to the streets after the lactation period, even though such registries were rare. in the study of morters et al. [61] , as well as in the present study, recruitment was driven, predominantly, by the arrival of adult animals. the recruitment contingent may comprise dogs born in the region and not identified as puppies, dogs from other regions that migrated to the study region or were relocated by residents who raised them unrestrictedly, previously restricted dogs that changed status to being freely raised, or dogs abandoned nearby that later joined the population. the study areas are geographically isolated from their neighboring regions and are located next to a highway where dogs were frequently abandoned. therefore, the latter mechanism seems more plausible to account for the increase in population size rather than the spontaneous immigration of dogs. although there are heterogeneities [67] , free-ranging dogs are territorial animals that, in general, do not move across long distances, unless forced by unfavorable environmental conditions [1] . the low mobility of dogs in a favorable environment is supported by our data, since there were no animal movements between the areas a and b. the replacement of a great number of dogs that died or emigrated by dogs that are born or immigrate, as observed in our study populations, drives the population structure and gives rise to health problems that result from these structures. a population with a high turnover may be more susceptible to diseases [24] . a high population turnover is the major obstacle for the success of control strategies against rabies in developing countries [68] . vaccination strategies under such population dynamics must occur in short intervals and achieve high coverage in order to maintain proper levels of immunization. on the other hand, the replacement of euthanized dogs by susceptible animals and new individuals entering the reservoir compartment are the main causes of the low effectiveness of the euthanasia of seropositive dogs, a control strategy adopted in brazil against leishmaniasis [69] . in addition, the population also becomes younger and more likely to acquire other infections under the high turnover regime [70] . the field of mammal ecology identifies two main reproductive strategies driving population size, each focusing on specific stages of the life cycle. the so-called "slow breeding" animals experience late maturation and their reproductive strategy depends on the survival of juveniles and young adults. on the other hand, "fast breeding" mammals complete their reproductive cycle within their first year of life and place emphasis on fertility as their survival strategy as a species [71, 72] . control of the population size of "fast breeding" animals, such as dogs [73] , is more effectiveness when relying on measures that restrict entry of new individuals into the population as opposed to subjecting animals to euthanasia, a practice that reduces adults' survival. the fast versus slow breeding rationale, the sensitive ethical issues, and the low effectiveness of euthanizing animals observed in regions where this practice has been applied [20, 61, 74] prompted us to only consider sterilization, and not culling, as an alternative control strategy in our study. its use as a population control measure against hydatid disease in developing countries, however, has been recommended [19] . it is worth noting that in the present study sterilization did not affect the canine population dynamics. after one year and two months, we observed no difference in survival, entrance or recruitment probability between the control region and the intervention area where 88% of the dogs were sterilized. the impact of sterilization takes place slowly as suggested by modeling exercises. it might take up to five years for the first impact of sterilization to become apparent and up to 30 years of uninterrupted efforts to reach its maximum impact [75] . reece and chawla [24] evaluated a program that surgically sterilized 19,129 neighborhood dogs in jaipur, india, for eight years and showed that the population declined by only 28 per cent. on the other hand, frank and carlisle-frank [76] observed only a small impact of a sterilization program on the number of dogs joining a shelter in the united states. amaku et al. [77] , based on results from a mathematical model developed specifically for stray dogs, concluded that sterilization becomes inefficient in the presence of high abandonment rates, even after prolonged periods of use. natoli et al. [78] reached the same conclusion after studying for 10 years the impact of a castration and devolution program on a non-restricted cat population. continuous negligent practices of animal ownership, including abandonment, had a negative impact on the sterilization strategy rendering it ineffective and countering the effect of 8,000 surgical interventions undertaken in that study. finally, in a study conducted in brazil, dias et al. [79] concluded that it is counter-productive to invest in sporadic sterilization campaigns of owned dogs, the currently strategy adopted in most of brazilian municipalities. the small impact in controlling the population size, especially in areas with high abandoning rates as in our case, the need to reach high coverage rates without interruptions, the absence of behavioural benefits for castrated dogs [80] , high costs and null impact on a shortterm perspective, minimize the relevance of sterilization of free-ranging dogs in managing the population and controlling diseases. in this context, it becomes apparent that public health services and non-governmental organizations must develop and prioritize more effective strategies against abandonment practices. in countries where free-ranging dogs are considered a humanitarian or a public health issue the implementation of educational programs addressing responsible animal owning at different levels, the registration of dogs and their owners and the improvement of legislation aimed at those who wish to have a pet becomes imperative [81] . probability of capture is a useful parameter in the identification of essential population features [82] . it is known to vary in space, time and among individuals [83] . although we observed no significant differences in this parameter between genders and areas, it varied over time even in the presence of standardized procedures. such fluctuations may be attributable to social organization features not yet investigated in the population, or to environmental and climatic factors. dias et al. [84] showed that weather exerts an influence on dogs' activity, and consequently influences the probability of finding a dog in a given capture effort. in our study, even with the vehicles driving around in all the streets of the target regions, there was a large number of animals present but not visualized in all captures. our observations indicate that individual counts based on a census do not adequately estimate the abundance of unrestricted dogs and that the majority of the estimates available in the literature show important biases. different studies aimed at estimating the abundance of free-roaming dogs did not model or even consider the existence of differences in the probabilities of dog detection [28] . as pointed out before [28] , counting techniques should be carried out only in short periods of time and when no other alternative becomes available considering logistics, geography and culture of the study region. values of capture probabilities obtained in the present study are similar to those estimated by kalati in a population of urban free-ranging dogs in kathmandu [85] and may be used as correction factors for the previously published estimates of abundance. in addition to the limitations already mentioned regarding the observation and sampling techniques applied in capture and recapture studies, issues related to the choice of appropriate analytical methodology deserve mentioning. environmental and individual variables, relevant to helping understand the population dynamics [30] were not included in our models. the logistics of fieldwork turned out to be complex and difficult, requiring the participation of at least six individuals in each capture effort and extensive fieldwork journeys. direct contact with animals was unavoidable since assessing the effectiveness of sterilization was one of the study objectives. studies assessing the population dynamics of dogs, however, could rely on only photographic methods, these being less complex and onerous [86, 87] . our choice of modeling procedures for open populations allowed for the estimation of survival and recruitment probabilities of unrestricted dogs. in addition, we tested the data for the statistical assumptions required by each model. model selection followed the aic technique, which compares favorably with the classic statistical and hypothesis testing [37, [88] [89] [90] . lastly, our parameter estimates and confidence intervals express more faithfully the sources of uncertainty present in the whole estimation process, due to the use of the "model averaging" technique. the analytical procedures adopted here addressed methodological limitations of previous publications and propose a new starting point for future studies. in our view, longer periods of observation, larger sample sizes, and the choice of more variable study settings including different social, cultural and geographic characteristics are important topics that need the attention of researchers in the field of unrestricted dogs' ecology. the agenda of the investigation of factors influencing the canine population dynamics must consider the variables addressed in the present study, and further consider the stratification of these population parameters by age groups, as well as by intrinsic animal features and environmental conditions not yet investigated. our estimates of population size in the studied regions in general were small compared to previous estimates in the literature. survival probability was small and probability of animal entry in the population was high during the 14 months period of follow-up. high turnover, attributed mostly to the abandonment of pet dogs, has important implications to the population composition and the control of zoonosis. estimates of survival, recruitment and capture probabilities varied over time. survival and recruitment showed an increasing tendency. mortality patterns did not differ between genders. the probability of entry in the population was higher among males. the observed population dynamics seem to be driven by density-independent factors. sterilization, in turn, had no influence upon the parameters analyzed. our observations are useful for a better understanding of the population dynamics of free-roaming dogs and may aid in the planning, designing and evaluation of population control actions. in this context, it becomes imperative that public health services and nongovernmental organizations develop educational training programs addressing responsible animal ownership and better strategies against abandonment practices. parameter estimates may also be used as input to new predictive mathematical models. even though our study generated important answers and new hypotheses, the scarcity of existent knowledge and the misuse of the proper methodology raise numerous relevant questions yet to be elucidated about the population dynamics of free-roaming dogs. the ecology of stray dogs: a study of free-ranging urban animals a 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overpopulation of companion animals: do adoption and low-cost spay/neuter programs merely cause substitution of sources? dynamics and control of stray dog populations management of feral domestic cats in the urban environment of rome (italy) dog and cat management through sterilization: implications for population dynamics and veterinary public policies effects of surgical and chemical sterilization on the behavior of free-roaming male dogs in stray dog and cat laws and enforcement in czech republic and in italy is heterogeneity of catchability in capture-recapture studies a mere sampling artifact or a biologically relevant feature of the population? revisiting the effect of capture heterogeneity on survival estimates in capture-mark-recapture studies: does it matter? size and spatial distribution of stray dog population in the university of são paulo campus, brazil street dog population survey, kathmandu: final report to wsp spot the match-wildlife photo-identification using information theory mark-recapture and mark-resight methods for estimating abundance with remote cameras: a carnivore case study choosing among generalized linear models applied to medical data null hypothesis testing: problems, prevalence, and an alternative model selection in ecology and evolution the authors are grateful for the invaluable support provided by members of staff of the municipal health service and by veterinarians who participated in this study. key: cord-023689-r03j5u18 authors: scott, danny w.; miller, william h.; griffin, craig e. title: viral, rickettsial, and protozoal skin diseases date: 2009-05-15 journal: muller & kirk's small animal dermatology doi: 10.1016/b978-0-7216-7618-0.50011-0 sha: doc_id: 23689 cord_uid: r03j5u18 nan syncytial-type giant cells in the epidermis. cowpox virus is a member of the orthopoxvirus genus that has sporadically caused infections of domestic and exotic cats in various european countries." cats with an uncharacterized poxvirus infection have been recognized in india and the united states, so the geographic area of involvement may be increasing. 1 in one study of 104 healthy cats, 13.5% were serologically positive for poxvirus antigen, suggesting a higher frequency of disease than recognized." the natural reservoir is small wild mammals. the virus circulates in different rodent species, especially voles and wood mice in western europe.v 11a cats typically become infected through wounding while hunting these wild mammals. cat-to-cat, cat-to-human, or cat-to-dog transmission can also occur. g • 45 there appears to be no age, breed, or sex predisposition to infection, and many cases are recognized in the fall of the year." the primary lesion in the disease is an infected "see references i, 5, 8, 19a, 27, 34, 35, 43-45 . partial alopecia with scaling. papularto-plaquelike lesions were also present but are not visible. bite wound, typically on the head, neck, or forelimb. local viral replication worsens the primary lesions and is the point source for the subsequent viremia. during the viremic phase, some cats develop mild pyrexia, inappetence, and depression. ten to 14 days after the primary lesion, multiple secondary lesions develop over the body. the secondary or pox lesions are initially macular but progress to ulcerated, papular to nodular lesions that crust rapidly (see fig. 7 -ie and d). pruritus is variable, and approximately 20% of infected cats develop oral vesiculation or ulceration. i. 5 lesions heal slowly over 3 to 4 weeks and may be permanently scarred. during the development and course of the secondary lesions, systemic signs of illness are uncommon unless the cat has some intercurrent immunosuppressive disease. one cat died of necrotizing pneumonia.s> the differential diagnosis includes bacterial and fungal infections, eosinophilic granuloma, and neoplasia (especially mast cell tumor and lymphoma). definitive diagnosis is made by skin biopsy, serologic testing, and virus isolation." dermatohistopathologic findings include hyperplasia, ballooning degeneration, reticular degeneration, microvesicle formation, and necrosis of the affected epidermis and the outer root sheath of the hair follicle (see fig. 7 -ie). eosinophilic intracytoplasmic inclusion bodies are found within keratinocytes of the epidermis, hair follicles, and sebaceous glands. the diagnosis of cowpox is based on the results of diagnostic tests. serum samples and fresh biopsy or scab material in viral transport medium are submitted to an appropriate diagnostic laboratory for serologic examination and viral isolation, respectively. serologic tests cannot differentiate cowpox from other orthopox viruses. histopathologic examination of secondary lesions usually supports the diagnosis, and orthopoxvirus involvement can be demonstrated by immunohistochemical techniques'' or electron microscopy. virus isolation is the only method of making a precise diagnosis. there is no specific therapy for cowpox infection. if secondary bacterial infections of the skin or other organs occur, appropriate antibacterial therapy should be instituted. severely ill animals, which typically have an underlying immunosuppressive disorder, require intense supportive care and may have to be euthanized. glucocorticoids are contraindicated. feline cowpox has zoonotic potential for contact cats, dogs, and humans.v-!4, !9a. 44, 45, 45a in humans, these contact infections are uncommon; they can be serious, however, especially if the individual is immunologically compromised. lesions are typically nodular and occur most commonly on the hands and arms. a fatality in a human receiving corticosteroids has been documented.r' accordingly, all infected cats should be isolated and handled carefully. the cowpox virus can remain viable under dry temperature conditions for several years, but it is susceptible to various disinfectants, especially to hypochlorite solutions." dogs develop solitary, asymptomatic, self-healing ulcerated nodules.v45 no data are available to indicate whether infection confers long-lasting immunity. to prevent possible reinfection, hunting should be prohibited. feline infectious peritonitis is a systemic viral disease caused by strains of coronaviruses. effusive and noneffusive forms are most common.' skin lesions other than those associated with debility have not been reported. several cats that have been experimentally infected have developed ulcerative lesions around the head and neck ( fig. 7-7 ).2 histopathologic tests showed changes typical of a superficial vasculitis, and viral antigen was demonstrated in blood vessel walls by immunohistochemical techniques. canine distemper is caused by a paramyxovirus.! in addition to severe respiratory, gastrointestinal, and neurologic disorders, the virus may produce skin lesions in some animals. because of their general debility, some dogs, and especially very young puppies, develop widespread impetigo (see fig. 7 -if). the classic skin manifestation of distemper is the so-called hard pad disease, in which the dog develops nasal ( fig. 7-8 ) and footpad ( fig. 7-9 ) hyperkeratosis of varying severity. although a variety of diseases (e.g., pemphigus foliaceus, lupus erythematosus, drug eruption) induce nasodigital hyperkeratosis, animals with those disorders are often not as systemically ill as dogs with distemper and have more widespread skin lesions. distemper, leishmaniasis, necrolytic migratory erythema (see chap. 10), and generic dog food skin disease (see chap. 17) all can produce the nasodigital lesions and similar systemic signs of illness. the index of suspicion for distemper should be high when the pads are much harder to the touch than the degree of hyperkeratosis would suggest and the dog's vaccination history is poor. histologically, the skin or pad surface is covered with marked orthokeratotic and parakeratotic hyperkeratosis, and acidophilic cytoplasmic inclusion bodies are commonly seen in keratinocytes." the inclusion bodies are variable in size and are round to irregular in outline. nuclear inclusions are rare. occasional multinucleated syncytial giant cells can be seen in the epidermis. immunohistochemical detection of canine distemper virus in haired skin and footpad epithelium was reported to be very reliable for the antemortem diagnosis of distemper.!" contagious viral pustular dermatitis (orf, contagious ecthyma) is a disease that is found primarily in sheep and goats and is caused by a parapoxvtrus.f contagious viral pustular dermatitis was reported in a pack of hounds allowed to feed on sheep carcasses.t" lesions consisted of circular areas of acute moist dermatitis, ulceration, and crusts, typically around the head. skin biopsy revealed epidermal hyperplasia, ballooning degeneration, acantholysis within the stratum spinosum, and marked infiltration of neutrophils. saline suspensions of skin biopsies were applied to the scarified skin of a normal sheep. crusts removed from the inoculation sites were processed for electron microscopy, and parapoxvirus virus particles were readily seen. one cat with parapoxvirus infection has been documented.f the cat had multiple large crusted lesions over the face and back. further details were not provided. therapy for contagious viral pustular dermatitis is topical and varies according to the symptoms involved. the usual course of the disease in animals is 1 to 4 weeks. the disease may be transmitted to humans if broken skin is exposed to lesion material or contaminated objects. generally, contagious viral pustular dermatitis is a benign disease in humans and results in the formation of a solitary lesion, especially on the hands. lesions in humans are characterized by maculae that progress through a papular, nodular, and papillomatous stage. the lesions are usually centrally umbilicated and occasionally are bullous. complications of contagious viral pustular dermatitis in humans include regional lymphadenopathy, lymphangitis, secondary bacterial infection, and rarely, generalized or systemic disease. pseudorabies is an acute, fatal viral disease caused by an a-herpesvirus. 1 pigs are the main reservoir of infection. dogs and cats can be infected by contact with an infected animal or, more typically, by eating raw pork products or offal. incubation periods range from 2 to 10 days,20a,23 and death typically occurs within 48 hours of the onset of clinical signs. 32 early work suggested that intense, maniacal upper body pruritus was the cardinal feature of the disease in dogs. 23 more recent work, however, showed that this sign occurred in only 52% of affected dogs. 32 ptyalism was a universal finding, followed by restlessness, anorexia, ataxia, and a variety of other neurologic abnormalities. when present, the pruritus is intense and leads to self-mutilation, typically of the head and ears. in cats, the neurologic signs seem to predominate and pruritus is rare. 1, 20a the diagnosis can be confirmed by virus isolation. treatment is usually not attempted; when it is, the result is unrewarding. prevention by means of strict hygienic procedures is of paramount importance. mumps is a human viral disease caused by a paramyxovirus, and there are reports of clinical disease in dogs from households where humans had mumps.' one author (whm) examined a dog with parotid salivary gland enlargement, a probably vesicular cheilitis, and positive antibody titer to the mumps antigen. only epidermal collarettes were present on the lips, so biopsies were not performed. the skin lesions resolved spontaneously as the salivary gland returned to normal. feline rhinotracheitis is an infection with an a-herpesvirus resulting in upper respiratory disease." occasionally, a cat develops oral and cutaneous ulcers. 1, 16. 38 the cutaneous ulcers are usually superficial and multiple, and can occur anywhere on the body, including the footpads. stress or trauma to the skin might precipitate the development of the ulcers. skin biopsies reveal epidermal ulceration with subjacent dermal necrosis and a mixed inflammatory infiltrate. basophilic intranuclear inclusion bodies may be visualized in the keratinocytes or dermal histiocytes. herpesvirus can be cultured from the skin; more diagnostically, it can be seen in the keratinocytes via electron microsoopy.p an ulcerative and necrotizing facial dermatitis or stomatitis has been associated with herpesvirus 1 infection in cats.s'42c, 47 affected cats mayor may not have active or historical ocular or respiratory signs. the disorder is recognized most often in adult cats but kittens can be affected. typically, crusted skin lesions involve the nasal planum, bridge of the nose, and periocular skin (see fig. 7 -ig). when the crusts are removed, the exposed skin is inflamed and ulcerated (see fig. 7-1h ). similar lesions can be found elsewhere on the body. exfoliative erythema multiforme has occurred in cats following upper respiratory infections. generalized exfoliation and erosions occur that histologically showed individual cell apoptosis and lymphocytic epitheliotropism. the lesions spontaneously resolve after the infection is cleared." with intercurrent respiratory signs, the diagnosis is straightforward. in the absence of respiratory signs, the differential diagnosis would include the felv dermatitis, drug reaction, erythema multiforme, pemphigus vulgaris, and systemic lupus erythematosus. diagnosis is via skin biopsy. serologic test results do not confirm active infection nor that the skin disease is due to the virus. in skin biopsies, an ulcerative, often necrotic, dermatitis and suppurative folliculitis and furunculosis is seen ( fig. 7-10 ). demodex cati mites may be visible within the follicular lumen.f" there is a perivascular-to-interstitial mixed inflammatory cell dermatitis with many eosinophils. in the surface and follicular epithelium, multinucleated keratinocytic giant cells can be seen ( fig. 7-11 ) and amphophilic intranuclear (cowdry type a) inclusion bodies can be seen in the giant cells ( fig. 7-12 ) and other keratinocytes. a unique feature of this disease is necrosis of epitrichial sweat glands ( fig. 7-13 ). ultrastructural studies demonstrate intranuclear virions consistent with herpesvirus. polymerase chain reaction (pcr) testing in affected cats was strongly positive for herpesvirus 1,21. 42c however, the use of pcr as a diagnostic test for feline herpesvirus-associated disease is of limited value because of the occurrence of healthy carriers." an immunohistochemical test was reported to be accurate.f" in adult cats, the disorder can be triggered by stress or corticosteroid usage. correction of these problems with the use of antibiotics and other symptomatic treatments may allow for spontaneous healing. other agents suggested include lysine (250 mg of the formulation without propylene glycol orally q24h), a-interferon, and acyclovir. 38 • 47 these treatments mayor may not be beneficial. oral ulceration is reported to be more common with calicivirus infection than with rhinotracheitis.' sporadic reports associate infection with skin lesions of the feet or perineum.p: 25 the tissues are swollen, tender, and ulcerated. although calicivirus was isolated from the skin in one case, no histopathologic tests were performed to demonstrate whether the virus was causal or a contaminant. because some infected cats can develop a presumed immune-mediated arthropathy." it is reasonable to assume that the virus can induce primary skin lesions. papillomaviruses belong to the papovavirus family and are either known to cause or suspected to cause oral papillomatosis, cutaneous papillomas, and cutaneous inverted papillomas in dogs (see chap. 20).34b at least two strains of virus and probably more exist in the dog. 24a in classic oral papillomavirus infection, self-cure is the rule, provided that the host is immunocompetent.w two additional syndromes have been recognized that are probably associated with papillomavirus infection in dogs. the first syndrome involves the development of multiple warts on the footpads of young dogs." affected dogs are 1 to 2 years of age at the onset of symptoms. they develop discrete, firm, hyperkeratotic, often hornlike lesions on multiple pads of two or more paws (fig. 7-14a ). in the dogs studied, lesions were not detected elsewhere. if the lesions are large or involve the weight-bearing surface of the pad, lameness can occur. the lesions wax and wane in severity, and individual lesions may spontaneously resolve but new ones develop. histologically, the lesions have the characteristics of viral papillomas; to date, however, efforts to demonstrate the virus have been unrewarding. treatment with topical keratolytic or softening (e.g., water and petrolatum) agents removes the hyperkeratotic debris, softens the lesions, and decreases the dog's discomfort, but it does not appear to alter the course of infection. topical dimethyl sulfoxide (dmso) and oral etretinate have been of no benefit in the few dogs treated. spontaneous resolution of all lesions has not been recognized and it is unknown whether immunotherapy would be of benefit." the second syndrome involves the development of multiple discrete and pigmented papules, plaques, or nodules ( fig. 7-15 ). the cases recognized have occurred in young adult dogs (3 to 5 years) with no prior history of skin disease.v 17, 24a, 33 in one dog, lesions developed while the dog was receiving a corticosteroid and spontaneously regressed within 3 weeks of drug withdrawal." lesions can be singular but typically are multiple from the onset, involved any skin surface, and became more numerous with time. histologically, the lesions are sharply demarcated and characterized by surface and infundibular follicular epithelial pseudocarcinomatous hyperplasia and dysplasia. no koilocytosis or inclusion bodies were seen. a novel papillomavirus was demonstrated in one dog37 and probably is responsible for the lesions in the other dogs. the lesions can persist unchanged for over 18 months, but transformation to intraepidermal carcinomas may occur.l": 33 no effective treatment is reported. although papillomas are occasionally recognized in cats, there was no evidence for their viral induction in cats until 1990. 1o , 42b two aged persian cats were described with multiple hyperkeratotic plaques (see fig. 7 -14b).9 the lesions were of variable size, predominately truncal in location, and hyperpigmented in one case. histopathologic studies showed surface and follicular infundibular epithelial hyperplasia and dysplasia with koilocytosis. intracytoplasmic inclusion bodies were seen and papillomavirus-like particles were demonstrated on electron microscopy. immunohistochemical staining demonstrated papillomavirus antigen that had characteristics of a novel feline papillomavirus. other cases with similar features were subsequently reported.12. 15 the cats were assumed to be immunocompromised. many cases of multicentric squamous cell carcinoma in situ have been recognized in the cat since 1990 4 . 18, 24, 31 (see chap. 20). the lesions from these cats also showed surface and follicular hyperplasia and dysplasia, but there was far more cellular atypia and 24 ultrastructural studies in six cats showed intranuclear particles that were compatible with papillomavirus, these data would suggest that the feline papillomavirus induces long-lasting dysplastic lesions that eventually become neoplastic. therapeutic options are limited. surgical removal is usually impractical because of the numbers of lesions, their location, or both, and because new lesions appear after surgery. topical treatment with 5-fluorouracil, which can be effective in humans and dogs,31 is contraindicated in cats because of its neurotoxicity. preliminary work suggests that bradiation therapy (strontium-btl plesiotherapy) is an effective treatment for early iesions'", as with surgery, however, it does not prevent new lesions. anecdotal reports suggest that treatment with interferon alfa may be effective (see chap. 20). papovaviruses cause cutaneous and mucosal papillomas (warts) in the dog (see chap. 20). feline sarcoma virus produces cutaneous fibrosarcomas in young cats (see chap. 20). felv and feline sarcoma virus have been associated with the development of lymphosarcoma, liposarcoma, melanoma, hemangioma, and multiple cutaneous horns in cats (see chap. 20). rocky mountain spotted fever is caused by the rickettsial agent rickettsia rickettsii and is transmitted by ticks.i. 52. 55, 56 it is a seasonal disease in the united states, with cases occurring between april and september. infected dogs develop fever, anorexia, lethargy, peripheral lymphadenopathy, and signs of neurologic dysfunction. approximately 20% of cases will develop skin lesions of erythema, petechiation, edema, and occasionally, necrosis and ulceration of the oral (see fig. 7-14c) , ocular, and genital mucous membranes and the skin of the nose, pinnae, ventrum, scrotum (see fig. 7-14d) , and distal limbs and feet. 51 . 56 edema of the extremities is frequently seen and is the earliest cutaneous sign. the epididymis of male dogs may be painful and swollen. hematologic changes may include anemia, leukopenia, or leukocytosis and thrombocytopenia. skin biopsy reveals necrotizing vasculitis (fig. 7-16) . dogs with rocky mountain spotted fever have a fourfold rise in serum antibody titer to r. rickettsii. direct immunofluorescence testing for r. rickettsii antigen in formalinfixed skin biopsy specimens is often positive (antigen seen within vascular endothelium)'! therapy includes tetracycline (22 mglkg q8h orally), doxycycline (10 to 20 mglkg q12h orally), or chloramphenicol (20 mglkg q8h orally) for 1 to 2 weeks and supportive care. 1. 59 intercurrent use of high-dose corticosteroids to treat the skin lesions can prolong the rickettsemia and course of treatment but otherwise has no detrimental effects/" the dog presents a potential public health danger when infested with r. rickettsii-infected ticks; there is also a danger when blood or tissues from rickettsemic dogs are handled without suitable protection. ehrlichiosis is caused by the tickbome rickettsial agent ehrlichia canis. 1 systemic signs of illness predominate and include fever, weight loss, depression, lethargy, and anorexia. hematologic and clotting abnormalities, vasculitis, and monoarthropathy or polyarthropathy also can be seen. skin lesions are very rare and include a crusting facial dermatitis involving the bridge of the nose.p' pustular and purpuric lesions due to vasculitis, and an intensely pruritic papulocrustous dermatitis." with its immunosuppressive nature, some german shepherd dogs develop recurrent german shepherd pyoderma until the ehrlichiosis is resolved (see chap. 4). the facial dermatitis had histologic features seen in lupus erythematosus, but the dog was antinuclear antibody (ana) negative and positive for ehrlichia canis. doxycycline, an antibiotic with very little direct effect on the skin, resulted in resolution of the skin lesions this highlights the need to consider infectious agents when the skin lesions clinically and histologically resemble those seen in lupus. feline haemobartonellosis (feline infectious anemia) is an acute or chronic disease of domestic cats characterized by fever, depression, anorexia, and macrocytic hemolytic anemia.' it is caused by the rickettsial agent haemobartonella felis. cutaneous hyperesthesia and alopecia areata have been reported to occur in cats with acute and chronic haemobartonellosis'p, however, no pictures, photomicrographs, or details of any kind were provided to substantiate these cutaneous diagnoses . toxoplasmosis is a multisystemic disease caused by the coccidian toxoplasma gondip' 99 toxoplasmosis has been rarely reported to cause various cutaneous lesions in humans'" and in cats.v 78 histopathologic findings in cats were reported to be necrotizing dermatitis and vasculitis with toxoplasma ( fig. 7-17 ). pcr-based techniques are available for the rapid and accurate diagnosis of toxoplasmosis.p" coccidia of the genus caryospora have a complicated life cycle involving rodents, reptiles, and raptors.'-76 infection occurs by ingestion of an infected host and results primarily in diarrhea. these organisms have been suspected'<-123 or identifled?" in puppies that developed pustules, plaques, or nodules on the skin of the trunk. the tissue reaction was pyogranulomatous with eosinophils, and numerous organisms in various stages were identified in macrophages and connective tissue cells (fig. 7-18 ). neosporosis is caused by neospora caninum.s58, 75, 80, 99.,108,120 because its tachyzoites and tissue cysts resemble those of toxoplasma gondii, the organism has doubtless existed unrecognized for years. its complete life cycle is unknown. infection occurs via vertical transmission or postnatal inoculation, the latter being most important,58, 117 exposure to cats may increase the risk of infection.'!" in one study, about 15% of normal dogs were serologically positive. sporozoites penetrate cells and change into tachyzoites, which divide rapidly and cause tissue damage. tachyzoites then become bradyzoites within tissue cysts. dogs of any age can be infected, but clinical signs are more severe in young dogs. neurologic and muscular signs predominate, but pneumonia, hepatitis, myocarditis, or dermatitis can also be seen. 89. 120 skin disease has been described in a small number of dogs. most had widespread draining nodules.?" 87. 99a, 112, 113. 115 but one had a rapidly spreading, ulcerative dermatitis of the eyelids, neck, thorax, and perineurrr" the lesions may be pruritic. histologically, nodular lesions are characterized by a pyogranulomatous dermatitis. tachyzoites can be seen within keratinocytes, macrophages, neutrophils, and rarely, in endothelial cells. the dog with ulcerative lesions had an eosinophilic necrotizing dermatitis with severe congestion, thrombosis, and infarction." to differentiate neosporosis from toxoplasmosis, immunohistochemical or ultrastructural studies are needed. serologic tests are available to determine the rate of infection.u? a titer of~1:800 by indirect fluorescent antibody testing is considered strongly suggestive of active infection.p" susceptibility testing of n. caninum to various antimicrobial agents has been performed but detailed studies on the in vitro versus in vivo correlation of these data are not available." susceptibility testing suggests that sulfamethoxazole, azithromycin, canthromycin, erythromycin, doxycycline, minocycline, and clindamycin hydrochloride can be of benefit. several cases were treated with a 21-day115 or 45-day79 course of clindamycin (12.5 to 18.5 mglkg, orally, ql2h) with resolution of the lesions.79.99a combination therapy with pyrimethamine (0.25 to 0.5 mglkg, orally, ql2h) and sulfadiazine (30 mglkg, orally, ql2h) is also reported to be effective. 99a when this combination is used, folinic acid (5 mg/day) or brewer's yeast (100 mg/kg/day) is given to prevent bone marrow suppression.p'" sarcocystis organisms are widespread in nature, especially in cattle and sheep.' dogs and cats become infected by ingesting tissue cysts (sarcocysts). sarcocystis species are typically not pathogenic for dogs and cats, although there has been one report of a dog with chronic diarrhea who developed multiple cutaneous abscesses over the whole body and especially on the hind limbs."? biopsy showed severe necrotizing, fibrinosuppurative dermatitis with numerous neutrophils and fewer eosinophils and macrophages. vessels were congested and plugged, and numerous protozoal organisms were seen within macrophages and, occasionally, in endothelial cells of vessels. canine babesiosis is a tickborne hematozoan disease caused by three species of babesial. 101 : babesia canis, which is worldwide in its distribution, and babesia gibsoni and babesia vogeli, which are more restricted. infection induces a parasitemia that results in varying clinical signs. asymptomatic carriers exist. aside from the oral or cutaneous petechial and ecchymotic hemorrhages associated with thrombocytopenia or disseminated intravascular coagulation, skin lesions are rare. skin lesions are due to subjacent leukocytoclastic vasculitis with or without vascular necrosis." clinical signs include edema, ecchymosis, ulceration, and necrosis (see fig. 7-14e ), which can be seen on the pinnae, axillae, groin, limbs, or scrotum.f" 69 as in ehrlichiosis, dogs with babesiosis can have some of the clinical features of systemic lupus erythematosus and may have a positive ana titer. accordingly, all dogs with suspect lupus erythematosus should have serologic tests for appropriate rickettsial or protozoal diseases before the diagnosis of systemic lupus is made. treatment with pentamidine isothionate resolved the skin lesions in the reported case/" other babesiacides may also be effective. leishmaniasis is a serious protozoal infection caused by a variety of leishmania spp.! 82, 94, 122 disease is most common in humans and dogs but can be seen in cats and other domestic animals. the disease is worldwide in distribution. in the old world, most cases in dogs occur in the mediterranean basin and portugal, but reports have originated in france, germany, switzerland, the netherlands, and other countries. in the new world, the disease is endemic in south and central america; endemic foci have been reported in texas, oklahoma, ohio, michigan, and alabama." dogs imported from endemic areas may develop the disease months or years later, so cases could be recognized anywhere. the disease is transmitted to humans and animals by bloodsucking sandflies of the genus lutzomyia in the new world and phlebotomus in the old world. the frequency of infection increases during warm months when the vector load is high. 57 domestic and wild dogs, rodents, and other wild mammals are the reservoir. twenty percent of seropositive asymptomatic dogs have leishmania organisms in clinically normal skin. 84a because of the occurrence of open lesions, some investigators have expressed concern regarding the possibility of direct or mechanical transmission from dog to dog or from dog to humans. leishmaniasis in hiv-positive humans is an emerging disease.p'" the possibility that humans act as a reservoir for other humans and animals has been put forth." in general, tissue damage in leishmaniasis is due to granulomatous inflammation and immune complex deposition. it has been hypothesized that dogs with subclinical or latent leishmaniasis may develop cutaneous lesions at the sites of external trauma and resultant inflammatory processes, because amastigotes in blood cells are transported to the inflamed areas. 1l 5a this mechanism could partly explain the distribution of inflammatory and ulcerative lesions at pressure points, which is common in canine leishmaniasis. the incubation period varies from weeks to several years with a gradual onset of signs and continual progression. the disease primarily affects dogs less than 5 years old. rural animals, especially those who spend the night outdoors, are at an increased risk 133 from 10 to more than 50% of seropositive dogs have no clinical signs of disease and may remain healthy for prolonged periods of time, if not perrnanently.sv-94a. 123a skin lesions occur in over 80% of dogs with visceral involvement.1, 72, 73, 94. 110, 116 the most common finding is an exfoliative dermatitis with silvery white, asbestos-like scaling. the exfoliation can be generalized but usually is most pronounced on the head, pinnae, and extremities (see fig. 7-14f ). nasodigital hyperkeratosis may accompany the scaling, and the involved skin can be hypotrichotic to alopecic. periocular alopecia (lunettes) is common. the next most common presentation is an ulcerative dermatitis (see fig. 7-14g and h) . other findings include onychogryposis (fig. 7-19) , paronychia, sterile pustular dermatitis, nasal depigmentation with erosion and ulceration, and nodular dermatitis.s" 94a secondary bacterial pyoderma occurs in about 25% of the dogs. 94a systemic signs of illness are many and varied. over 50% of involved dogs show decreased endurance, weight loss, and somnolence. i, 71, 72, 94, 94a because of the parasitemia and the host's immunologic response to the organism, physical abnormalities are varied, generalized lymphadenopathy and hepatosplenomegaly are common findings. other abnormalities include muscle wasting, cachexia, intermittent fever, keratoconjunctivitis, and lameness. because of the leishmania-induced cell-mediated immunodeficiency, these dogs can be predisposed to generalized demodicosis in all of its forms. i06a cats are resistant to experimental infection, and reports of spontaneous cases are rare,59, 93, 96, iii the majority of cases had a nodular or crusting dermatitis of the lips, nose, eyelids, and pinnae. a generalized exfoliative dermatitis can also be seen. because immunodeficiency is not a prerequisite for infection, dogs with leishmaniasis show an immunologic response to the organism. resistance or susceptibility to clinical leishmaniasis appears to be associated with the stimulation of a t helper-lor t helper-2 cell response, respectively.p" il-2 and tnf-a seem to playa protective role,94a with infection, serum levels of anti-leishmania igg, igm, iga, and circulating immune complexes increase and, with high titer, predispose to renal disease. 102, 103, 105 with infection, the number of c021 +, c05+, c04+, and c08+ cells decreases and the degree of incompetence seems to influence the severity of clinical signs. 62, 63, 66, 106 early in experimental infection, the dog develops a cell-mediated immune response to the organism but this can disappear with the onset of clinical signs.ll b with a persistent cell-mediated response, the dog's clinical signs are absent or milder and the number of organisms found in the tissues is fewer. 62, 66, 85 the differential diagnosis includes pemphigus foliaceus, systemic lupus erythematosus, zinc-responsive dermatosis, necrolytic migratory erythema, sebaceous adenitis, and lymphoma. laboratory findings usually include nonregenerative anemia, hyperglobulinemia, hypoalbuminemia, and proteinuria. tests for immune-mediated diseases (coornb's tests, ana, lupus erythematosus preparation, rheumatoid factor) can be positive in dogs with ielshmaniasis.p: 94a the frequency of a clinically insignificant positive ana titer varies from 16% to over 80% of dogs tested.p 94, 104 because many of the clinical signs of leishmaniasis overlap with those of systemic lupus erythematosus, immunodiagnostic test results must be interpreted carefully when a dog comes from an endemic area. demonstration of anti-leishmania antibodies, positive skin test reaction," or the organism itself confirms the diagnosis. although dogs can have positive serologic test results in the absence of clinical disease, spontaneous elimination of the parasite is rare; therefore, positive test results indicate infection.' various serologic tests are available (ifa, elisa, dot-elisa) and the published sensitivity and specificity vary with the test, population being studied, and investigator.50, 84, 94, 118., !27, !28 no test is 100% accurate, with false-negative results reported in more than 10% of infected dogs tested. the test method also dictates whether the test can be used to monitor response to treatment. for that purpose, the indirect immunofluorescence test (ifa) is recommended.s-!3! amastigotes are most easily seen with ciemsa's stain and are found most often in smears from lymph nodes or bone marrow (fig. 7-20) . identification in other tissues is more difficult and often unrewarding. lymph node cytology is positive in about 85% of the clinically ill dogs, whereas ifa is positive in about 97%.94. in early clinical disease, cytology may be positive when serology is negative. 94 ' there is no correlation of severity of clinical signs with serological titer. 94 • skin biopsy findings vary considerably, orthokeratotic and parakeratotic hyperkeratosis are usually prominent; the inflammatory infiltrate typically consists of macrophages with fewer numbers of lymphocytes and plasma cells. granulomatous perifolliculitis (fig. 7-21 ), interstitial dermatitis, superficial and deep perivascular dermatitis, lichenoid interface dermatitis, nodular dermatitis, lobular panniculitis, suppurative folliculitis, and intraepidermal pustular dermatitis ( fig. 7-22 ) are the nine inflammatory patterns that have been recognized in leishmaniasis; this large number reflects the clinical variability of the disease.?' the three most common patterns are granulomatous perifolliculitis, superficial and deep perivascular dermatitis, and interstitial dermatitis. it is common for a dog to have more than one pattern of inflammation present. in the perifollicular pattern, total obliteration of the sebaceous glands occurs in approximately 45% of the cases. this sebaceous destruction no doubt contributes to the high frequency of clinical exfoliation. the leishmania organisms are found intracellularly and extracellularly in approximately 50% of cases. they are round to oval, 2 to 4 /-lm in size, and contain a round, basophilic nucleus and a small, rodlike kinetoplast. although they are visible in routine stains, leishmania organisms are best seen when giemsa stain is used. immunohistochemical techniques facilitate the identification of the organlsm." per is at least as sensitive as immunohistochemical detection of leishmania in biopsy specimens, and may be positive when the latter is negative.n 9 • dogs with visceral leishmaniasis show increased levels of igg-2 specifically directed against o-acetylated sialic acids during active disease, and these antibodies have 96.6% sensitivity and 75% specificity." at present, canine leishmaniasis is considered an incurable disease in the vast majority of cases. treatments can bring about a clinical cure, but relapses months to years after treatment are to be expected. these relapses are probably due to incomplete eradication of the parasite, but could also represent reinfection. accordingly, with the poor prognosis for cure and the possible reservoir status of the dog for human infection, euthanasia may be indicated. in endemic areas, insect control measures can be beneficial in reducing the rate of insect feeding and, hopefully, infection.p when treatment is indicated, the most widely used treatment is meglumine antimonate. 1, 82 dosages vary with the study and range from 20 to 50 mglkg given subcutaneously twice daily to 200 to 300 mglkg given intravenously every other day.73., 118b, 124, 126 remission rates approaching 85% can be achieved with a large number of injections.p: 124 but parasitologic cure rates are much lower. studies in humans and dogs have suggested that the antileishmanial activity of liposome-encapsulated meglumine antimonate is vastly superior to that of the unencapsulated drug. 70, 125 other drugs that have been used in canine leishmaniasis include aminosidine, 114, 132 amphotericin b,95 allopurinol,67.,97, 129, 130 metronidazole, and ketoconazole. allopurinol has been receiving widest attention because of its low cost and safety."? with daily dosages of 11 to 15 mglkg, clinical cure is achieved in most cases, but parasitologic cure is rare. various drug combinations have been studied, with meglumine antimonate and allopurinol receiving widest usage. 73, 73., 88, 109 in one study, the combination of meglumine and allopurinol was more effective than either product alone.p' the addition of allopurinol improves the clinical response rate, and its use on a daily basis for 1 week each month appears to prevent relapse. however, the rate of parasitologic cure is not impacted. the most helpful new treatment is an admixture of chemotherapy and immunotherapy with lif2 antigen, an antigen derived from l. infantum. 107 simultaneous use of this antigen and meglumine resulted in parasitologic cure in all cases tested. there is no correlation between serologic titers and clinical signs in treated or untreated dogsy4. high titers often persist in clinically cured animals and cannot be used to monitor progress of therapy or to confirm complete cure. 94• antigen-specific lymphoproliferative responses do reappear in successfully treated dogs.ll s, leishmania organisms can often be demonstrated cytologically, by culture, or by per in successfully treated dogs. 67., 11sb thus, these dogs remain important carriers and reservoirs. successful treatment of feline leishmaniasis has not been reported. greene ceo infectious diseases of the dog and cat muller and kirk's small animal dermatology autovaccination as a treatment in 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polymerase chain reaction canine neosporosis: a case report and literature review coccidia associated with cutaneous nodules in a dog leishmaniasis in the united states a coccidia-like organism associated with subcutaneous granulomata in a dog asymptomatic canine leishmaniasis in greater athens area, greece the effect of intravenous or subcutaneous administration of meglumine antimonate (glucantime) in dogs with leishmaniasis. a randomized clinical trial pharmacokinetics of liposomeencapsulated meglumine antimonate after intramuscular and subcutaneous administration in dogs pharmacokinetics of meglumine antimoniate after administration of a multiple dose in dogs experimentally infected with leishmania infantum development of a slide elisa for canine leishmaniasis and comparison with four serological tests a sensitive and specific 30-min dot-elisa for the detection of anti-leishmania antibodies in the dog first evaluation of the use of allopurinol as a single drug for the treatment of canine leishmaniasis treatment of canine visceral leishmaniasis with allopurinol antibody kinetics during allopurinol treatment in canine leishmaniasis clinical recovery and limited cure in canine visceral leishmaniasis treated with aminosidine (paromomycin) epidemiological patterns of canine leishmaniosis in western liguria (italy) key: cord-302261-vpspaxmx authors: whitley, e.m. title: comparative pancreatic pathology date: 2014-08-21 journal: pathobiology of human disease doi: 10.1016/b978-0-12-386456-7.03415-8 sha: doc_id: 302261 cord_uid: vpspaxmx spontaneous pathologies of the pancreas are important causes of morbidity and mortality in some veterinary species and rare in others. as in human beings, the pancreas of most domestic and exotic animals is a composite organ with both endocrine and exocrine functions. the similarities between structure and function of porcine, canine, and human pancreata are such that the pig and dog serve as valuable models in basic and translational studies, most recently for efforts aimed at modeling pancreatitis and diabetes, developing functional and sustainable replacement of endocrine functions, and in imaging and manipulation studies. this article will provide a brief review of spontaneous veterinary diseases and their underlying mechanisms and the morphological features that reflect these alterations. several speciesor breed-specific conditions and the effects of selected systemic diseases on the pancreas are also discussed. the contributions to our knowledge of pancreatic physiology and pathology by small mammal (rodent) and engineered animal models and the in-depth mechanisms homologous to those in the human pancreas are covered in other sections of this article. the pancreas is an abdominal tissue that is an essential component of two organ systems, the digestive and endocrine systems. originally identified by an ancient egyptian anatomist-surgeon, herophilus, born in 336 bc, it was later named descriptively 'all-flesh' when written in greek. there is little variation in the arrangement of the exocrine pancreas among most mammals, but differences in body structure and metabolism among other members of the animal kingdom are reflected both anatomically and functionally. among mammals, including human beings, most physiological mechanisms are believed to be homologous. there are, however, important differences in certain diseases, notably pancreatic cancer. as a component of the digestive system, the pancreas functions as an exocrine gland, secreting digestive enzymes to support release of energy from ingested foods, peptides that modulate the microbiome, and anionic fluids to buffer the ph of the duodenum. as in humans, inflammation of the pancreas has life-threatening or life-ending consequences and is a relatively common condition in carnivores. neoplasia of the exocrine pancreas is an important disease in dogs and cats but is relatively unknown in horses and most livestock. the pancreas also has a central, critical role in endocrinologic regulation of multiple metabolic pathways. control of glucose homeostasis is a central role of the islets of langerhans, aggregates of endocrine cells that secrete insulin, glucagon, and other hormones into the bloodstream. as in human beings, loss of insulin production or cellular resistance to its actions results in elevated systemic levels of glucose, which manifest as diabetes mellitus (dm). animals with diabetes do not, however, exactly mimic the disease in humans because of differences in underlying physiology or anatomy. neurohormonal regulation of the secretory functions of both endocrine and exocrine components of the pancreas involves many different receptors and signaling pathways, which are becoming better characterized during health and disease. while advancing, our knowledge of the relationships between the pancreas, intestinal tract, and gut microbiome is incomplete. with recent advances in molecular techniques able to query host cell activities and provide metagenomic data regarding metabolism and growth of uncultured intestinal microbes, refining details regarding control of pancreatic secretion and factors that induce inflammation and neoplasia can be expected in the coming years. although the pancreas has a central role in survival, it is somewhat an enigma, often relegated to a short section at the end of articles on the gastrointestinal system or liver. there is still much to be learned about this organ, especially in veterinary species. much of the available information regarding pancreatic function and pathogenesis is derived from investigations of human disease or the use of animals as models for human diseases. the human pancreas is a retroperitoneal organ with head, neck, body, and tail, while in many companion animals (dogs and cats) and ruminants, the pancreas is a bilobed, vshaped organ, supported by the mesoduodenum. in dogs and cats, there are usually two pancreatic ducts, one opening at or near the bile duct and a larger duct opening more distally into the duodenum, and several pancreaticoduodenal lymph nodes that are often inconspicuous in health. the horse has a triangular pancreas that is partially retroperitoneal. a thin band of pancreatic tissue, termed the 'portal ring,' wraps around the portal vein in some species (figure 1) . among wild or exotic mammals, birds, fish, and reptiles, there is a wide variety of anatomical arrangements, even among relatively closely related species. in the sea otter, for example, the pancreatic parenchyma is distributed in the mesoduodenal adipose tissue while the north american river otter has a distinct bilobed organ. the octopus has a single organ for pancreatic and hepatic functions, the 'hepatopancreas,' from which secretions flow into the pyloric cecum, an appendage of the stomach. the pancreas develops from the fusion of two endodermal diverticula of the embryonic gut in the region of the duodenum. the two diverticula eventually give rise to the individual lobes of the pancreas. the development of a ductular system is the result of coordinated interplay between the endodermal cells and the local mesenchymal elements. endocrine components of the pancreas, cells composing the islets of langerhans and endocrine cells scattered individually between and within acini, are also endodermal in origin. cell populations that serve as progenitors within the mature pancreas (mesenchymal stem cells) are being defined. the presence of acinar and ductular structures typical of exocrine pancreas within islet cell tumors is likely the manifestation of maturation of progenitor cells along different pathways of differentiation. understanding the histogenesis and differentiation of islet cells from mesenchymal stem cell populations has developed new importance in recent years as minimal success is met by efforts to transplant insulin-producing beta cells to cure dm. dogs have been used to study plasticity of pancreatic cell populations and respond to an islet neogenesis-associated protein, an initiator of islet development. the product of the pancreatic and duodenal homeobox-1 gene is transcription factor that has critical roles in the development of both exocrine and endocrine cells and is expressed by cells of both functional units. with maturation, high levels of expression are limited to endocrine populations producing insulin and somatostatin, while exocrine acinar and ductal cells express low levels of this gene product. the canine homologue has recently been determined to reflect high levels of sequence identity and gene structure, pattern of gastrointestinal and pancreatic expression, and ability to induce insulin biosynthesis with orthologs from other species. developmental anomalies of the pancreas that are severe are usually not compatible with life, with animals dying in the neonatal period. because of a low level of postmortem examinations of neonatal companion and production animals, the incidence of developmental anomalies may be underestimated. minor developmental anomalies of the pancreas are often clinically silent, due to the large reserve capacity of this organ. minor anomalies are often identified as incidental findings during examination of tissues for other reasons. the exocrine portion of the pancreas is a compound tubuloalveolar gland, with acinar cells that produce digestive enzymes and ducts and ductules that convey secretory products to the duodenum. the ductular epithelium also has intrinsic metabolic functions critical to the proper functioning of the digestive system. acini are spherical to elongated and are connected by stemlike ductal system that are supported by increasing amounts of connective tissue as intralobular ducts come together to form interlobular ducts. the parenchyma is covered by a thin peritoneum and is not surrounded by a distinct capsule. lobules are separated by thin connective tissue septa and are relatively loosely arranged. acinar cells are pyramidal, are oriented radially around a tiny, central lumen, and have intracytoplasmic, membrane-bound zymogen granules in the apical region. the more basophilic perinuclear region, containing the rough endoplasmic reticulum, gives the acinar cell a two-toned, apical-basal polarized appearance. catalytically inactive precursor proteins (trypsinogen, chymotrypsin, procarboxypeptidase, proelastase, and kallikreinogen) are synthesized and stored in the zymogen granules, ready for release by granule fusion with the apical membrane when cells are stimulated by cholecystokinin and acetylcholine. under normal physiological conditions, precursor enzymes are activated once they reach the lumen of the figure 1 postmortem image of the grossly normal right lobe of the pancreas (arrow) of a 4-month-old female lamb. the lobe of pancreatic tissue is present in the mesoduodenum. descending duodenum, arrowhead; caudate lobe of the liver, star. duodenum, through the sequential activation of trypsinogen to trypsin, followed by trypsin-mediated cleavage of other proenzymes (figures 2 and 3) . the cell volume composed of zymogen granules is highly variable and dependent, in part, on the secretion state of the pancreas, which is related to the physiological need for digestive enzymes. often, acinar cells within a lobule or group of acini will have similar volume of zymogen granules, which may be different from adjacent lobules, so there may be some level of regional regulation of secretion. during fasting or starvation, autophagic removal of cytoplasmic proteins and decreased production of zymogens reduce the size of the acinar cell. other features of nutritional exocrine atrophy include loss of apical-basal polarity and vacuolation of the cytoplasm. in contrast, high levels of dietary carbohydrate and protein will result in hypertrophy and hyperplasia of the acinar cells. in some animal species, there is seasonal variation of pancreatic morphology. for example, in free-living geckos (gekkonidae), there is a reduction of acinar cell numbers and therefore exocrine volume during the dry season, when food supplies are scarce. pancreatic ducts are lined by columnar cells with luminal microvilli and glycocalyx and small apical cytoplasmic mucin droplets. in large ducts, many epithelial cells also have cilia, which function to aid the downstream movement of exocrine secretions (figures 4 and 5) . metabolic functions of ductal epithelial cells also are critical to the exocrine function of the pancreas. in addition to serving as a conduit for the transfer of acinar secretory products to the lumen of the duodenum and acting as a barrier between pancreatic parenchyma and its enzyme-rich secretions, the epithelial cells lining the ducts secrete abundant fluid containing bicarbonate and chloride ions. in human beings, epithelial cells lining the ducts may undergo a series of progressively malignant changes that may result in pancreatic ductal adenocarcinoma. secretion of bicarbonate ions by ductal epithelial cells, giving secretions an alkaline ph (ph 8), is critical for homeostatic control of the gut microbiome, because this fluid buffers the acidity of the chyme flowing from the stomach into the duodenum. immortalized cell lines derived from pancreatic ducts from various animal species, including dogs, cattle, and mice, have been developed as a resource for the study of ductal physiology and carcinogenesis. important differences in the physiology of pancreatic ductal epithelium between human and veterinary species (which may be important in the selection of model species for human disease) include the absence of a spontaneous cystic fibrosis-like disease among animals and the ductal epithelium as a frequent site of carcinogenesis in humans, but only rarely in veterinary species. the endocrine function of the pancreas is realized through the hormonal secretions of endodermally derived cells arranged in groups (islets of langerhans) or scattered individually or in small numbers between the exocrine tissues. hormones secreted by these cells have critical roles in homeostasis and control metabolism of dietary carbohydrates, lipids, and proteins. a more complete description of the endocrine physiology of the pancreas is presented elsewhere in this encyclopedia. briefly, several functionally different populations of endocrine cells in the islet produce and secrete hormones directly into the capillaries that course through the islet. the alpha, beta, delta, gamma (pp), and epsilon cells produce glucagon, insulin and amylin (islet amyloid polypeptide), somatostatin, pancreatic polypeptide, and ghrelin, respectively. insulin secretion from the islets of langerhans promotes glucose absorption, lowering blood glucose levels, whereas glucagon has an opposite effect, resulting in elevation of blood glucose levels. amylin functions to regulate gastric emptying, thus affecting the availability of gastric contents for digestion in the small intestine and affecting the glycemic response. ghrelin, secreted by epsilon cells of the islet and in the mucosa of the gastric fundus, plays a significant role in the control of appetite, thereby affecting availability of dietary carbohydrate. in the pancreas, pancreatic polypeptide functions to regulate the secretions of pancreatic cells in both endocrine and exocrine compartments. somatostatin, produced by delta cells and other locations in the digestive system, regulates the release of a variety of hormones, both within the islets and in other organs. blood flow through the islet and exocrine portions of the pancreas is arranged to sequentially distribute hormones to different environmental niches to coordinate appropriate hormonal control of digestion and metabolism. immunostaining is used to identify the different populations of endocrine cells ( figure 6 ). small numbers of endocrine cells reside in extrainsular sites, scattered in groups of one to three cells in the thin connective stroma between acini. immunostaining sections of canine pancreas reveals small numbers of insulin-producing cells in acini. there is the potential for much plasticity among cells in the pancreas, even among highly differentiate populations, with in vitro acinar-to-beta-cell transdifferentiation reported, so it may be that this acinar population of extrainsular endocrine cells is derived from acinar epithelial cells. there is nonhomogenous distribution of the islets of langerhans within the pancreas and there are differences in distribution of cells within the islets, with different patterns among species. patterns of distribution of cells and islets match the unique metabolic needs of different species of animals. for example, alpha and beta cells of lizards and snakes alternate in alignment along vascular spaces, instead of the alpha cells surrounding beta-cell architectural islet arrangement as in many other species. the endocrine cells may also be distributed within the spleen of some snakes. the water monitor (varanus spp.) and some lower snakes have a juxtasplenic body that is a large collection of endocrine cells, effectively a large islet, attached to one lobe of the trilobed pancreas. in animals that rapidly ingest carbohydrate-rich meals that generate large amounts of glucose, such as fruit bats, an expanded beta-cell population appears to provide a timely and sufficient insulin response to maintain glucose homeostasis (figures 7 and 8) . arterial supply is usually from the celiac artery or a subsidiary, venous drainage is to the portal veins, and innervation is from the celiac and mesenteric plexuses. afferent blood flows first through small arterioles that penetrate the islets and then serves adjacent acinar glands, effectively integrating the delivery of hormones to the populations of endocrine cells of the islet and to the exocrine cells ( figure 9 ). the extrinsic innervation of the pancreas converges on a plexus of intrinsic parasympathetic (cholinergic) pancreatic ganglia embedded in the pancreatic parenchyma that serve to control pulsatile secretion of insulin. intrapancreatic ganglia are frequently observed microscopically in pancreatic sections from dogs and cats. in felines, pacinian corpuscles are frequently encountered embedded in the pancreatic parenchyma and mesenteric stroma. pacinian corpuscles may be visualized grossly as 1-2 mm diameter, clear nodules that are usually located near the margin of lobules. these viscoelastic mechanoreceptors are well-demarcated, multilamellar structures surrounding a central afferent nonmyelinated nerve ending that detect changes in pressure and vibrations (figures 10 and 11 ). in adult cattle, the pancreatic duct may incidentally contain calcium-rich concretions. the presence of pancreatic calculi in cattle does not appear to be associated with pancreatic inflammation. accumulations of inspissated secretory material, often admixed with debris of inflammatory cells, are, however, a feature of damage to ductal elements in chronic pancreatitis in many other species. adipose infiltration within and between pancreatic lobules, in the absence of clinical signs or histological features of dm, is a common finding in obese and aged animals and has long been considered an incidental finding as no overt clinical signs have been associated with this condition. as a better understanding of the relationships between adiposity and systemic disease (such as dm and low-grade systemic inflammation) is developed, the view of this condition as incidental may change, particularly in species prone to type 2 dm (discussed in the succeeding text in this article). increased amounts of lipid are stored as intracellular triglyceride in cells usually located at the periphery of lobules or in septa, suggesting storage in mesenchymal cells of the septal stroma. apparent expansion of the volume of mesenteric adipose tissue also occurs in exocrine pancreatic insufficiency (epi) (discussed later in this article), concurrent with loss of acinar volume ( figure 12 ). groups of pancreatic cells in nontraditional locations, including the wall of the stomach, intestine, or gallbladder, figure 6 endocrine cells are located in distinct groups (islets of langerhans (a)) and scattered in smaller numbers between acini (extrainsular endocrine cells). immunohistochemical staining with antibodies directed to insulin (b) and glucagon (c) highlights individual populations that are producing hormones. diaminobenzidine chromogen and hematoxylin counterstain (b), (c). mesentery, or in the liver or spleen, are reported occasionally in animals. animal species in which heterotopic pancreatic tissue have been reported include the dog, cat, horse, cynomolgus macaque, and rat. ectopic pancreatic tissue usually consists of well-differentiated acini with ducts, sometimes with interspersed islets. the presence of aberrantly located pancreatic parenchyma is probably due to heteroplastic differentiation of embryonic endoderm or, in the case of pancreatic tissue in the wall of the duodenum, incomplete migration of primordial endodermal cells followed by differentiation to pancreatic cells. heterotopic pancreatic tissue is theoretically susceptible to a range of pathologies, but reports of such are rare ( figure 13 ). the distribution and amount of endocrine tissue within the pancreas vary among species. in the pancreas of an egyptian fruit bat, whose diet contains abundant, readily digestible carbohydrate, the islets of langerhans are large and comprise a high percentage of the pancreatic parenchyma. case material courtesy of dr. michael yaeger, iowa state university. afferent blood to the pancreatic parenchyma flows first through a capillary in the center of the islets of langerhans (arrow), then peripherally around the islet, and eventually to the nearby acini. this glomeruloid pattern of blood flow allows hormones secreted directly into the blood by endocrine cells in the islet to rapidly coordinate local endocrine and exocrine activities. figure 10 intrapancreatic ganglion. innervation of the pancreas is via the celiac and mesenteric ganglia, with intrinsic ganglia such as this one that provides local regulation of insulin secretion. pancreas from an 8-year-old, male, golden retriever dog. rarely, the pancreatic parenchyma is the site of nodules of functional splenic tissue in rabbits, nonhuman primates, cats, and dogs. intrapancreatic accessory spleens are small, single or multiple nodules, are usually considered to be congenital, and are incidental findings. microscopic features are consistent with normal splenic tissue with red and white pulp contained within a partial or complete connective tissue capsule. differential diagnoses include primary pancreatic neoplasia or metastatic neoplasia (especially when concurrent with splenic hemangiosarcoma, a relatively common malignancy in dogs) ( figure 14) . abundant adipose tissue is present as sheets between pancreatic lobules and as small groups of cells within lobules. also in this image, many islets are expanded by eosinophilic material (consistent with amyloid), but other clinical or histological features of diabetes mellitus (dm) were absent. tissue from a 10-year-old, domestic shorthaired cat that died of cerebral lymphoma. heterotopic pancreatic tissue in the wall of the duodenum in an adult, mixed-breed dog with acute pancreatitis. a well-demarcated island of exocrine tissue is present between muscular tunics (arrow) and is undergoing early degeneration, similar to the normally located lobules. suppurative inflammation and fat necrosis also are present in the adjacent mesentery (arrowhead). duodenal mucosa, star. histological examination of biopsy or postmortem (necropsy) samples is required for the definitive diagnosis of many pancreatic diseases. during collection of pancreatic tissue for microscopic evaluation, tissues should be handled gently and, in the case of postmortem samples, collection should be performed as soon after death as practically possible, to reduce artifactual changes. increasingly common in recent years since laparoscopic methods for abdominal exploration have become commonplace, pancreatic biopsy specimens from dogs and cats are seen more frequently among surgical biopsy samples. these samples are often tiny and may not be representative of pancreatic lesions, but as exploratory laparotomy is contraindicated in many severely ill patients, these samples sometimes provide otherwise unobtainable information. laparoscopic punch biopsy does not appear to alter the clinical course of veterinary patients that undergo this procedure or to elevate serum enzyme levels, but biopsy sites in patients that later undergo postmortem examination are noted to be foci of variably intense hemorrhage and inflammation ( figure 15 ). the pancreas is notoriously susceptible to postmortem autolysis, and light microscopic interpretation of pancreatic sections must take into consideration this possibility. importantly, differentiation of post-and antemortem autolytic changes is critical to the diagnosis of pancreatic inflammation, which has an autolytic component. early autolytic changes include sloughing of the ductal epithelium and disruption of acinar architecture, followed by loss of cytoplasmic definition, with apparent lysis of zymogen granules, and condensation of nuclear chromatin. karyolysis and cytolysis soon follow. due to the relationship between the pancreatic duct system and intestinal tract, bacteria are sometimes present in autolytic regions, usually in highest concentrations near vessels and ducts, suggesting retrograde movement from the portal tract or pancreatic ducts ( figure 16 ). exocrine pancreatic insufficiency (epi), a clinical maldigestion syndrome of dogs characterized by weight loss, diarrhea, steatorrhea, is caused by failure of the pancreas to synthesize and secrete adequate amounts of digestive enzymes. in dogs, epi appears to be caused most frequently by the atrophy and loss of enzyme-producing acinar cells, termed pancreatic acinar atrophy (paa), as the result of an autoimmune mechanism directed to acinar epithelial cells. clinical diagnosis is based on characteristic clinical signs of weight loss in the face of a voracious appetite, abundant, gray, malodorous feces, and low levels of serum trypsin-like immunoreactivity (tli) or fecal hydrolase ( figure 17 ). heterotopic spleen within the pancreas of an adult, domestic short-haired cat (a). this lesion was an incidental finding at necropsy. note the red pulp with abundant blood and scattered, thin, fibromuscular trabeculae and white pulp consisting of periarteriolar lymphocytic sheaths (arrows) within a lobule of pancreatic parenchyma (b). an islet (arrowhead) is visible in (a). pancreatic (arrow) and intestinal (arrowhead) biopsy was performed during exploratory laparotomy in an 8-year-old, male, dachshund dog with a history of melena, anorexia, and vomiting. microscopic features of acute necrotizing pancreatitis and enteritis with early biopsy site dehiscence were present in tissues examined later at necropsy. epi affects dogs of many breeds, with distinctly enhanced risk of occurrence in the german shepherd, rough-coated collie, chow chow, and cavalier king charles spaniel breeds of dogs. originally believed to be a simple autosomal recessive trait, recent work examining the canine major histocompatibility complex in affected german shepherd dogs points to a more complex mechanism of inheritance, probably as the result of a combination of genetic and epigenetic/environmental factors. protective and high-risk haplotypes of dog leukocyte antigen (dla) have been identified. this disease is slowly progressive, with underlying pathology for an extended time before overt disease is noticed, because of the large reserve capacity of the pancreas for the secretion of digestive enzymes. prior to clinical signs, the subclinical phase of pancreatic inflammation is characterized by infiltration of exocrine lobules by t lymphocytes, with fewer plasma cells and macrophages. individual lymphocytes are present in acini and ductal epithelium or may form lymphoid follicles. low levels of serum tli, a pancreas-specific marker, support a diagnosis of exocrine insufficiency. with continued infiltration of lymphocytes, acinar epithelial cells undergo degeneration and necrosis, which results in loss of the pancreatic parenchyma. sharply demarcated border zones between affected and minimally affected regions are typical. electron microscopic changes include dilation of the rough endoplasmic reticulum, mitochondrial swelling, and fusion of zymogen granules within acinar cells. scattered apoptotic acinar cells and autophagocytic vacuoles also are present. during the subclinical phase, these changes are reflected in reduced serum tli levels. eventually, sufficient parenchyma is lost to result in clinical signs of maldigestion, namely, steatorrhea. in late stages of the disease, the pancreas consists of small numbers of disorganized acini infiltrated by fewer numbers of lymphocytes, with prominent residual islets containing insulin-producing cells, and mature adipocytes that replace lost parenchyma. fibroplasia is usually scant. in some areas of damaged exocrine parenchyma, regenerative duct-like structures appear to be formed from a progenitor cell population. failure or concomitant loss of exocrine and endocrine pancreatic function is reported rarely in young dogs. in one series of greyhound dogs, marked loss of pancreatic parenchyma was characterized by widespread atrophy of lobules with degenerative changes in acinar cells, including apoptosis, loss of zymogen granules, intracytoplasmic vacuolation, and loss of islet cells. in animals available for antemortem evaluation, concurrent exocrine insufficiency and insulin-dependent dm were present. the cause of this syndrome is not currently known. except for two siblings evaluated, the genetic relationship of affected dogs was not reported. ductal damage or periductal inflammation was not observed, ruling out the possibility of an ascending etiology. a variably intense infiltrate of lymphocytes and plasma cells was present in some lobules, suggesting the possibility of an immune-mediated mechanism. a similar concurrent loss of exocrine and endocrine elements has been described in a small number of young beagle dogs. loss of pancreatic parenchyma and the clinical appearance of a small, firm, nodular pancreas, diffusely or regionally, are relatively common among dogs and cats and are usually the sequel of chronic, fibrosing pancreatitis. in contrast to the apparently inherited, primary pancreatic atrophy described earlier, damage to the parenchyma during acute pancreatitis or pancreatic necrosis results in degeneration or loss of acinar structures and is usually followed by proliferation of fibrous tissue. as a physiological mechanism, atrophy of acinar cells without fibrosis also occurs with inappetence or starvation, as described earlier in this article. hyperplastic exocrine nodules are very common incidental findings in older dogs and cats and occur sporadically in cattle. multiple hyperplastic nodules are usually interspersed between figure 16 autolytic pancreatic tissue from an adult horse that died of colic (intestinal rupture with endotoxic shock). note the disruption of acinar architecture, loss of zymogen granules, and presence of rodshaped bacteria without a neutrophilic response. normal lobules and consist of slightly raised, discrete, whitetan nodules randomly scattered throughout the organ. affected lobules are similar in size to adjacent normal lobules and are minimally expansile. varying degrees of acinar cell hyperplasia are usually present among the multiple hyperplastic nodules in a single patient. hyperplastic acinar cells have features consistent with their normal counterparts and often have more or fewer zymogen granules than adjacent apparently normal lobules. hyperplasia of ductular cells is uncommon. the stimulus (or stimuli) for proliferation is not known, but does not appear to be related to regeneration after pancreatic injury, because evidence of prior damage is usually absent or minimal. additionally, because of the high frequency of spontaneously occurring hyperplastic nodules and the low incidence of pancreatic carcinoma in animals, it is not likely that hyperplastic nodules represent an early stage of carcinogenesis ( figure 18 ). benign neoplasia of the exocrine pancreas (pancreatic adenoma) is not commonly reported. as adenomas do not appear to progress to malignancy and therefore have low clinical significance, they are considered an incidental finding. exocrine adenomas are thinly encapsulated and have an expansile growth pattern, sometimes causing compression and atrophy of adjacent lobules. cells comprising acinar adenomas resemble normal acinar cells. the molecular events that lead to the formation of exocrine adenomas in domestic animals are not known ( figure 19 ). malignant neoplasms arising from acinar or ductal elements of the pancreas are uncommonly reported among veterinary species, with carnivores (pet dogs and cats) having the highest incidence. pancreatic adenocarcinoma is rarely reported in horses, cattle, swine, and exotic animals. as with most neoplasms, incidence increases with age. adenocarcinomas may be solitary or multiple nodules that efface local pancreatic parenchyma. by the time of diagnosis, pancreatic adenocarcinomas have invaded locally and undergone transcoelomic or vascular/lymphatic metastasis. similar to ductal adenocarcinomas of humans, invasion and metastasis occur early in canine and feline patients, with destructive invasion locally and regionally and metastasis across the coelomic cavity and to distant organs such as the lung, liver, and other organs. concurrent inflammation and atrophy of adjacent, nonneoplastic parenchyma and mesenteric steatitis with fat saponification are usually present (figure 20) . in contrast to this disease in human beings, where infiltrating ductal adenocarcinomas represent the majority of carcinomas, pancreatic carcinomas of carnivores have a range of morphologies that reflect acinar and ductal differentiation, within and between tumors. carcinomas composed of cells with differentiation reminiscent of zymogen-filled acinar cells are designated acinar cell carcinomas. other carcinomas have a tubular architectural arrangement of neoplastic cells with formation of lumens in some groups of cells, reflecting duct-like differentiation. cytological features of malignancy are also variable among neoplastic cells, ranging from mild to marked nuclear atypia, few to many mitoses, and nuclear to cytoplasmic ratios that usually reflect the degree of differentiation. differences in morphology with regard to acinar or duct-like appearance do not appear to affect prognosis, which is grave. a dense fibrous (desmoplastic) response commonly present surrounding poorly differentiated pancreatic carcinomas may result in a firm texture. there is a subtype of pancreatic cancer in dogs, hyalinizing pancreatic carcinoma (described in the following text in this article), that appears to have a less aggressive clinical course (figures 21-23) . exocrine pancreatic cancer may be derived from cells with ductal or acinar differentiation or perhaps from progenitor populations in the pancreas. the histogenesis of exocrine carcinomas in veterinary patients is not defined. likewise, precursor lesions have not been identified. molecular defects that cause loss of regulation of differentiation and proliferation of figure 19 exocrine adenoma. a single, focal, thinly encapsulated pancreatic adenoma is present in the pancreas of a 22-year-old, maleneutered, domestic short-haired cat. exocrine nodular hyperplasia. scattered through the pancreatic parenchyma in this 13-year-old, male-neutered, domestic long-haired cat are multiple, firm, tan-white nodules of exocrine hyperplasia. precursor cells are not currently known. the disease does not appear to have an inherited (breed-related) cause. a report of an apparent increased risk in the airedale terrier dog from the 1970s has not been confirmed or refuted. there is scant research in veterinary fields to identify the underlying molecular basis for malignancy. a recent study of canine exocrine pancreatic acinar cell carcinomas demonstrated loss of claudin-4, an integral membrane protein of intercellular tight junctions, from cells comprising acinar cell carcinomas, which may be related to local invasiveness. a rare form of exocrine pancreatic carcinoma with abundant, hyaline extracellular material has recently been reported. tumors were usually present as a singular mass in the right lobe of the pancreas. microscopically, groups of neoplastic figure 20 a poorly differentiated, ductular pancreatic carcinoma effaces the architecture of the pancreas and mesoduodenum (a) of a 10-year-old, male-neutered, domestic long-haired cat and has undergone transcoelomic metastasis (carcinomatosis) to the mesentery of the jejunum (arrow) and parietal peritoneum (arrowhead) (a), (b). in a minimally affected region of this pancreas, a nodule of neoplastic cells effaces lobular architecture in the mesoduodenum (c). this neoplasm is composed of groups of proliferating, poorly differentiated polygonal cells forming scattered duct-like structures (d). there is abundant apoptotic debris (arrows) and a mitotic figure (arrowhead) in this image. cells with apical zymogen granules are arranged as tubules and acinar structures. abundant, brightly eosinophilic material of unknown composition fills the lumens of tubules and expands the stroma. the hyaline material is not congophilic and immunonegative for amyloid, immunoglobulin light chains, islet amyloid polypeptide, laminin, or alpha-1-antitrypsin. features of malignancy, such as absence of a capsule, mild cellular atypia, frequent mitotic figures, and stromal invasion, were present. an extended survival time (>15 months), even in the presence of metastatic disease, was reported in two dogs. the development of paraneoplastic panniculitis appears to be frequent among patients with this neoplasm (figure 24 ). inflammation of the subcutaneous adipose tissue has been reported as a comorbidity with pancreatic disease, including neoplasia, in dogs, cats, and human beings. patients present with multifocal, draining, truncal cutaneous nodules. the underlying lesion is chronic destruction of the subcutaneous adipose tissue, with an intense inflammatory response composed of neutrophils and macrophages. the pathogenesis appears to be related to the release of activated digestive enzymes, that is, lipase, from damaged pancreatic tissues, and speculated mechanisms include systemic release of lipolytic enzymes from neoplastic tissue, perhaps through 'leaky' intratumoral blood vessels. once in the systemic circulation, circulating enzymes are speculated to damage blood vessels, escape into the surrounding tissues, and cause fat necrosis. alternatively, or perhaps concurrently, a deficiency of enzyme inhibitors such as alpha-1-antitrypsin or alpha-2-macroglobulin may predispose patients to this rare condition (figure 25) . nesidioblastosis is defined as the proliferation of both ductular and islet cells, with hypertrophy of beta cells in islets and the formation of ductuloinsular complexes (closely associated groups of proliferating endocrine cells and small exocrine ducts). in human beings, this condition is associated with hyperinsulinemia and hypoglycemia in the absence of an insulinoma. well-defined groups of irregularly or haphazardly arranged cells are present within exocrine lobules. this condition is reported in the veterinary literature occasionally, but usually without clinical signs associated with hyperinsulinemia. ductuloinsular complexes typical of nesidioblastosis were recently described in a simmental calf with arthrogryposis. proliferating beta cells expressed low levels of insulin. similar lesions composed of proliferating endocrine and exocrine ductular epithelial cells have been reported in dogs. twenty nine of three hundred and thirty-two dogs in a cohort of young beagle dogs were reported to contain this change. the condition was most prominent in young dogs. hyperglycemia and progressive weight loss have been reported in two squirrel monkeys with immunohistochemical evidence of glucagon production by proliferating endocrine cells (figure 26 ). inflammation of the pancreatic parenchyma may occur as a specific entity (usually in the dog) or as a component of a variety of systemic or multifocal diseases or conditions that impact this organ. pancreatitis may be an acute, temporary condition or, if the inciting stimulus remains or damage is sufficiently severe, may result in permanent loss of exocrine and/or endocrine function. pathogenic mechanisms of acute and chronic inflammation of the pancreatic exocrine tissues are believed to be similar among mammals, so information derived from studies of human patients is often applied to veterinary patients. the clinical diagnosis of pancreatic inflammation depends heavily on the evaluation of serum levels of pancreatic enzymes. as new biomarkers of pancreatic injury are identified, retrospective evaluation of the specificity of serum assays, involving correlation of clinical signs, clinicopathologic data, and necropsy finding provide information regarding prognostic value of these tests. canine pancreas-specific lipase levels are useful in determining clinically the presence of pancreatic inflammation, except for pancreatic infiltration by lymphocytes. tli, measured independently for each animal species, is a useful test for pancreatic inflammation. limited information is available for serum enzyme activities, tissue sources, and reference ranges in nondomestic animal species, but reference ranges for various biomarkers of pancreatic pathology are becoming available for exotic animal species. acute necrotizing pancreatitis is a condition generally seen in small companion animals (dogs and cats). recent work has highlighted this condition as previously underdiagnosed in cats. the cause of acute pancreatitis in dogs is not known, but patients with acute pancreatitis are typically older, obese, female, or female-spayed, miniature breed dogs. dog breeds at increased risk of developing acute pancreatitis include miniature schnauzer, yorkshire terrier, and cocker spaniel dogs, but genetic studies to identify specific mechanisms have not been performed. the labrador retriever dog appears to have a reduced risk of acute pancreatitis. other risk factors include ingestion of a high-fat meal, endocrine disease (hyperglucocorticoidism or hypothyroidism), hypercalcemia, uremia, trauma, or treatment with the immunosuppressive drug, azathioprine. damage or disease of the biliary tree or obstructions of the pancreatic duct may cause pancreatitis. mechanical injury to the pancreas may also incite pancreatitis, especially if there is damage to the ductal system. in small companion animal species, focal iatrogenic pancreatitis may result from biopsy or rough handling during laparotomy, or may be more widespread or regional after more intense trauma such as vehicle impact. the pathogenesis of acute pancreatitis is complex and includes the intra-acinar activation of digestive enzymes resulting in degeneration and necrosis of acinar cells, leading to autodigestion of the pancreatic parenchyma and an intense concurrent inflammatory response. damage to the acinar cells and cells lining the ductules and ducts is usually avoided because digestive enzymes are sequestered in membranebound intracytoplasmic vesicles and are produced as catalytically inactive proenzymes. it appears that events early in the process include the intra-acinar activation of trypsinogen and signaling in support of a local and systemic inflammatory cascade. the trigger for these processes is not known, but a variety of cellular mechanisms are likely involved, including altered calcium signaling, autophagy, and mitochondrial function (figures 27-30) . a recently identified consequence of pancreatitis is damage to the intestinal mucosal barrier leading to translocation of gut microbes across the mucosa, resulting in sepsis and multiple organ failure. pancreatitis may also predispose patients to the formation of thrombosis of splenic and portal veins. these complications are important causes of mortality among patients with acute pancreatitis. fibrosing pancreatitis is a chronic inflammatory process characterized by irreversible destruction of the architecture of the parenchyma and ductal system, with production of increased amounts of fibrous tissue. this chronic inflammatory process results in loss of both exocrine and endocrine functions. dog breeds with increased risk of developing chronic pancreatitis are the cavalier king charles spaniel, collie, and boxer. grossly, lobules are firm and white, with expansion of the interlobular stroma and thickening of the serosal tissues. dystrophic mineralization may occur in parenchyma and adjacent mesenteric adipose tissue damaged by the release of pancreatic enzymes. the architecture of affected lobules is distorted by sheets and strands of fibrous tissue that dissect between and within affected regions, with marked distortion of acinar structure and compression of acinar cells. abundant, paucicellular (mature) fibroplasia is often present around ducts, with hyperplasia of ductal epithelium and, sometimes, duct stenosis or luminal obstruction by condensed secretory material. the inter-and intralobular spaces and periductal adventitia are also infiltrated by varying numbers of neutrophils, lymphocytes, plasma cells, and macrophages (figures 31-33) . in some lobules with early or mild fibrosis, ducts may be irregularly dilated and have flattened epithelial cells without microvilli; these changes probably reflect impaired secretion of anionic fluid, leading to precipitation and eventual inspissation of proteins secreted by acini and ultimately resulting in loss of secretory flow (figure 34 ). early, acute, necrotizing pancreatitis. within this lobule, there are multifocal to coalescing regions of acinar necrosis (arrows) characterized by loss of cellular detail and replacement by amorphous eosinophilic cell debris. a mild neutrophilic inflammatory infiltrate is present (arrowheads). tissues are from a 9-year-old, male, labrador retriever dog. an intense suppurative inflammatory infiltrate expands the interlobular stroma, covers the peritoneal surface, and extends into this degenerating pancreatic lobule. the pancreas is susceptible to secondary damage by a wide variety of agents that impact this organ via direct, hematogenous, or ductular routes. multisystemic eosinophilic epitheliotrophic disease (meed) in horses is characterized by infiltration of eosinophils, cd3ã¾ lymphocytes, and fewer plasma cells and the formation of eosinophilic granulomas in various internal organs, including the pancreas and the skin. this condition has some similarity to hypereosinophilic syndrome of human beings. clinical signs usually include weight loss and skin lesions, with or without fever. the cause of this syndrome is unknown, but suggested mechanisms include a hypersensitivity to intestinal parasites or other antigen resulting in secretion of interleukin-5 ( figure 35 ). damage to a ductule during pancreatic inflammation has resulted in degenerative changes to the ductular epithelium, resulting in loss of their secretory function and subsequent condensation of intraluminal proteinaceous secretory material. atrophic ductular epithelial cells also have lost surface cilia, which also impairs luminal flow. there is mild adventitial fibroplasia and ongoing local acinar damage, indicating that this is a subacute to early chronic process in this image. infectious agents and animal species where pancreatic damage causes clinically important disease. endoparasites may cause disease by occluding pancreatic ducts or inducing an inflammatory response that causes periductal fibrosis. pancreatitis secondary to endoparasitism is reported to be an important cause of death in free-ranging giant pandas. eurytrema spp. flukes are common parasites of cattle in south america and asia and may be found in primates, carnivores, pigs, birds, and other animals. trematode eggs are common findings within the pancreatic parenchyma and other organs of many turtle species. inflammatory responses surrounding the eggs are usually minimal. disseminated toxoplasmosis is an important cause of acute multifocal pancreatic necrosis in many mammals, especially figure 36 the pyogranulomatous inflammatory response in feline infectious peritonitis often affects the pancreas, contributing to clinical signs in this progressive, fatal disease of domestic cats caused by a variant feline coronavirus. multifocal to coalescing fibrinous and pyogranulomatous inflammation (arrows; pancreas arrowhead) and an abundant, protein-rich effusion are present in the abdomen of a 1-year-old, female-spayed, domestic short-haired cat (a). multiple lobules of the pancreas are infiltrated or replaced by inflammatory cells (b), (c). immunohistochemical staining using antibody directed at porcine coronavirus (a related virus) demonstrates the presence of coronavirus antigens in inflammatory cells (d). faint, nonspecific (background) staining is present in acinar cells. diaminobenzidine chromogen and hematoxylin counterstain (d). felines (domestic and exotic cats). toxoplasma gondii, a zoonotic protozoan, may cause disseminated granulomatous and necrotizing inflammation in many organ systems. feline infectious peritonitis is a fatal disease of domestic cats that is caused by infection by a virulent feline coronavirus (fcov). fcov is a close relative to the human sars coronavirus. under some conditions, the host immune system mounts an intense response to fcov antigens resulting in damage to serosal surfaces, including the mesentery, and widespread pyogranulomatous inflammation. serosal damage may lead to accumulation of abundant protein-rich fluid in the abdomen. the pancreas frequently is affected, with multiple foci of surface and parenchymal damage (figure 36) . infection by ophidian paramyxovirus is an important cause of death in viperidae snakes due to severe lung pathology. the pancreas and liver are also affected, with pancreatic acinar and ductal hyperplasia and intracytoplasmic inclusion bodies. the pancreas is uncommonly the site of metastatic neoplasia. effects of metastasis include replacement of pancreatic parenchyma by neoplastic tissue with loss of that functional capacity, as well as the potential for damage to the parenchyma or duct system with secondary pancreatitis. hemangiosarcoma in dogs and, less frequently the visceral form in cats may undergo metastatic spread to the pancreas. the pancreas may be infiltrated widely by neoplastic lymphocytes in patients with disseminated lymphoma. other systemic effects of neoplasia, such as metabolic imbalances, hypercalcemia, or prothrombotic conditions, may also affect the pancreas (figures 37-39) . the pancreas is a target organ for zinc toxicosis. zinc toxicosis manifests as degeneration of acinar cells, without damage to cells in islets or forming exocrine ducts. damage to acinar cells ranges from mild atrophy with loss of zymogen granules to more severe loss of acinar architecture. toxicosis is seen among diving ducks that swim on shallow bodies of water, such as those at fountains and parks, into which humans throw pennies containing zinc. dm is a group of metabolic diseases characterized by abnormal glucose metabolism resulting in chronically elevated levels of blood glucose. the cause of dm in humans and animals appears to be a complex interplay between genetic and epigenetic (environmental) factors. two basic underlying mechanisms reflect the different forms of the disease, namely, defects in production of insulin by the pancreas (type 1 dm) and resistance by target cells to the effects of insulin coupled with abnormal secretion of insulin (type 2 dm). in addition, metabolic changes during pregnancy are sometimes related to the development of elevated levels of blood glucose and are termed 'gestational diabetes.' there are important differences in the incidence, pathogenesis, and consequences of this disease among animal species, although similarities to the disease in human beings exist. risk factors include increasing age. obesity is important in some animals (cats), whereas it does not appear to predispose others (dogs) to developing dm. diabetes and metabolic syndrome resulting from insulin resistance is being recognized as an important disease in horses. the incidence of reporting of dm among exotic animals is increasing, probably due to enhanced medical care of these animals. reports of dm among livestock are unusual, perhaps due to population dynamics and economic considerations. in contrast to cats that develop type 2 dm most frequently, domestic dogs with dm have underlying destruction of beta figure 37 hemangiosarcoma, a malignancy of endothelial cells common in dogs, effaces local pancreatic parenchyma in this image. similar masses composed of neoplastic endothelial cells forming blood-filled channels were widely disseminated throughout the body of this dog. multiple nodules of neoplastic lymphocytes, grossly evident as soft, white masses (arrows), infiltrate pancreatic parenchyma in the pancreas of a 10-year-old, female-spayed, siamese cat with disseminated t-cell lymphoma. multiple nodules of nodular hyperplasia (arrows; grossly indistinguishable from lymphoma) and regions of chronic, periductal inflammation also are present. abundant adipose tissue is present in the mesoduodenum (star). case material courtesy of dr. ronald myers, iowa state university. cells with permanent loss of the ability to produce insulin. it is usually a slowly progressive disease that affects older adult dogs. the association of a specific dla haplotype has been associated with the development of dm in dogs and is common in dog breeds that have increased incidence of dm (samoyed, cairn terrier, and tibetan terrier) and reduced incidence in dog breeds that are relatively resistant to dm (boxer, german short-haired pointer, and golden retriever). pathogenic mechanisms associated with dm in dogs appear to be centered on the development of autoimmunity, with reports of autoantibodies to insulin and other islet antigens, similar to latent autoimmune diabetes in human beings. domestic cats with dm usually have features most consistent with type 2 dm of human beings, sometimes with histological features consistent with concurrent pancreatitis. besides domestic short-and long-haired cats in which the disease is common, some populations of burmese cats have increased risk of developing type 2 dm. in addition, cats sporadically present with hyperglycemia due to causes that would be classified as 'other specific types' under the human classification scheme. obesity, the accumulation of excess amounts of lipid within adipocytes and associated metabolic, inflammatory, and hormonal changes, has reached epidemic proportions among companion animals (dogs and cats) and to a lesser extent among horses in western countries, similar to the situation in human beings. obesity-induced insulin resistance occurs in dogs, cats, and horses, but dogs have not been documented to progress from this state to type 2 dm. obese dogs do have elevated postprandial levels of insulin, glucose, and triglyceride, but members of a small cohort of obese dogs followed for several years did not develop clinical signs of dm. dogs maintain high fasting concentrations of insulin in order to compensate for insulin resistance and are able to maintain normal blood glucose levels. leptin, a hormone involved in monitoring energy levels in the body and produced in the white adipose tissue, appears to be involved in this compensation, as serum levels are elevated in obese dogs with insulin resistance. microscopic changes that support a diagnosis of dm include loss of islets, infiltration of islets by inflammatory cells, and marked cytoplasmic vacuolation of islet cells. ductular epithelium often has basally located intracytoplasmic vacuoles. in cats and cynomolgus macaques, deposition of amyloid in islets is common. the development of amyloidosis of the islets is almost universal in type 2 dm in human beings and cynomolgus macaques and is also a common finding in wild and domestic felids with this disease. it is, however, also present at a low incidence in some clinically normal cats, so it is not pathognomonic for type 2 dm in cats. the deposition of amyloid occurs due to altered beta-cell function whereby overproduction of amylin (islet amyloid polypeptide) results in the formation of amyloid fibrils. other aspects of altered beta-cell function are believed to accompany the overproduction of amylin (figures 40-42) . cells in the islets of this 10-year-old, male-neutered, domestic long-haired cat with dm and chronic pancreatitis exhibit marked cytoplasmic vacuolation (arrows). acinar cells are atrophic, with few intracytoplasmic zymogen granules. focally, a ductule (arrowhead) is dilated by secretory material. case material courtesy of dr. amanda fales-williams, iowa state university. tumors may arise individually in each of the endocrine cell populations in the pancreas, sometimes with overproduction of an endocrinologically active hormone such as somatostatin, pancreatic polypeptide, and others. excess levels of a tumorsecreted hormone usually result in disrupted homeostasis. the most common tumors arising in islet cell populations that are clinically evident, and therefore diagnosed, are benign or malignant neoplasms of the beta cell. neoplasms arising from a beta cell that has retained the ability to produce insulin are termed a 'functional' beta-cell tumor. these neoplasms are hormonally active and may secrete high concentrations of insulin, which result intermittently in marked hypoglycemia, due to insulin-induced uptake of glucose by target cells of the body. although not specific for these neoplasms, episodic and progressively severe clinical signs associated with hyperinsulinism include hindlimb weakness, exercise intolerance, ataxia, reduced mental capacity, and, late in the disease, seizures. adenomas and carcinomas arising from beta cells are the most frequent type of endocrine tumor of the pancreas, with carcinomas reported to be more common than adenomas in dogs. dog breeds with an increased incidence of beta-cell tumors include boxers, fox terriers, standard poodles, and german shepherds. beta-cell tumors, 'insulinomas,' are common in ferrets and have been reported occasionally in other animal species. the nomenclature used to describe benign versus malignant and productive versus nonproductive tumors seems to vary between veterinary clinicians and pathologists, so 'insulinomas,' that is, insulin-producing beta-cell tumors, usually have a malignant clinical course, with frequent metastasis outside the pancreas. macroscopically, adenomas and carcinomas of the islets usually have a similar color and consistency to the surrounding pancreatic parenchyma. adenomas are usually small, welldemarcated nodules confined by a connective tissue capsule, while carcinomas are larger and have very invasive growth characteristics, invading into the adjacent parenchyma, migrating within lymphatic vessels, and forming secondary tumors in distant sites. functional islet tumors producing severe clinical signs may be small and difficult to identify. cells lining this interlobular duct of a 10-year-old, maleneutered, domestic long-haired cat with type 2 dm and chronic pancreatitis have prominent, basilar, intracytoplasmic vacuoles (arrow). case material courtesy of dr. amanda fales-williams, iowa state university. insular accumulation of amyloid is a common finding in cats with dm (homologous to human type 2 dm). this material (red orange in this congo red-stained section (a)) is derived from islet amyloid polypeptide (amylin), a hormone secreted by beta cells of the islet. amyloid fibrils have apple green birefringence under polarized light (b). a compendium of canine normal tissue gene expression morphology of the pancreas of some species belonging to the genera phelsuma and gecko (family gekkonidae): evidence of apoptotic process during the seasonal cycle immunocytochemical identification of endocrine cells in the pancreas of the fruit bat, rousettus aegyptiacus specificity of a canine pancreas-specific lipase assay for diagnosing pancreatitis in dogs without clinical or histologic evidence of the disease the role of islet neogenesisassociated protein (ingap) in pancreatic islet neogenesis insulin and glucose sensitivity, insulin secretion and beta-cell distribution in endocrine pancreas of the fruit bat artibeus lituratus cdna cloning and mrna expression of canine pancreatic and duodenum homeobox new advances in pancreatic cell physiology and pathophysiology a comparative immunohistochemical study of pancreatic islets in laboratory animals (rats, dogs, minipigs, nonhuman primates) juvenile pancreatic atrophy in greyhounds: 12 cases current status of genetic studies of exocrine pancreatic insufficiency in dogs figure 43 this section of the pancreas from a 5-year-old, male, european ferret contains a poorly demarcated, expansile, and mildly infiltrative neoplasm (a) composed of groups of beta cells supported by fine strands of fibrovascular tissue (b) and demonstrating immunoreactivity to insulin (c) diaminobenzidine chromogen and hematoxylin counterstain (c) hyalinizing pancreatic adenocarcinoma in six dogs nesidioblastosis in a simmental calf multisystemic, eosinophilic, epitheliotropic disease with intestinal lymphosarcoma in a horse histologic assessment and grading of the exocrine pancreas in the dog portal vein thrombosis in 33 dogs: 1998-2011 spontaneously occurring extra-islet endocrine cell proliferation in the pancreas of young beagle dogs alleles of the major histocompatibility complex play a role in the pathogenesis of pancreatic acinar atrophy in dogs exocrine pancreatic insufficiency in the dog: historical background, diagnosis, and treatment canine diabetes mellitus: from phenotype to genotype canine diabetes mellitus: can old dogs teach us new tricks pancreatic endocrinopathies in ferrets diabetes, insulin resistance, and metabolic syndrome in horses pathogenesis of feline diabetes endocrine diseases in dogs and cats: similarities and differences with endocrine diseases in humans insulin resistance in cats veterinary immune reagent networkrepository for information regarding genes, antibodies, and cytokines for veterinary species key: cord-297724-xoqrc3xo authors: miyaji, kazuki; suzuki, aki; shimakura, hidekatsu; takase, yukari; kiuchi, akio; fujimura, masato; kurita, goro; tsujimoto, hajime; sakaguchi, masahiro title: large-scale survey of adverse reactions to canine non-rabies combined vaccines in japan date: 2012-01-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2011.12.023 sha: doc_id: 297724 cord_uid: xoqrc3xo canine non-rabies combined vaccines are widely used to protect animals from infectious agents, and also play an important role in public health. we performed a large-scale survey to investigate vaccine-associated adverse events (vaaes), including anaphylaxis, in japan by distributing questionnaires on vaaes to veterinary hospitals from april 1, 2006 through may 31, 2007. valid responses were obtained for 57,300 vaccinated dogs at 573 animal hospitals; we obtained vaaes information for last 100 vaccinated dogs in each veterinary hospital. we found that of the 57,300, 359 dogs showed vaaes. of the 359 dogs, death was observed in 1, anaphylaxis in 41, dermatological signs in 244, gastrointestinal signs in 160, and other signs in 106. onset of vaaes was mostly observed within 12 h after vaccination (n = 299, 83.3%). in this study, anaphylaxis events occurred within 60 min after vaccination, and about half of these events occurred within 5 min (n = 19, 46.3%). furthermore, where anaphylaxis was reported, additional information to support the diagnosis was obtained by reinvestigation. our resurvey of dogs with anaphylaxis yielded responses on 31 dogs; 27 of these demonstrated collapse (87.1%), 24 demonstrated cyanosis (77.4%), and both signs occurred in 22 (71.0%). higher rates of animal vaaes, anaphylaxis, and death were found in japan than in other countries. further investigations, including survey studies, will be necessary to elucidate the interaction between death and vaccination and the risk factors for vaaes, and thus develop safer vaccines. moreover, it may also be necessary to continually update the data of vaaes. canine non-rabies combined vaccines, containing zoonotic pathogen leptospira, are widely used in small animal veterinary medicine. vaccination is aimed at fig. 1 . the questionnaire form used for the investigation of vaccine-associated adverse events (vaaes). the questionnaire was distributed by the japan small animal veterinary association to veterinary hospitals in japan from april 1, 2006 through may 31, 2007 . et al., 1995 roth, 1999) occasionally observed in humans and dogs after vaccination, and which sometimes causes death (brooks, 1991; sakaguchi et al., 2000) . according to large epidemiological studies on canine vaaes, the incidences of anaphylaxis were 0.018/10,000 vaccinated dogs in the united kingdom (gaskell et al., 2002) and 0.65/10,000 vaccinated dogs in the united states (moore et al., 2005) . based on a survey of a small number (35) of japanese veterinary hospitals, the anaphylaxis rate in japan appeared to be somewhat higher, at 0.17% (6/3477 vaccinated dogs) (fujimura, 2006) . however, to date, no large-scale investigation had been carried out on vaaes associated with canine non-rabies combined vaccine in japan. moreover, although incidences of anaphylaxis were provided, none of the previous studies (gaskell et al., 2002; moore et al., 2005; fujimura, 2006) considered important details, such as concurrent symptomatic states. here, we report the results of a large-scale survey of vaaes in japan, based on diagnosis made by experienced veterinarians in their practices. our results reveal useful information on vaaes, including critical detail on anaphylaxis and death caused by canine non-rabies combined vaccines. it is useful to record vaccine reactions routinely, and report vaaes to specific agents such as local governments in veterinary medicine as well as human medicine. the questionnaires about adverse reactions to canine non-rabies combined vaccines were distributed by the japan small animal veterinary association to veterinary hospitals in japan from april 1, 2006 through may 31, 2007. the questionnaires not only recorded standard information: date of birth, breed, sex and neuter status, weight, and date of vaccination, but also included important factors associated with adverse reactions, such as type of vaccination, signs, and time of their onset since vaccination (fig. 1) . practicing veterinarians diagnosed adverse reactions and classified them into 5 groups according to clinical signs: death, anaphylaxis, dermatological signs (swelling of face, pruritus, urticaria, flush, and erythema), gastro-intestinal signs (vomiting and diarrhea), and other signs (including hypodynamia and anorexia). in cases of anaphylaxis, additional information was obtained by reinvestigation to support the diagnoses. to define the population, each responding veterinarian also stated the number of affected cases per last 100 vaccinated dogs. the following non-rabies combined vaccines for dogs are commonly used in japan: combined live vaccines composed of canine parvovirus, canine distemper virus, canine adenovirus type 2, and canine parainfluenza virus (group 1); adding live or inactivated coronavirus to group 1 (group 2); adding inactivated leptospira to group 1 (group 3); adding inactivated coronavirus and leptospira to group 1 (group 4); adding live coronavirus and inactivated leptospira to group 1 (group 5). duramune mx5 (kyoritsu seiyaku corporation, tokyo, japan; fort dodge animal health, iowa, usa), eurican 5 (merial animal health, lyon, france), and nobivac dhppi (intervet, boxmeer, netherlands) were classified as group 1; canine-6ii (kyoto biken laboratories, kyoto, japan), duramune dx6, and vanguard plus 5/cv (pfizer animal health, new york, usa) were classified as group 2; canine-8, eurican 7, and nobivac dhppi+l as group 3; duramune dx8 and vanguard plus 5/cv-l as group 4; and canine-9 (ii) as group 5. sex and neuter status were analyzed as categorical data. continuous variables of age and weight were converted to categorical variables because nonlinear trends were detected in the model-fitting process. dogs were grouped on the basis of age at date of vaccination as follows: 2-9 months, 9 months to 1.5 years, 1.5-2.5 years, 2.5-3.5 years, 3.5-5.5 years, 5.5-8.5 years, and >8.5 years. weight was converted from continuous to categorical data of 0-5 kg, 5-10 kg, and > 10 kg. the p values for anaphylaxis in total vaaes were evaluated using a multivariate unconditional logistic regression model. the variants included sex and neuter status, weight, age, and vaccine group. the model was assessed for significance by use of the le cessie-van houwelingen test. a value of p < 0.05 was considered significant. statistical analysis was performed with r version 2.11.1 (www.r-project.org/). valid responses were obtained for 57,300 vaccinated dogs, from 573 animal hospitals; of these, 359 dogs were diagnosed with vaae (62.7/10,000 vaccinated dogs). unfortunately, the detailed information regarding dogs with no adverse events could not be obtained in the present study. of the 359 dogs, anaphylaxis was observed in 41, dermatological signs in 244, gastrointestinal signs in 160, and other signs in 106 (table 1) . a single death (0.2/10,000 vaccinated dogs) was reported within a few days after vaccination. in decreasing order of frequency, 181 (50.4%) miniature dachshunds, 37 (10.3%) chihuahuas, 18 (5.0%) mixedbreeds, and 17 (4.7%) toy poodles were reported to have vaaes in the present survey; other breeds were involved less frequently. the population with vaaes included 145 (40.4%) sexually intact males, 156 (43.5%) sexually intact females, 24 (6.7%) castrated males, 24 (6.7%) spayed females, and 10 (2.8%) unknown. almost half these dogs (n = 164, 45.7%) were between the ages of 2 and 9 months. dogs in the weight category of 0-5 kg had the highest frequency of adverse reactions (n = 249, 69.4%). most adverse events (n = 299, 83.3%) were observed within 12 h after vaccination (fig. 2) . more detailed information about vaccine-associated anaphylaxis is provided in tables 2 and 3, as well as fig. 3 . miniature dachshunds (n = 13) accounted for approximately 30% of the anaphylaxis cases. anaphylaxis occurred in four miniature schnauzers out of a population of the eight with vaaes. of 41 dogs diagnosed with anaphylaxis, 17 (41.5%) were sexually intact males, 18 (43.9%) were intact females, 3 (7.3%) were castrated males, and 3 (7.3%) were spayed females. according to age and weight, the greatest frequency of anaphylaxis was recorded among dogs aged 2-9 months (n = 20, 48.8%) and among dogs weighting less than 5 kg (n = 27, 65.9%), respectively. a multivariate logistic regression model, including sex and neuter status, weight, and age satisfied requirements for goodness of fit (p = 0.12). in the final model, there was no significant relationship between anaphylaxis and the factors of sex and neuter status (p = 0.58), weight (p = 0.15), age (p = 0.24), or vaccine group (p = 0.96). all cases of anaphylaxis occurred within 60 min after vaccinations, and about half occurred within 5 min (n = 19, 46.3%) (fig. 3) . when we attempted to resurvey the 41 dogs that experienced anaphylaxis, responses were received for 31 of these dogs (recovery ratio = 75.6%). as shown in table 3 , collapse and cyanosis were observed in almost all 31 of the dogs, with 27 (87.1%) demonstrating collapse, 24 (77.4%) demonstrating cyanosis, and 22 (71.0%) showing both signs. a single death (chihuahua, intact female, 2 months, 0.9 kg), occurring 38 h after vaccination, was reported in our questionnaire. the risks involved in vaccination of dogs were highlighted by previous large epidemiological studies (gaskell et al., 2002; moore et al., 2005) ; however, surveys on vaaes had only been performed on a small scale in japan (ohmori et al., 2002 (ohmori et al., , 2005a fujimura, 2006) . here, we present the results of a large-scale investigation of vaaes at japanese veterinary hospitals. we found a vaae rate of 62.7/10,000 vaccinated dogs, which is much higher than the rates reported in the united kingdom (0.093/10,000 vaccinated dogs) (gaskell et al., 2002) and the united states (38.2/10,000 vaccinated dogs) (moore et al., 2005) . previous reports suggested that small breed dogs (<10 kg), especially dachshunds, were more prone to vaaes than larger dogs (gaskell et al., 2002; moore et al., 2005; moore and hogenesch, 2010) . according to the japan kennel club (http://www.jkc.or.jp), miniature dachshund, chihuahua, and toy poodle are the most popular breeds in this country, and account for just over half of the total registrations in the club (50.3%). this popular breed bias and the proposed breed susceptibility may contribute to higher vaaes rate in japan than in other countries. factors known to cause vaccine reactions include the primary vaccine agent or antigen, adjuvants, preservatives, stabilizers, and residues from tissue culture used in vaccine production (hogenesch et al., 1999; roth, 1999; georgitis and fasano, 2001) . our previous study reported large amounts of bovine serum albumin (bsa) and bovine igg contents in canine vaccines (ohmori et al., 2005b) . furthermore, we found ige reactivity against fetal calf serum (fcs) components in dogs with allergic reactions after vaccination, suggesting that most of these reactions might be caused by fcs components derived from the culture media used to produce vaccines (ohmori et al., 2005b (ohmori et al., , 2007 . many vaccines that are commonly used in japan are imported from abroad, although a few are made in japan. multilateral studies are necessary to clarify the relationship between vaaes and vaccine by-products, and to ascertain whether certain vaccines are more likely to give rise to vaaes. among the many clinical signs of adverse reactions to vaccines, anaphylaxis is the most dramatic (roth, 1999) . in the present investigation, the incidence of anaphylaxis (7.2/10,000 vaccinated dogs) in dogs was remarkably higher than those reported in previous studies (gaskell et al., 2002; moore et al., 2005; moore and hogenesch, 2010) . the dogs showing anaphylaxis in the present study included many small breeds such as miniature dachshund and miniature schnauzer. this implies that a breed predisposition may play a key role in anaphylaxis after vaccination of dogs, just as in total vaaes, and that genetic factors may be involved in these clinical signs. moore et al. (2005) have indicated that the risk of vaaes was significantly increased for small and neutered dogs, highest for dogs approximately 1-3 years old, and least for dogs ≥6 years of age. unfortunately, as the information regarding dogs with no adverse events could not be obtained in the present study, the odds ratios of vaaes could not be estimated; however, we did estimate the p values for anaphylaxis according to adverse reactions after vaccine administrations using a logistic regression model. additionally, we were not able to analyze the relationship between breed and anaphylaxis due to small sample numbers for many breeds. however, no significant differences were observed in the anaphylaxis risk by sex and neuter status, weight, and age; thus, our data seem to show the same tendency of potential risk factors for vaaes in the results of moore et al. (2005) . the results of the present study suggest that various factors such as weight and age could predict the potential risk of adverse reactions, including anaphylaxis after vaccination, which may allow practicing veterinarians to allay the anxiety of at least a few owners regarding vaccination. as only a single death was reported in this investigation, a direct association between death and vaccine administration was not clear; however, as an incidence of 0.02/10,000 vaccinated dogs was reported in the united state (moore et al., 2005) , the mortality rate also appeared to be increased in japan. this case did not show anaphylactic signs, although other clinical sign was observed. further extensive surveys are needed to uncover the predictive factors and clinical signs that precede such outcomes, in order to prevent such unfortunate events. moreover, to clarify the interaction between death and vaccination, comparison with randomized unvaccinated dogs may be effective. we present data regarding vaaes, and in particular, on anaphylaxis, based on a large-scale study in japanese veterinary hospitals. higher rates of vaaes, anaphylaxis, and death were observed in japan compared to other countries. further international studies aimed at obtaining similar information will be necessary to elucidate the risk factors, including breed predisposition, the quality of vaccine products, and prior sensitization with variable allergens, for vaaes, with a view to developing safer vaccines. in addition, as our study was not current, it may be also be necessary to continually update the data of vaaes. adverse reactions to canine and feline vaccines a survey on the adverse reactions to combined vaccines in a small group of veterinary hospitals (35 hospitals) veterinary products committee working group report on feline and canine vaccination allergenic components of vaccines and avoidance of vaccination-related adverse events vaccine-induced autoimmunity in the dog adverse events diagnosed within three days of vaccine administration in dogs adverse vaccinal events in dogs and cats a retrospective study on adverse reactions to canine vaccines in japan suspected allergic reactions after vaccination in 85 dogs in japan ige reactivity to vaccine components in dogs that developed immediate-type allergic reactions after vaccination immunoblot analysis for ige-reactive components of fetal calf serum in dogs that developed allergic reactions after non-rabies vaccination mechanistic bases for adverse vaccine reactions and vaccine failures ige antibody to gelatin in children with immediate-type reactions to measles and mumps vaccines minimum estimated incidence in japan of anaphylaxis to live virus vaccines including gelatin the authors would like to thank all staff of participating veterinary hospitals for their kind assistance in responding to the questionnaires. we also thank the japan small animal veterinary association for its support. we thank dr. sakae inouye in otsuma women's university for critical review of this manuscript. this research was partially supported by the promotion and mutual aid corporation for private schools of japan, grant-in-aid for matching fund subsidy for private university, and by a project grant awarded by the azabu university research services division. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord-304385-864o4buq authors: fiorello, christine v.; noss, andrew j.; deem, sharon l. title: demography, hunting ecology, and pathogen exposure of domestic dogs in the isoso of bolivia date: 2006-06-01 journal: conserv biol doi: 10.1111/j.1523-1739.2006.00466.x sha: doc_id: 304385 cord_uid: 864o4buq abstract: disease is increasingly recognized as a threat to the conservation of wildlife, and in many cases the source of disease outbreaks in wild carnivores is the domestic dog. for disease to spill over from a domestic to a wild population, three conditions must be satisfied: susceptibility of the wild species, presence of the disease agent in the domestic population, and contact between the two populations of interest. we investigated the potential for disease spillover from the domestic dog population to the wild carnivore population in the isoso of bolivia, an area of tropical dry forest contiguous with a national park. using questionnaires and discussions with residents, we gathered data on the demography of dogs in the isoso, including adult and neonatal mortality, litter size, and hunting frequency. we analyzed a large data set containing self‐recorded information on hunting in various communities of the isoso to determine the extent of dog participation in hunting and the duration of hunting trips. finally, we took blood samples from dogs in the isoso for a serosurvey of common canine pathogens. more than 95% of dogs had positive titers to canine distemper virus and canine parvovirus. there was also a high seroprevalence in dogs for other pathogens, a high population turnover of dogs (which may allow diseases to be maintained endemically), and frequent opportunities for contact between domestic and wild carnivores. based on our results and the susceptibility of wild species previously reported in the literature, domestic dogs represent a disease risk for wildlife in the bolivian isoso. vestre, y en muchos casos el perro doméstico es la fuente del desencadenamiento de enfermedades de carnívoros silvestres. para que una enfermedad pase de una población doméstica a una silvestre, se deben satisfacer tres condiciones: susceptibilidad de la especie silvestre, presencia del agente patógeno en la población doméstica y contacto entre las dos poblaciones de interés. investigamos la potencial transmisión de enfermedades de la población de perros domésticos a la población de carnívoros silvestres en el isoso de bolivia, unárea de bosque tropical seco contigua a un parque nacional. mediante cuestionarios y discusiones con residentes, reunimos datos de la demografía de perros en el isoso, incluyendo mortalidad de adultos y neonatos, tamaño de camada y frecuencia de cacería. analizamos un extenso conjunto de datos conteniendo información sobre cacería en varias comunidades del isoso para determinar la extensión de la participación de perros en la cacería y la duración de los viajes de cacería. finalmente, tomamos muestras de sangre de perros en el isoso para un muestreo de suero para buscar los patógenos caninos más comunes. más de 95% de los perros fueron positivos a moquillo y parvovirus caninos. también hubo seroprevalencia alta para otros patógenos, una alta productividad en la población de perros (lo que puede mantener a las enfermedades endémicamente) y frecuentes introduction disease in wildlife is increasingly recognized as a conservation concern (e.g., mccallum & dobson 1995; woodroffe 1999; funk et al. 2001) . domestic animals often act as reservoir species that can maintain infectious diseases in their populations and periodically transmit these diseases to naïve wildlife populations (cleaveland & dye 1995; rhodes et al. 1998; lafferty & gerber 2002) . more attention is now being paid to the diseases of domestic dogs that live in areas surrounding protected areas and other regions with significant wildlife populations (e.g., alexander et al. 1993; laurenson et al. 1998; cleaveland et al. 2000) . knowing which diseases are present and their prevalences are not the only important pieces of information; it is also necessary to understand the ecology of the dogs to determine their potential impact on the neighboring wildlife. many protected areas are surrounded by buffer zones and transition areas, which are used for hunting and other forms of resource extraction (neumann 1997) . theoretically, buffer zones are considered areas where only lowimpact activities, compatible with protection of the central core of the reserve, occur (shafer 1999; smith 2003) . in practice, however, buffer zones tend to be used for multiple purposes and are often the site of both sustainable and unsustainable resource exploitation (sánchez-azofeifa et al. 2003) . in bolivia protected areas include national parks and integrated management areas (ima). the imas generally surround parks and may include or be contiguous with indigenous territories (tierra comunitaria de origen, or tco). each tco establishes its own zonation and management plan, defining regulations on resource extraction and land use that respect relevant national legislation. bolivia's kaa-iya del gran chaco national park and ima are contiguous, but nonoverlapping, with the isoseño tco, which is considered part of the "zona de influencia," or zone of influence, of the park (kaa-iya project 2001) (fig. 1) . although it does not have legal status that defines it as part of the park buffer zone, for practical purposes it functions as such. about 8000 isoseños inhabit 23 communities clustered along the rio parapetí in the tco and use this 3000-4000 km 2 region west of the park boundary for subsistence and commercial hunting (kaa-iya project 2001) . dogs are abundant in the communities, and may come into contact with wildlife in or near the communities. in addition, because nearly all hunting activities include dogs (noss & cuéllar 2001; kaa-iya project, unpublished data) and hunters from the communities utilize much of the tco, the transmission of infectious diseases from the domestic dogs to wildlife is a potential risk. previous research in bolivia indicates that many domestic dogs have been exposed to pathogens of conservation concern ( widdowson et al. 2002; fiorello et al. 2004) . many wild carnivores inhabit this area, including protected species, such as jaguars (panthera onca), ocelots (leopardus pardalis), geoffroy's cats (oncifelis geoffroyi), and other felids and canids, such as jaguarundis (herpailurus yagaurondi), pumas (puma concolor), crab-eating foxes (cerdocyon thous), and pampas foxes (pseudalopex gymnocercus) (taber et al. 1997; cuéllar 2000; maffei et al. 2003 maffei et al. , 2004 . the disease susceptibility of captive south american carnivore species to domestic dog diseases is known from the literature. for example, heartworm disease has been reported in jaguars and jaguarundis (otto 1974) ; canine parvovirus (cpv) has been reported in crab-eating foxes (mann et al. 1980; barker & parrish 2001) ; and canine distemper has been reported in a jaguar (appel et al. 1994 ) and a crab-eating fox (rego et al. 1997) . there is no reason to suppose that these species would not be susceptible in the wild; in fact, sarcoptic mange has been reported in free-ranging pampas foxes in the isoso (deem et al. 2002) . our goal was to further our understanding of disease and population ecology of hunting dogs in a national park buffer zone in south america. we used a combination of blood sampling, informal discussions with local residents, a questionnaire survey, and a large data set on hunting to study domestic dogs in indigenous communities of southeastern bolivia. our specific aims were to determine whether domestic dogs serve as potential reservoirs of disease agents pathogenic to wild carnivores and have regular opportunities to come into contact with wild carnivores or their environment. we developed a questionnaire to obtain basic information on the number, age, and mortality rates of domestic dogs. informal discussions were also conducted on an ad hoc basis, and the age, vaccination status, and hunting status of sampled dogs were recorded (when known). the questionnaire was administered orally in guaraní and spanish by researchers (including local assistants) during sampling (see below) or by a hunting monitor from iyobi. we focused on the community of iyobi, but also collected data opportunistically at hunting camps or on occasional trips to other communities. copies of the questionnaire are available from c.v.f. the kaa-iya project is a collaborative effort of the wildlife conservation society ( wcs) and the capitanía del alto y bajo isoso (cabi), an indigenous organization in bolivia that administers the kaa-iya del gran chaco national park, ima, and the isoseño tco. the kaa-iya project employs members of the communities to serve as hunting monitors as part of their mission to manage the natural resources of the area. data on hunting are collected to monitor harvests of game species, upon which the local people rely for protein. hunters who volunteer to participate provide their community hunting monitor with information on location, date, time, and duration of each hunt and on weapons used, number of dogs present, the weather, and number and characteristics (age, sex, condition) of each animal harvested. not all hunters in a given community participate, and those who do participate do not necessarily record complete data from every hunt. individual communities differ in their level of hunter participation; in some, all hunters participate, whereas in others very few participate . however, despite these drawbacks, the data set does contain useful information. based on hunts (n = 140) when researchers from the kaa-iya project accompanied local hunters, we estimated that between 25 and 30% of hunts were successful (kaa-iya project, unpublished data). therefore, the data collected on successful hunts likely represent about 25% of the total hunting effort by participating hunters in the area. extensive blood sampling of dogs was done in iyobi, rancho viejo, and yapiroa ( fig. 1) , three communities that vary somewhat in size, location, and economic base (table 1) (noss & cuéllar 2001) . iyobi and yapiroa, two of the largest communities (kaa-iya project, unpublished data), have a more diverse economy and their inhabitants are less likely than those in small communities to rely on seasonal migratory work and hunting. residents of rancho viejo, an intermediate-sized community, rely heavily on hunting during the months when seasonal agricultural work is unavailable. situated in the northern part of the isoso on the east bank of the river, rancho viejo and iyobi are more isolated than yapiroa. yapiroa is located farther south, on the west bank of the river, closer to the nearest town and railroad station. dogs living in yapiroa might be expected to have more contact with dogs from outside the isoso because this area is closer to main roads and it is not necessary to cross the river to reach them. accompanied by a community member, we walked from house to house, asking owners for permission to take blood samples from their dogs. to ensure that the antibodies detected were endogenous and not of maternal origin (greene 1998a) , only dogs 5 months of age and older were sampled. we drew blood from the jugular vein. on the few occasions when dogs were too fractious to handle, they were sedated with standard intramuscular doses of acepromazine (boehringer-ingelheim, ingelheim, germany). in addition to sampling in the villages, we opportunistically sampled hunting dogs at research stations in the buffer zone. these stations sometimes double as hunting camps, where hunters and their animals spend the night during longer hunting trips. if hunters came through when we were present, we spent the evening talking with them and sampling their dogs. blood was placed into serum separator tubes and centrifuged for 10 minutes. sera were drawn off, stored in cryotubes, and placed in liquid nitrogen. the samples were transported to the united states on ice and stored in a freezer at −80 • c until analysis. analyses were performed by commercial laboratories in the united states and switzerland (table 2) . with the exception of the sarcoptes scabiei test, assays for antibodies document infection at some point in the past. antibodies to s. scabiei wane within 2 months of the infection clearing, so when they are found they indicate current or very recent infection (lower et al. 2001 ). the assay for dirofilaria immitis, the causative agent of heartworm disease, detects antigen and therefore indicates active infection or a recently (3-5 months) cleared infection (goodwin 1998 ). we used statview (sas 1999) for all statistical analyses. unpaired t tests and analyses of variance (anova) were used for comparing ages for both sexes and across towns; chi-square tests were used to compare sex ratios across towns. we used anova to compare length of hunts and number of dogs and chi-square tests to compare the percentage of dogs with positive titers to different pathogens among different towns, differences between the sides of the rio parapetí, and to explore correlations among disease agents. where sample sizes were too small for chisquare tests, g tests were used. we also used g tests to compare percentage of male and female dogs with positive titers. we used unpaired t tests to compare the ages of dogs with positive titers for various disease agents. significance was accepted at p < 0.05. thirty-nine households from iyobi (slightly over 25%) completed the questionnaire, providing information on 155 dogs (table 3) . partially completed questionnaires were obtained from nine households from other communities. information on sex, age, vaccination status, and hunting status of dogs was obtained during sampling of 85 dogs (37 from iyobi, 28 from rancho viejo, 9 from yapiroa, and 11 from other communities; table 1 ). sex and age data on dogs obtained during sampling both including and excluding iyobi were compared with data obtained from the survey. the results were nearly identical and thus are not reported here. based on the surveys and discussions with residents, we concluded that essentially all households had dogs. the rare household without a dog resulted from the temporary absence of adult men-who took their dogs with them when they left to do seasonal work-or the recent death of dogs that had not yet been replaced. dogs in the isoso are all owned, and are considered necessary for hunting all terrestrial species except brocket deer (mazama gouazoupira). we did not collect data on the source of dogs, but informal discussions indicated that most dogs were received or purchased from friends or relatives in the isoso, with an occasional dog being purchased in nearby cities. there were on average 3.8 adult dogs/household (table 3 ) and 4.9 people/household (kaa-iya project, unpublished data). there was roughly one dog for every 1.5 persons. based on these estimates and the population census, a community the size of iyobi has approximately 522 dogs (table 1) . data from the questionnaires indicated that 17 of 161 dogs (11%) were vaccinated against rabies. during sampling, however, rabies vaccination status was recorded for 60 dogs, and only 2 of these (3%) were vaccinated. eightysix percent ( based on questionnaires) or 90% ( based on sampling data) of dogs were reported to hunt, which in a village of iyobi's size equals 448-470 dogs ( table 3) . the average age and annual mortality of the dog population indicated that turnover was quite high. the sex ratio of dogs indicated a male bias, although the average age of male and female adult dogs was similar (table 3) . when data from the questionnaire were compared with those collected during sampling, average age of dogs was nearly identical (mean ± sd: 3.1 ± 1.7 from questionnaire vs. 2.9 ± 2.3 from sampling, p = 0.945, unpaired t test). average age of dogs did not differ among communities ( p = 0.912, anova; data not shown), or between the east and west banks of the river ( p = 0.272, unpaired t test, data not shown). the average annual mortality of adult dogs, based on the number of dogs that owners reported dying in the past year, was 34%. average litter size was 4.2, and females were reported to have 1 litter/year. an average of 73% of puppies died as neonates. when questioned about the cause of death of puppies, dog owners mentioned diarrhea (48.1%), mange (44.4%), worms (25.9%), vomiting (22.2%), anorexia (3.7%), and infanticide (3.7%); 34.7% said they did not know. dogs of both sexes and all ages (starting at approximately 8-10 months of age) hunted; in addition, younger dogs often accompanied their dams into the forest on hunts, although their owners claimed that these dogs do not actively hunt. because these young dogs were not included when owners were asked how many hunting dogs they have, the estimate that 86-90% of dogs hunt is actually an underestimate of the number of dogs that go into the forest. eighty-two percent of hunting dogs were reported to hunt weekly or more often (i.e., a minimum of 367 dogs from iyobi entering the forest each week, or 52 dogs/day). data were available for 6466 hunts between august 1996 and november 2002. slightly less than half, or 42.5% (2745 hunts), had information regarding dog participation. for just under 12% of hunts, the number of dogs was recorded simply as "one or more." the vast majority (2633/2745, or 95.9%) of hunts included at least one dog, and the average number of dogs/hunt was 2.7 ± 1.6 (n = 2421). of hunts with dogs, 14.3% involved one dog, 33.1% involved two dogs, 25.7% involved three dogs, and 26.9% involved four or more dogs. successful hunts included significantly more dogs (2.8 ± 1.5, n = 2264) than unsuccessful hunts (2.2 ± 1.9, n = 157) (unpaired t test, p < 0.0001). information on duration of hunt was available for 3721 (57.5%) of hunts. most (92.9%) hunts lasted for <24 hours, and the average duration was 10 hours (fig. 2) . nearly half (47.7%) of all hunts were between 6 and 12 hours. the mean duration of hunts that included dogs (9.4 ± 9.4 hours) did not differ from the duration of hunts in which dogs did not participate (9.0 ± 8.7 hours) (unpaired t test, p = 0.6311). similarly, mean duration was not different for successful (9.5 ± 9.3 hours) and unsuccessful (9.5 ± 12.1 hours) hunts (unpaired t test, p = 0.9830). in general, as hunts got longer, more dogs were present (anova, p < 0.0001). sufficient quantities of serum for at least one analysis were collected from 98 dogs. the number of dogs sampled from each town is included in table 1 . not all dogs were tested for all disease agents because of a lack of serum and financial limitations. exposure to canine distemper virus (cdv) and cpv was extremely common (fig. 3) . positive titers to s. scabiei, canine herpesvirus, and canine coronavirus were present in 50% or more of dogs tested. all dogs tested positive for exposure to at least one pathogen. with the exceptions of toxoplasma gondii and canine adenovirus, the percentage of dogs positive for a given pathogen did not differ among towns on the east and west sides of the river. positive titers for t. gondii were more common on the east side of the river, but this was due to the fact that dogs positive for this agent were more commonly found in rancho viejo (48%) or a neighboring ranch (100%, compared to 26% in all other communities; p = 0.007). when these two locations were removed from the analysis, t. gondii titers did not differ between sides of the river ( p = 0.10). similarly, positive titers to adenovirus were very common in the village of yapiroa (73% vs. 25% in all other communities; p = 0.002). when yapiroa was excluded from the analysis, there was no difference between villages on the east and west sides of the river ( p = 0.230). the seroprevalence of most diseases increased with age. antibodies to t. gondii, cpv, adenovirus, coronavirus, and brucella canis were found more commonly in older dogs. this effect was particularly strong for coronavirus (fig. 4) . all four of the dogs negative for cpv were under 1 year of age, and three out of four of the dogs negative for cdv were <1 year old. there were no significant differences in disease prevalence between male and female dogs. three conditions must be satisfied for disease spillover from domestic to wild carnivores to occur: (1) the wild carnivores of interest must be susceptible to the disease agent, (2) the disease agent must be present-and preferably endemic-in the domestic carnivore population, and (3) there must be an ecological mechanism for contact between the two populations (or their infectious material) to occur. these conditions appear to be in place in the isoso. wild carnivore susceptibility to disease agents is known from the literature, and our results suggest that many diseases are maintained in the domestic dog population and that opportunities for contact between domestic and wild carnivores are plentiful. the seroprevalence of common canine diseases was quite high in domestic dogs in the isoso when compared with similar surveys performed in other regions. for example, in the serengeti area of tanzania, seropositivity to cdv ranged from 10 to 80%, with a higher proportion of positive dogs in the older age classes (cleaveland et al. 2000) . in the brazilian amazon, seropositivity to cdv and cpv was 9% and 13%, respectively, (courtenay et al. 2001) . a study in ethiopia found 100% seropositivity to cpv in 18 dogs sampled, but seroprevalence of other viruses was lower. for cdv it was approximately 32%, whereas for canine adenovirus it ranged from 0 to 90%, depending on the region (laurenson et al. 1998 ). antibodies to b. canis were found in 7% of stray dogs in são paulo, brazil (larsson et al. 1981) , and antibodies to leptospira spp. were found in 14% of dogs in southern bolivia (ciceroni et al. 1997) . a survey of dogs in northwestern bolivia found seroprevalences similar to those in the isoso for many canine pathogens . the presence of these disease agents-at least during the period of our study-appears certain. seropositivity was highest for cdv and cpv, the most common canine pathogens worldwide, and major threats to endangered wildlife elsewhere (mech & goyal 1993; laurenson et al. 1998; cleaveland et al. 2001; hedrick et al. 2003) . other viruses are also relatively common in this population. although these pathogens may be of less conservation concern than cdv or cpv, they may still represent a risk for wild carnivores (evermann et al. 2005) . first, because exposure to these less prevalent pathogens is not uniform, some dogs are becoming infected during their adulthood, and it is adult dogs that are most likely to encounter wildlife. second, coinfection with some viruses, including canine coronavirus and canine adenovirus, can cause much more severe disease than single infection (pratelli et al. 1999 (pratelli et al. , 2001 , and it may increase the amount and duration of viral shedding of one or both pathogens (pratelli et al. 2001) . most dogs had antibodies to more than one disease agent. although we have no data to demonstrate whether these are sequential or simultaneous infections, it is reasonable to assume that at least some of these dogs had simultaneous infections. in general, seropositive animals were found in all communities and on both sides of the river, although there were some interesting differences that may have a biolog-ical basis. the finding that antibodies to t. gondii were more common in dogs living near a cattle ranch may reflect their proximity to cats. domestic cats are rare in the isoso; however, several cats lived on or around this ranch. felids are the definitive host for t. gondii, and so the dogs living near the ranch may be more likely to be exposed to infectious cysts shed in cats' feces. although dogs may become infected from eating wild prey, they are rarely given meat by their owners and do not seem to hunt much on their own. it is also possible that they are being infected by cysts in the feces of wild felids, although this would not explain the higher seroprevalence in dogs near the ranch. regardless of how they are being exposed, these dogs share an environment with their human caretakers, who are also susceptible to this zoonotic pathogen. in contrast, there is no obvious reason why canine adenovirus exposure was more common in the village of yapiroa. this virus may persist in the environment and can be shed for prolonged periods of time in the urine of recovered dogs (greene 1998b) . it is possible that the environment in yapiroa is more contaminated with this virus than other areas. because the data we collected for this study were cross-sectional, it is possible that an outbreak was beginning in yapiroa and will continue to spread to other communities. if additional sampling is done in the future, we may be able to determine whether the pattern of higher exposure in yapiroa is persisting or whether it represented an epidemic wave that spread through the isoso. based on the data from other pathogens, it does not appear that the river is serving as a geographic barrier to disease transmission among domestic dogs. the finding that, for several pathogens, seroprevalence increased with age is typical for many disease agents (giesecke 2002) . the longer an individual organism is alive, the more opportunities it has to be exposed to pathogens. in the isoso, nearly all dogs that reach the age of 1 year will have been exposed to cdv and cpv (fig. 4) . this is reassuring because those that survive the infection are likely to be immune for several years, and given the short lifespan of most isoseño dogs, they are probably immune for life. thus, only a small proportion of hunting dogs would be exposed to pathogens as adults, and would be actively shedding virus during their working years. showing that antibodies to disease agents were common in the dog population at the time of sampling is not equivalent to demonstrating that these diseases are endemic in the population. however, we do have compelling evidence that isoseño dogs are ideal disease reservoirs (i.e., able to maintain virulent pathogens in their population). first, isoseño communities have an abundance of dogs. these dogs are infrequently vaccinated and receive no other preventive health care, an unsurprising finding in a place where people are often unable to obtain adequate health care for themselves and their families. second, the high mortality of dogs, and especially of puppies, suggests that mortality from disease is playing a large role in regulating the canine population. owners' descriptions of the clinical signs exhibited by puppies support the conclusion that many of the pups are suffering from infectious diseases. third, the apparent high turnover of the population-over 1000 puppies born just in iyobi each year-provides for many new susceptible hosts to contract and shed pathogens, thus preventing disease agents from dying out. contact between wild and domestic carnivores occurs regularly in this habitat. hunters with dogs capture and kill wild cats when encountered inside and outside communities, and presumably some cats escape after physical contact with dogs (noss 1998; cuéllar 2000; noss et al. 2003) . although hunters do not target foxes, they report frequent encounters with foxes on hunting trips, and foxes are observed near communities on a daily basis (kaa iya project, unpublished data; c.v.f., unpublished data). both local residents and researchers have observed foxes and wild cats enter communities to take chickens and goats (c.v.f., unpublished data). this anecdotal evidence, however, does not provide a quantitative measure of contact. using our questionnaire results and the hunting data set, we estimated the amount of time dogs were spending in the forest. if there are 8000 people in the isoso and 1 dog for every 1.5 persons, if 86% of dogs hunt, and if 82% of these dogs hunt at least weekly, the result is over 3000 dogs entering the forest each week in an area smaller than 4000 km 2 . if each of these dogs is in the forest for an average of 11 hours, the total is 33,000 dog-hours in the forest. even if only a small fraction of dogs were infectious at any given time, it nonetheless represents a large number of infectious hosts that have the potential to encounter susceptible wild carnivores. fifteen percent of all dogs sampled (only dogs >5 months old were sampled) were <1 year old. if only 5% of these young dogs were shedding virus, it amounts to over 20 dogs/week, or 220 infectious dog-hours. these numbers may overestimate the amount of time infectious dogs are likely to spend in the forest because very sick dogs are unlikely to go hunting. however, dogs seem to greatly enjoy hunting and so are highly motivated to follow their owners if they are able. dogs also shed virus before they are symptomatic and/or after clinical recovery. and, puppies that are not yet hunting often accompany their dams into the forest. these puppies are the most likely to be actively infected with pathogens, especially cdv and cpv. the hunting range of the isoso is between 3000 and 4000 km 2 . assuming that 3000 hunting dogs "occupy" this range each week, the dog density is between 0.75 and 1.0 dog/km 2 . however, the effective density is much higher, because dogs and wild carnivores are not evenly distributed across the landscape. isoseños and their dogs hunt by walking roads and trails, and camera-trap data confirm that wild felids ( jaguars, pumas, ocelots) spend a disproportionate amount of their time on roads and trails (maffei et al. 2003 (maffei et al. , 2004 . other carnivores, especially foxes, are commonly seen using roads, and carnivore scats are deposited along trails, presumably to mark territories. the potential for contact-especially indirect contactis greatly magnified because of the preference of both domestic and wild carnivores for roads and trails. roads represent an area of concentrated use, whereas wildlife is likely to encounter dogs and the infectious materials that dogs may leave behind. disease spillover can only occur if infected domestic dogs are encountering susceptible wild carnivores. our results demonstrate that domestic dogs are commonly infected with pathogens and that they regularly contact wild carnivores. although some serology data are available from wildlife (fiorello 2004) , as yet no data are available regarding the pathogenicity of the local viral strains for wild carnivores in this habitat or on whether the conservation biology volume 20, no. 3, june 2006 strains circulating in the dog population are in fact the same as those circulating in the wild carnivore populations, as has been shown in other systems (battilani et al. 2001) . research on those questions is urgently needed to inform conservation management planning. however, if a cautionary stance is adopted and the evidence from the literature that these carnivores are susceptible to canine pathogens is accepted, it must be acknowledged that the possibility for disease spillover in the isoso exists. the isoseños rely heavily on their dogs during hunting, which is a major contributor to their economy. it is therefore likely that domestic dogs will continue to live in the isoso and enter the forest and the habitat of wild carnivores, and that domestic and wild carnivores will continue to overlap in and around communities. although hunters apparently do not regularly move between the tco and the park, wild carnivores (especially jaguar and puma) presumably do-and when they do, they almost certainly use the same roads and trails that hunting dogs are frequenting. these dogs represent a risk to wildlife in the buffer zone of kaa-iya del gran chaco national park, and in the park itself. additional work is needed to investigate methods of reducing the disease risk to the wild carnivores of the area. such methods may include vaccination of domestic dogs, prohibition on ill dogs participating in hunting activities, and sterilization campaigns to limit the number of dogs in the community. serologic survey for selected microbial pathogens in african wild dogs (lyacon pictus) and sympatric domestic dogs 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disease ecology of wild and domestic carnivores in bolivia seroprevalence of pathogens in domestic carnivores on the border of madidi national park the role of disease in carnivore ecology and conservation. pages 443-466 in modern infectious disease epidemiology the serologic diagnosis of heartworm infection in dogs and cats immunoprophylaxis and immunotherapy. pages 717-51 in infectious canine hepatitis and canine acidophil cell hepatitis canine parvovirus enteritis, canine distemper, and major histocompatibility complex genetic variation in mexican wolves plan de manejo: parque nacional y area natural de manejo integrado kaa iya del gran chaco. published by capitanía del alto y bajo isoso, fundación ivi iyambae, wildlife conservation society, ministerio de desarrollo sostenible planificación/servicio nacional de areas protegidas good medicine for conservation biology: the intersection of epidemiology and conservation theory canine brucellosis in são paulo: serologic survey of kennel and stray dogs disease as a threat to endangered species: ethiopian wolves, domestic dogs, and canine pathogens evaluation of an elisa for serological diagnosis of sarcoptic mange in dogs jaguar and other mammal camera trap survey cerro ii, cerro cortado field camp (19 • 31 36 s, 61 • 18 36 w), kaa-iya del gran chaco national park 1000 jaguars in bolivia's chaco? camera trapping in the kaa-iya national park montali. 1980. canine parvovirus infection in south american canids detecting disease and parasite threats to endangered species and ecosystems canine parvovirus effect on wolf population change and pup survival primitive ideas: protected area buffer zones and the politics of land in africa el monitoreo comunitario de cacería en el izozog: datos preliminares community attitudes towards wildlife management in the bolivian chaco hunter self-monitoring as a basis for biological research: data from the bolivian chaco occurrence of the heartworm in unusual locations and in unusual hosts severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type 1 and canine coronavirus fatal coronavirus infection in puppies following canine parvovirus 2b infection distemper in brazilian wild canidae and mustelidae: case report rabies in zimbabwe: reservoir dogs and the implications for disease control integrity and isolation of costa rica's national parks and biological reserves: examining the dynamics of land-cover change sas institute. 1999. statview 5.0.1. sas publishing us national park buffer zones: historical, scientific, social, and legal aspects land-cover assessment of conservation and buffer zones in the boswas natural resource reserve of nicaragua a new park in the bolivian gran chaco-an advance in tropical dry forest conservation and community-based management epidemiology of urban canine rabies managing disease threats to wild mammals we gratefully acknowledge the enthusiastic field assistance of r. cuéllar, c. cuéllar, n. trepp, j. barrientos, j. ity, f. mendoza, f. soria, l. gonzález, f. leaños, j. perú, e. ity, j. segundo, and a. arambiza. logistical support was provided by the office of cabi, d. rumiz, o. vitingay, l. samuels, r.cuéllar, w. ayala, a. garcia in santa cruz, and l. starr, v. greco, k. poppe, and a. yang in new york. c. padoch helped with the questionnaire design. l. gonzález administered many of the questionnaires. k. mcfadden, m. gompper, and two anonymous reviewers made helpful comments on the manuscript. e. dubovi supervised the laboratory analyses at cornell. the wildlife conservation society ( wcs) field veterinary program, the kaa-iya project ( wcs, cabi), the wcs jaguar conservation program, and cerc provided financial support. we especially thank all of the parabiologists and the isoseño community as a whole for their participation in and support of this project. this publication was possible thanks in part to the support of the u.s. agency for international development (usaid)/bolivia cooperative agreement 511-a-00-01-00005). the opinions expressed here represent the authors and do not necessarily reflect the criteria of usaid. key: cord-298155-ou8vjogc authors: silva, vanessa; silva, joana; gonçalves, margarida; brandão, carlos; vieira e brito, nuno title: epidemiological survey on intestinal helminths of stray dogs in guimarães, portugal date: 2020-07-27 journal: j parasit dis doi: 10.1007/s12639-020-01252-2 sha: doc_id: 298155 cord_uid: ou8vjogc the new legislative framework on animal welfare brought increased responsibilities to municipal shelters, in particular in the collection of stray dogs, their sterilization and future adoption. these centers quickly became overcrowded, leading to high parasitism environmental contamination, to the easy spread of parasitic infections and to increased risks to public health. the prevalence of intestinal parasites was evaluated by examination of dog faecal sample, in the municipal control animal centre of guimarães (north portugal), identifying risk factors and transmission to man. the overall prevalence of gastrointestinal helminths was 57.2% (95% confidence interval 41.3–71.9%) and observed helminths of the gastrointestinal tract were recorded: ancylostoma caninum (33%), toxocara canis (29%), dipylidium caninum (6%), capillaria spp. (3%), trichuris vulpis (1.66%). it is important to point out that young dogs were significantly infected more frequently (p ≤ 0.1) than non-sterilized females and the higher occurrence of nematode infection occurred at the new arrival of stray dogs, in the third collection. with impact on public health, the higher prevalence (p ≤ 0.1) of t. canis in young dogs suggests the existence of real risk for human infection and demonstrate the necessity for a parasite control programme reinforcement at the municipal dog shelter. parasitic diseases are often underestimated in the eyes of civil, medical and scientific society, but they are of utmost importance, and comparable to infectious diseases. in portugal exists an increasing number of stray animals, both in urban and rural areas, where, during 2018, more than 40 thousand stray animals were collected from the streets and, in 2019, in an estimated population of 891.788 dogs, only 539.025 were identified and registered (dgav 2019; fecava 2019) . this represent a substantial public health risk factor, from an epidemiological point of view, due to the strong environmental contamination of stray dog's feces, without any deworming program and with a high probability of carrying pathogens and parasites, easily transmitted to man. as a potential reservoir of endoparasites, the contamination of public places increases the susceptibility of others animals and humans, in a distressing zoonotic chain (dado et al. 2012) . ''one health'' is a worldwide strategy whose purpose is the expansion of interdisciplinary communications, which link human, animal and environmental health. not all the mentioned categories are independent and, in fact, parasitism in pets, directly affects the community health, either through direct or environmental contact. it is critical that the control, both of internal and external parasites, occurs periodically, avoiding the parasitic zoonosis transmission (neto and coelho 2016) . as an animal shelter, the control animal centre (cac) has an important role in public health in the collection of stray animals, in their sterilization, in the implementation of medical and prophylactic control measures, as deworming, and in the promotion of adoption policies. in 2018, the law that forbids the euthanizing of pets due to economic incapacity or overcrowding was enacted (dre 2019). however, the law brought many inconveniences to the municipalities and the number of stray dogs has been progressively rising, leading to the overpopulation and the reduction of housing conditions. there are, in most cases, several animals living in tiny cages, inducing higher parasitism prevalence (bresciani et al. 2013) , especially when new animals arrive. in view of the high number of parasites with zoonotic potential, the awareness of the society is extremely important, as well as the correct provision of information to future guardians seeking to adopt animals at the cac. this lack of information generates great concern, since naivety in relation to the topic promotes an inappropriate use of deworming which, consequently, induces the parasitism resistance and inefficiency in parasite load elimination (pereira et al. 2016) . the most commonly detected dog parasites of the gastrointestinal tract are dipylidium caninum, toxocara canis, ancylostoma spp., trichuris vulpis, taenia spp. and cystoisospora spp. (mateus et al. 2014) . although less frequent, hammondia heydorni and cryptosporidium spp. are also observed as well as neospora caninum, a mandatory intracellular protozoan, is frequently found in the blood system (funada et al. 2007; taylor et al. 2016 ). there are no specific studies about parasitism in stray dogs gathered in official shelters, although a higher prevalence of a. caninum, t. canis, t. vulpis, cryptosporidium spp. and strongylidium spp. in portugal was reported (silva 2010; melo and lebre 2011; otero et al. 2014) . the main objective of this study is to determine the prevalence of gastrointestinal helminthes in stray dogs in guimarães, with especial attention to potential zoonotic diseases. this epidemiological study was carried out based on the collection of dogs faecal samples from the guimarães cac, a municipal shelter. the cac has a maximum accommodation capacity of 100 animals and perform the sterilization and the adoption of stray animals. an external and internal deworming plan is established, consisting in the administration of milbemycin oxime and praziquantel, in an age frequency dependence. the study, outlined for 1 year but interrupted by the covid-19 pandemic, occurred for a period of 3 months, from november 2019 to january, 2020 when the cac average population was 78 animals. a sample of 25% of the animals was considered and divided into the three cac groups: young dogs (under the age of 1 year); sterilized males and females (over the age of 1 year); and non-sterilized females (over the age of 1 year). feces, 7 samples from each group, were collected every 3 weeks, stored at 4°c and processed within 24 h in the department laboratory. fecal samples were first examined for macroscopic observation and, following, faeces were processed using the techniques of concentration by sedimentation (ritchie 1948) and flotation by centrifugation-flotation (maff 1986; bowman et al. 2004 ) and counted in a mcmaster chamber (vasconcelos-nóbrega et al. 2017 ). identification of eggs and larvae was based according to the literature keys and guidelines (yamaguti 1961; menezes et al. 2013) . analyses were performed with spps ò 22 software for windows. pairwise comparisons between categories of the same independent variable incorporated bonferroni's correction. a p value b 0.1 was considered as statistically significant. macroscopic analysis results are presented in table 1 . only 15 of the 63 total samples presented a non-normal consistency (6 liquids and 9 pasties), and relatively to color, 15 samples had a light brown color, including 6 samples with undigested food particles. no parasites adult forms were observed. feces with a pastier consistency were identified (table 1) , more evidently in the third collection and, in particular, in younger animals. in this specific group, was registered color change (light brown) and the presence of poorly digested foods. concerning the microscopic analysis (table 2) , several eggs parasitic forms were identified. the overall prevalence of parasitic infection with parasites was 57.2% [(95% confidence interval (ci) 41.3-71.9%)] and the most frequently observed parasite was ancylostoma caninum (33%), followed by toxocara canis (29%) and dipylidium caninum (6%). in line with the macroscopic analysis results, an important increase in parasitism in the third collection was verified, particularly in younger animals (86% prevalence), but also in sterilized ones (71.4% prevalence). at this third sampling, a. caninum (present in all samples) and t. canis revealed a higher prevalence (86% and 43%, respectively), in terms of group and/or reproductive status. in addition to the high parasite load verified (particularly, a. caninum 1146 fec) in the third collection, the presence of eggs of trichuris vulpi (1fec), capillaria spp. (1fec) and d. caninum were identified also mainly in young dogs. the presence of d. caninum (329 fec) was observed, only in this collection, in all groups analyzed, but particularly in sterilized animals (29% prevalence). regarding the first collection, a second-form l2 larvae of t. canis was identified in the group of non-sterilized females. also in this group, but in the second collection, eggs of capillaria spp. were observed in a small infestation (1fec). the mean and standard deviation parasite load values (fec) obtained by group and collection are observed in fig. 1 , stressing the high values verified in the youngest animals and third collection, comparing to the lowest levels of adult animals' infestation (sterilized or not), with the exception of the third smf sample. significant differences (p b 0.1) were found among collections (table 3) , with higher values between the third (73.190 fec), the first (2.143 fec) and the second (10.762 fec) collection, as well as groups with higher (p b 0.1) parasite load value in the youngest (66.81 fec) comparing to the nsf (3.38 fec). the average values (fec) per group, were estimated and higher parasite load of t. canis (10.62 ± 15.66 fec) and d. caninum (55 ± 156.57 fec) was found in younger animals while a. caninum (4.52 ± 20.271 fec) showed higher levels in sterilized animals. t. canis also presented significant differences between groups (p b 0.05), with a superior infestation (109) in the youngest animals (10.62 fec) comparing to the sterilized (1.14 fec) and nonsterile (1.71) groups. natural transmission of parasitic infections from dogs to man may occur, directly or indirectly, via environmental factors and represent a potential public health risk, particularly to individuals with close contact with those animals (pullola et al. 2006; becker et al. 2012; dado et al. 2012; zanzani et al. 2014) . similar values (54.3%) were found by katagiri and oliveira-sequeira (2008) in stray and domiciled dogs in brasil, also in stray dogs in nigeria (52.6%, okoye et al. 2011 ), malaysia (48%, mahdy et al. 2012 and canada (21%, joffe et al. 2011) . higher prevalence was reported, in stray dogs, in iran (86%, emamapour et al. 2015) , méxico (85%, eguía-aguilar et al. 2005) , south africa (76%, minner et al. 2002) , spain (71%, martínez-carrasco et al. 2007 ) and poland (68%, bajera et al. 2011) . our results indicate the requirement, in the official dog shelters, of an effective anti-parasite control programme, due to the continuous collection of stray dogs, with no health control measures and, because of their habits, exposed to natural infections. shelter dogs are exposed to a greater parasite load, to less effective anti-parasite treatments, and have a poorer nutritional status. this implies a change in shelter's routine, such as the entry of new animals, particularly pups, which has a direct influence on the increase in the parasite load and requires more frequent hygiene and prophylaxis measures, as quarantine or deworming. pups are infected by vertical transmission, transplacental and/or trans-mammary as well as horizontal transmission through the ingestion of embryonated eggs from the environment or ingestion of larvae via vertebrate and/or invertebrate paratenic hosts (overgaauw and van knapen 2013) . the t. canis trans-placental infection route results in egg excretion 16 days after parturition and lactogenic transmission, more limited, continues to occur for 5 weeks, shedding, after infection, millions of eggs per day into the environment (lloyd and morgan 2011) . in fact, parasitic infections were observed specially in the third collection, in young animals that had recently arrived and were dewormed, revealing a severe environmental contamination with a higher risk of zoonotic transmission from dogs. given this prevalence in pups, specific interventions with a focus in animal birth control, parasites control programs and public education to take wise action relating to the parasites and pets, need to be reinforced. moreover, animal shelters facilitate spread of gastrointestinal parasites to incoming animals and shelter staff if there is overcrowding and frequent exposure to a contaminated environment (raza et al. 2018) . according to similar results in different studies, the most prevalent species observed were a. caninum, and t. canis, widely known as potential zoonotic agents (emamapour et al. 2015; cociancic et al. 2018; suganya et al. 2019) . a. caninum is the primary hookworms' specie infecting dogs worldwide (traversa et al. 2014 ). hookworms may occur in dogs of all ages, independently of sex or season (coggins 1998) , even when under regular control programs (sager et al. 2006) , as verified in our study, where no significant difference were obtained between groups. the zoonotic importance of a. caninum, a historical association of humans, dogs and hookworms, resides in that its larvae survive in the environment for several months (shepherd et al. 2018) . adult dogs can become infected with environmental larvae or when hypobiotic stages are re-activated by drivers of stress (bowman et al. 2004) . hypobiotic larvae may survive for years in the tissues of adult dogs and when reactivated during oestrus and in the last 2-3 weeks of pregnancy, transmitted via milk to the litter (little et al. 2009; traversa et al. 2014) . although has been decreased significantly over time, presumably due to routine use of broad-spectrum anthelmintics, t. canis is the primary roundworm specie infecting dogs worldwide (robertson and thompson 2002) . eggs of t. canis are very resistant and can withstand harsh environmental conditions and is estimated that toxocara eggs contamination soil may be more than the 90% of the investigated areas worldwide (kirchheimer and jacobs 2008) . t. canis is more prevalent in puppies and can be fatal, especially when there is heavy prenatal infection (overgaauw 1997) . pups are infected in utero by reactivated somatic larvae of t. canis from the mother from day 42 of gestation resulting in egg excretion * 16 days after parturition (traversa et al. 2014) . puppies can also be infected through the transmammary route (robertson and thompson 2002) . once infected, pups shed millions of eggs per day into the environment, depending on the intensity of t. canis infection and host immune status (glickman and schantz 1981) . in our study, significant differences were observed between young and adult dogs infected with t. canis. these results are consistent with other findings revealing that adult worm infections are generally less common in dogs with less than 6 months of age, and that faecal egg counts are much lower than in pups (papazahariadou et al. 2007; xhaxhiu et al. 2011) . d. caninum is the most common cestode in the world, especially in dogs, frequently infested by fleas and biting lices and with a higher prevalence in adult animals (raza et al. 2018) . humans can become infected and very young children are the ones most often affected, associated with diarrhea and abdominal pain (molina et al. 2003) . the prevalence observed in this study is significantly lower than in others reported by different authors (xhaxhiu et al. 2011; emamapour et al. 2015) and was verified in all ages but only in the third collection, what can be attributed to an infestation by the eventual entry of new stray dogs. zoonotic parasitosis is a serious public health problem and parasitism in stray and shelter dogs is a relevant indicator of, both, the deficiencies in the sanitary and ecological conditions of their hosts, of the socio-environmental conditions and hygiene practices of the population (cociancic et al. 2018) . in order to reduce environmental contamination and parasitism in the shelter, the implementation of sanitation measures is mandatory, such as quarantine of new stray dogs collected or more regular faecal examination, and the appropriate use, particularly in terms of dose and frequency, of deworming agents, according to the animal's weight and the shelter parasitic load (raza et al. 2018) . anthelmintic therapy should be accompanied by flea control programs to minimize transmission of d. caninum 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shelter dogs: occurrence, pathology, treatment and risk to shelter workers an ether sedimentation technique for routine stool examination enteric parasitic zoonoses of domesticated dogs and cats coprological study on intestinal helminthes in swiss dogs: temporal aspects of anthelmintic treatment of dogs and hookworms: man's best friend and his parasites as a model for translational biomedical research. parasites vectors 11:59 rastreio de parasitas gastrointestinais, pulmonares, cutâneos e musulares em canídeos domésticos e silvestres no norte de portugal. (unpublised doctoral dissertation) prevalence and molecular characterization of zoonotic helminths in dogs veterinary parasitology, 4 a edn environmental contamination by canine geohelminths abc series on diagnostic parasitology part 2: the mcmaster method principal intestinal parasites of dogs in tirana intestinal parasites of owned dogs and cats from metropolitan and micropolitan areas: prevalence, zoonotic risks, and pet owner awareness in northern italy acknowledgements the authors are grateful to all the guimarães municipality and cac collaborators, in particular to the vmd msc guida brito, for providing necessary research facilities to carry out this work, especially in the sample collections and animals handling. we also want to extend our thanks to ms. andreia silva for the technical and laboratory support. to vmd msc elsa machado, from food and veterinary general directorate, our gratitude to the continuous support and incentive of this research. key: cord-008085-3ihuqvei authors: thomas, william b. title: nonneoplastic disorders of the brain date: 2005-07-06 journal: clin tech small anim pract doi: 10.1016/s1096-2867(99)80030-9 sha: doc_id: 8085 cord_uid: 3ihuqvei computed tomography (ct) and magnetic resonance imaging (mri) are helpful in the diagnosis of many nonneoplastic brain disorders in the dog and cat. the ability of ct and mri to depict normal and abnormal anatomy facilitates the identification of developmental anomalies, including hydrocephalus, chiari malformations, arachnoid cysts, and cerebellar hypoplasia. these imaging modalities also allow the detection of hemorrhage and infarction and are therefore useful in the evaluation of spontaneous cerebrovascular disorders and head trauma. finally, many inflammatory diseases, such as encephalitis, brain abscess, and parasite migration, cause abnormalities detectable by ct and mri. although more research on the imaging features of specific nonneoplastic brain disorders is needed, current information indicates that ct and mri are useful in the management of these disorders. imaging strategies for ct and mri are similar to those described for intracranial neoplasia. (refer to the article entitled "intracranial neoplasia" by kraft and gavin in the may 1999 issue.) throughout this chapter, the mri features of the various disorders are those depicted with spin echo imaging. readers should refer to the article entitled "advanced imaging concepts: a pictorial glossary of ct and mri technology" by tidwell and jones in the may 1999 issue for a description of other pertinent mri techniques such as gradient echo and inversion recovery pulse sequences, magnetic resonance angiography, and diffusion/perfusion imaging. disorders of brain development ecent advances in imaging techniques have greatly improved the ability to detect pathologic processes in the brain, localize lesions precisely, and predict the type of disease more accurately than ever before. 1 although most reports of computed tomography (ct) and magnetic resonance imaging (mri) of brain disease in veterinary medicine have focused on the diagnosis of brain tumors, these imaging methods are also valuable in the evaluation of various nonneoplastic brain diseases. the purpose of this article is to provide an overview for imaging some of the more frequently encountered nonneoplastic diseases of the brain. clinical features of each disease are included because imaging can supplement but never replace a careful history and thorough physical and neurological examination. typical pathological changes are also mentioned, because ct and mri reflect gross and microscopic pathology and an understanding of the expected pathological changes can be very helpful in predicting imaging findings. ideally, a review article such as this would be based on systematic research and clinical data. unfortunately, the current literature on imaging of nonneoplastic brain disease in veterinary patients is fairly sparse. therefore, the information presented in this article is based on the limited data regarding veterinary patients supplemented by the much more complete information on comparable diseases in human patients, as well as my own experience. as more information becomes available, the conclusions in this article may have to be modified. hydrocephalus is a pathological condition in which there is accumulation of cerebrospinal fluid (csf) within the cranium. 23 this usually occurs within the ventricular system (internal hydrocephalus) but can involve the subarachnoid space (external hydrocephalus)) hydrocephalus is not a specific disease, but rather a multifactorial disorder with a variety of pathophysiological mechanisms. csf is produced at a constant rate by the choroid plexuses of the lateral, third, and fourth ventricles; the ependymal lining of the ventricular system; and blood vessels in the subarachnoid space. the csf circulates through the ventricular system into the subarachnoid space, where it is absorbed by arachnoid villi. 3 hydrocephalus can be caused by a blockage of the flow of cse called obstructive hydrocephalus, or secondary to decreased volume of brain parenchyma, termed hydrocephalus ex vacuo. (increased formation of csf is virtually never a cause of hydrocephalus.) a number of conditions, such as infarction and necrosis, can result in decreased volume of brain parenchyma and hydrocephalus ex vacuo. obstruction to csf flow can occur anywhere along the pathway from its formation to the site of absorption in the cranial and spinal arachnoid villi. obstruction within the ventricular system or at its outflow through the lateral apertures is called noncommunicating hydrocephalus because the ventricular system does not communicate with the subarachnoid space. obstruction within the subarachnoid space or at the level of absorption in the arachnoid villi is called communicating hydrocephalus. ~ hydrocephalus can be classified further as congenital or acquired. congenital hydrocephalus is most common in toybreed dogs. the causes are diverse and include genetic factors, developmental anomalies, and intrauterine or perinatal infection or bleeding in the brain. congenital hydrocephalus may be associated with a wide range of other nervous system anomalies, including meningomyelocele, chiari malformation, dandy-walker syndrome, and cerebellar hypoplasia. clinical signs of congenital hydrocephalus include an enlarged, domeshaped head with persistent fontanelles and open cranial sutures. neurological deficits include abnormal behavior, disturbed consciousness, ataxia, circhng, blindness, seizures, and vestibular dysfunction. in mature dogs, diseases such as tumors and inflammatory diseases can cause acquired hydrocephalus. neurological deficits are similar to those m puppms, but if hydrocephalus develops after the cranial sutures have closed, malformation of the skull does not develop. 2, 3 ct and mri allow accurate assessment of ventricular size, extent of cortical atrophy, and the presence of any focal lesions that may account for the hydrocephalus. imaging is also useful in monitoring patients after surgical placement of ventriculoperitoneal shunts used for treatment. changes in ventricular size can be monitored, and the presence of complications such as subdural hematoma or hygroma can be evaluated. 4 ventricular size is usually assessed subjectively, noting the progressively greater proportion of the intracranial volume occupied by the lateral, third, and/or fourth ventricles 4,5 ( fig 1) . several investigators have provided quantitative measurements. when measured at the level of or caudal to the lnterthalamlc adhesion, hudson et al 6 state that lateral ventricles are considered enlarged ff lateral ventncular height exceeds 0.35 cm or the ratio between the height of the lateral ventricle and the width of the cerebral hemisphere (ventriclehemisphere ratio) exceeds 0.19. spaulding and sharp 7 consider the lateral ventricles enlarged if the ratio between lateral ventricular height and the dorsoventral height of the cerebral hemisphere exceeds 0.14. however, there is a poor correlation between clinical signs and ventricular size, and symmetric or asymmetric enlargement of the lateral ventricles is relatively common in normal adult dogs and puppies. 6q° therefore, diagnosis of hydrocephalus must be based on clinical features, not just ventricular size. hydrocephalus ex vacuo can be distinguished from obstructive hydrocephalus based on enlarged cortical sulci and subarachnoid space secondary to atrophy of brain parenchyma (fig 2) . in obstructive hydrocephalus, the site of obstruction may be identified by dilatation of csf spaces proximal to the obstruction, and normal or collapsed spaces distally. for example, obstruction at the level of the third venmcle would be expected to result in enlarged lateral ventricles but no enlargement of the mesencephalic aqueduct and fourth ventricle (fig 3) . dilatation of all the ventricles and the subarachnmd space implies an obstruction at or near the arachnoid vilh. unfortunately, this simplisuc approach has limited accuracy. for example, 25% to 35% of human patients with communicating hydrocephalus have little or no &latation of the fourth ventricle. 4 obstructing masses, such as tumors, granulomas, and cysts, may be identified, especially on postcontrast images (fig 3) . mri is more sensitive than ct in showing small focal lesions, especially those in the caudal fossa) periventricular edema may be identified in some patients with hydrocephalus. experimentally, acute obstructive hydrocephalus in dogs causes edema starting at the dorsolateral angles of the lateral ventricles and spreading into the adjacent white matter, n on ct, this is evident as blurring or loss of the normally sharp ventricular margins. 4 the edema is best appreciated on t2-welghted mri as increased intensity compared with normal white matter. 1~ periventrlcular edema is most frequently associated with acute hydrocephalus and increased intraventricular pressure, rather than chronic, relatively compensated hydrocephalus with normal intraventricular pressure. 4 imaging techniques make it possible to readily identify ventriculomegaly but may give little clue as to its clinical significance. it is therefore necessary to interpret the finding of ventriculomegaly in context with clinical features. chiarl described four unrelated types of malformations of the brainstem and cerebellum in human patients. the most common is chiari i malformation, which consists of caudal displacement of a portion of the cerebellum through the foramen magnum into the cervical portion of the vertebral canal. the cause is an underdeveloped occipital bone that induces overcrowding of the caudal fossa, which is accommodated by caudal displacement of the cerebellum. 12 many human patients with this malformation develop hydromyelia (a dilatation of the central canal of the spinal cord that is lined by ependyma) and/or syringomyelia (an accumulation of fluid within the spinal cord that is not lined by ependyma). the term syringohydromyefia is often used because it can be &fficult or impossible to tell whether the cavity is lined with ependyma except at necropsy. syringohydromyelia in chiarl i malformation is caused by obstruction of csf flow at the foramen magnum. the brain expands as it fills with blood during systole, inducing a pressure wave in the csf that is accommodated in normal subjects by rapid movement of csf from within the skull to the vertebral canal. with obstruction to rapid movement of csf through the foramen magnum, the caudal portion of the cerebellum moves downward with each systolic pulse, acting as a piston on the surface of the spinal cord. this relentless compression of the spinal cord propels csf into and within the syrinx. 13 some pauents also have hydrocephalus attributed to obstruction of csf flow in the crowded caudal fossa. clinical signs of chiari i malformation in human beings can develop in childhood or adulthood and include head and neck pain, myelopathy, or encephalopathy. 12, 13 a similar malformation has been reported m adult dogs with neck pain and tetraparesls. 1~a5 diagnosis is best made on sagittal mri of the craniocervmal junction (fig 4) . the caudal portion of the cerebellum is displaced into the vertebral canal to a variable degree3 4, 16 the cerebellomedullary cistern is small or absent and the caudal fossa may appear subjectively overcrowded. hydrocephalus and syringohydromyelia may be evident. 14-16 syringohydromyelia often involves the caudal cervical or cranial thoracic segments, so this entire region should be imaged. all patients with syrmgohydromyelia should have imaging of the craniocervical region to rule out a chiari i malformation or other obstruction of csf flow. 16 chiarl ii malformation, also known as arnold-chiari or cleland-chari malformation, involves caudal displacement of the brainstem and cerebellum through an enlarged foramen magnum. the fourth ventricle is elongated and extends caudal to the foramen magnum, and the brainstem may be kinked. 16 chiari iii malformation is displacement of the cerebellum through a cervical spina bifida resulting m a cervmal encephalocele. chiari ii and iii malformations are not well described in small animals. chiari iv malformation is severe cerebellar hypoplasia and is discussed separately.17 in human patients, the dandy-walker complex is a group of malformations consisting of an enlarged caudal fossa, hypoplasia of the cerebellar vermis, and cystic dilatation of the fourth ventricle that nearly fills the caudal fossa. the cerebellar hemispheres are usually hypoplastic as well. other anomalies are also common, including hydrocephalus, hypoplasia of the corpus callosum, and syringohydromyelia. dandy-walker malformation is caused by outflow obstruction of the fourth ventricle in the developing embryo. this causes dilatation of the fourth ventricle, which enlarges upward between the developing cerebellar hemispheres, preventing their fusion. when the cerebellar hemispheres do not fuse, the vermis does not form. 16 analogous malformations have been described in the dog and cat. ~<18-21 in contrast to human patients, most dogs and cats with this syndrome do not have obvious enlargement of the caudal fossa, ls-21 neurological deficits typically occur at a young age and primarily reflect cerebellar dysfunction, including ataxia, hypermetria, intention tremor, and vestibular dysfunction, ls-21 on ct and mri, dandy-walker malformation is characterized by an enlarged caudal fossa filled by an enormous fourth ventricle and a small cerebellum. 14,16,2°,21 the cerebellar vermis may be absent, producing a cleft in the middle of the cerebellum. hydrocephalus may also be evident.20.21 a variety of in utero msults and heritable defects may cause failure of normal development of the cerebellum, resulting in a hypoplastic or absent cerebellum. the most common cause in cats is in utero or permatal infection with the feline panleukopenia virus. cerebellar hypoplasia has also been reported in dogs, in which a heritable basis has been suspected in some cases, but in utero infections cannot be excludedy ,23 single or multiple animals within a litter may be affected. there is nonprogressive ataxia, hypermetria, and intention tremor, first apparent at about the time the animal begins to walk. ct or mri shows a small or absent cerebellum. the remainder of the caudal fossa is filled with cse other anomahes, such as hydrocephalus, may also be apparent. = arachnoid cysts (also called intra-arachnold cysts) are accumulations of fluid surrounded by a membrane resembhng the arachnmd mater. they carl develop in the subarachnold space anywhere along the neuraxis. congenital arachnoid cysts are developmental anomalies originating from a splitting or duplication of the arachnoid mater. the cyst wall is formed by several layers of arachnoid cells. acquired arachnoid cysts may result from postinflammatory loculauon of csf caused by trauma, infection, or hemorrhage. 24 in human beings, congenital intracranial arachnoid cysts may be asymptomatic or cause neurological deficits as the cyst progressively enlarges and compresses adjacent neural structures or interferes with csf circulation. enlargement of arachnoid cysts occurs because of fired production by the cyst wall or an anatomical communication functioning as a oneway valve between the cyst and the subarachnoid space. 2~ intracranial arachnoid cysts have been reported in the dog and cat. z5, 26 the most common locatmn is the subarachnoid space between the cerebellum and the tentorium cerebelli. clinical signs may occur in immature or mature animals and include smzures, paresis, abnormal behavior, and cranial nerve deficits. in some patients, the cyst is an incidental finding on imaging. 26 on ct, the cyst has well-defined margins and contains fired that is isodense wath csf (fig 5) . there is mal, or intraventricular. epidural, subdural, and subarachnoid hemorrhages are often associated with trauma and are discussed later. intraparenchymal hemorrhage can be primary or secondary and can extend into the ventricular system. the cause of primary intraparenchymal hemorrhage is incompletely understood. people with this disorder often have systemic hypertension and fibrinoid degeneration of arteries in the b r a i n y although intracranial hemorrhage caused by hypertension is poorly documented in dogs and cats, primary hypertension and hypertension secondary to other disorders, such as renal disease, hyperadrenocorticism, and hyperthyroidism, are well recognized. 28 these animals may be predisposed to cerebrovascular disease and intracranial hemorrhage. 28 secondary causes of intracranial hemorrhage include trauma, infarction, congenital vascular malformations, intracranial tumor, vasculitis, and coagulopathies. [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] the clinical signs of intraparenchymal hemorrhage consist of a sudden onset of neurological deficits referable to a focal brain lesion. signs may progressively worsen as the hematoma expands, compressing adjacent neural structures and increasing intracranial pressure. 30-33 36-39 computed tomography ct is exqmsitely sensitive to acute hemorrhage. because the attenuation of x-rays is primarily attributable to the globin portion of blood, there is a linear relationship between ct attenuation and hematocrit. 4° the attenuation of whole blood with a hematocrit of 46% is approximately 56 hounsfield units (hu). in comparison, normal gray matter has an attenuation of 37 to 41 hu, and normal white matter, 20 to 34 hu. 4° acute hemorrhage in a patient with a normal hematocrit is therefore immediately evident as increased attenuation on ct ( fig 6) .31,36 hemorrhage in patients with severe anemia may be less obvious. an acute hematoma may he surrounded by a hypodense region corresponding to edema, and there is often an associated m a s s effect. 31'36 41 the attenuation of extravasated blood increases for the first 72 hours with clot formation and extrusion of low-density serum and subsequent increase in hemoglobin concentration. 4° after that, the attenuation gradually decreases as the result of lysis and phagocytosls of erythrocytes, which starts at the periphery and progresses centrally. 4°~1 the hematoma eventually becomes isodense at about 1 month after onset. 4°-41 use of contrast agent is unnecessary in most acute hematomas and may obscure the inherent hyperdensity, confusing the diagnosis. however, use of contrast agent may be helpful if there is a small hemorrhage with mass effect out of proportion to the size of the hematoma. in this instance, the differential diagnosis includes bleeding into a tumor, and contrast may be necessary to visualize the tumor. 4° in hematomas not associated with underlying neoplasia, enhancement first appears at the periphery of the hematoma at approximately 4 to 6 days because of neovascularization.36.40-42 a ring pattern of enhancement may persist for 2 to 6 weeks. 4° although variations can occur, the mri features of most intracranial hematomas follow a predictable course over time. factors intrinsic to the lesion that affect images include the age of the hemorrhage, whether the bleeding is arterial or venous, the location of the bleeding, and the presence of any underlying lesions. operator-dependent factors that affect the images include the mare magnetic field strength, the exact pulse sequence parameters used, and the method of echo formation. 43 hemorrhage can be thought of as an iron salvage pathway, in which iron from the extravasated erythrocytes is mobilized from hemoglobin and converted to short-term iron-containing proteins for transfer to the reticuloendothelial system, or to long-term storage proteins for local deposition. 44 during this process, oxyhemoglobin is converted in a stepwise fashion to deoxyhemoglobin, methemoglobin, and finally ferritin and hemosiderin. these various forms of hemoglobin affect the appearance of hemorrhage in three ways: (1) protein effects that are present with all forms of hemoglobin; (2) paramagnetm relaxation caused by paramagnetic molecules, which is significant only with methemoglobin; and (3) inhomogeneous susceptibility effects induced by paramagnetlc forms of hemoglobin (deoxyhemoglobin and methemoglobm) within erythrocytes. 45, 46 protein effects. relaxation times are shortened in the presence of proteins in water solutions. 45 this is caused by the changing magnetic fields of nearby nuclei as the molecules undergo brownian motion. 45 protein effects are generally proportional to protein concentrations and result in shortening of t1 and t2; that is, increased intensity on tl-weighted images and decreased intensity on t2-weighted images, compared with pure water. 45 paramagnetic effects. biological substances containing unpaired electrons have magnetic properties dominated by the magnetic effects of these unpaired electrons. the magnetic fields of the unpaired electrons are normally oriented randomly such that there is no net magnetization. however, when an external magnetic field is applied, the unpaired electrons tend to align parallel to that field, resulting in enhancement (increased magmtude) of that applied field. substances that have no intrinsic magnetic field m the absence of an applied magnetic field, but align with and thus enhance that applied field, are termed paramagnetic. 46 some forms of hemoglobin contain iron with paramagnetic properties, which greatly affects the mri appearance. if water molecules approach a paramagnetic center, there is an interaction between the nuclear magnetic dipoles of the water (protons) and the magnetic dipoles of the paramagnetic center (unpaired electrons). this proton-electron dipoledipole interaction shortens relaxation ume of the water protons. for proton relaxation enhancement to occur, however, the water proton must come within about 0.3 nm of the paramagnetic ions' unpaired electrons, because the magnitude of this interaction is inversely proportional to the sixth power of the distance between the dipoles. 46 these paramagnetic effects are more prominent on tl-weighted images than on t2-weighted images. 43 they will therefore result in an increased signal on tl-weighted images, similar to the effects of gadolinium-based contrast agents. (paramagnetism of mri contrast medium is also discussed in the tidwell & jones article in the may 1999 issue.) inhomogeneous susceptibility effects. when paramagnetlsm is confined to a small region (such as within erythrocytes), there is an inhomogeneous distribution of paramagnetic susceptibility that creates microscopic field gradients. 45 water molecules diffusing across the erythrocyte membrane experience a fluctuating magnetic field that results in dephasing and shortened t2 relaxation, a process called inhomogeneous susceptiblhty effect. in spin-echo sequences, inhomogeneous susceptibihty effects result in signal loss because of shortened t2 relaxation. this leads to decreased intensity on t2-weighted images. 46 hematoma evolution. these three relaxation mechanisms along with proton density contribute to image contrast during the various stages of hematoma formation (table 1) . because numerous factors can effect the appearance of intracranial hemorrhage on mri, the following summary is necessarily oversimplified but generally applicable to spin-echo sequences at 0.5 to 2.0 t. (refer to the tidwell &jones article in the may 1999 issue for a discussion on gradient echo imaging of hemorrhage.) furthermore, the timing of various stages may vary significantly, and different stages often overlap. 43, 45 the precise dating of specific hematomas is notoriously inaccurate, so the nomenclature of the temporal stages of hematomas is somewhat arbitrary. 43 oxyhemoglobm. freshly extravasated blood from arterial bleeding is composed of intact erythrocytes containing primarily oxyhemoglobin. 43 the iron in oxyhemoglobin is in the ferrous form (fe+2). because there are no unpaired electrons in the iron (or other atoms), oxygenated blood is not paramagnetic. 43 in the absence of paramagnetism, peracute hematomas appear as a high-proton-density region, with slightly shortened relaxation times, compared with water, mainly because of the protein content of blood. 43 this peracute stage of a hematoma usually persists for fewer than 24 hours. because of their transient nature, peracute hematomas are rarely seen in clinical practice. they are isointense to slightly hypointense on t1weighted images and hyperintense on t2-weighted images, and therefore appear similar to many other brain lesions. 43,.7 thus, ct is the preferred imaging modality if peracute hemorrhage is suspected. deoxyhemoglobin. after a few hours, the oxygen tension within the hematoma is reduced, resulting in the formation of deoxyhemoglobin (acute hematoma). deoxygenation is usually complete by 24 hours. 45 removal of molecular oxygen changes the distribution of electrons in the ferrous ion, leaving four unpaired electrons in the outer shell. these unpaired electrons confer deoxyhemoglobin with paramagnetic properties. the paramagnetic iron of deoxyhemoglobm is contained within a hydrophobic crevice in the hemoglobin mdlecule. this prevents water from coming close enough to the paramagnetic ion to induce paramagnetic (dipole-dipole) interaction, thus, the hematoma is still isointense to slightly hypointense on t 1-weighted images. 43 as long as the erythrocyte membrane is intact, the paramagneuc deoxyhemoglobln is localized within the cell. this results in inhomogeneous suscepub]lity effects# 3 acute hematomas that contain intracellular deoxyhemoglobin consequently appear very hypointense on t2-we]ghted images. 43 at this stage, any surrounding edema appears as a hyperintense perimeter on t2-weighted images) 3 intracellular methemoglobin. as oxygen tension within the hematoma falls further, deoxyhemoglobin is oxidized to methemoglobin (subacute hematoma). in whole blood, methemoglobin begins to accumulate at the rate of about 10% per day, after the first 2 days. 4s conversion to methemoglobin is maximal when the p02 is approximately 20 mm hg. if the oxygen tension is higher or lower, conversion to methemoglobin is slowed, affecting mri contrast. 46 the iron in methemoglobin is in the ferric (fe +3) state, which has five unpaired electrons in its outer shell and is highly paramagnetic. also, one of the coordination sites of the methemoglobln molecule is occupied by a water molecule that is rapidly exchanged with other water molecules in solution. as a result, water is positioned within 0.3 nm of the paramagnetic iron. this allows paramagnetic (dipole-dipole) interaction to shorten t1 relaxation, causing methemoglobin to be hyperintense on tl-weighted images. this hyperintensity begins at the periphery of an intraparenchyreal hematoma and progresses inward, probably because the outer rim of the hematoma has the optimal oxygen tension for the oxidation of hemoglobin. 43,45 at this stage the hematoma is still hypointense on t2-welghted images23 other causes of t1 hyperintensity or t2 hypointenslty include fat, calcification, mucinous material, intratumoral melanin, flow effects, and enhancement with paramagnetic contrast agents23 extracellular methemoglobin. soon after methemoglobin formation begins, glucose reserves in the erythrocytes are depleted and hemolysis ensues. this eliminates the inhomogeneous susceptibility effect as the methemoglobin becomes uniformly distributed. this prolongs t2, and the hematoma is once again hyperintense on t2-weighted images (rebound hyperintensity). 45 on the other hand, tl-weighted images are not affected and therefore continue to show hyperintensity. 45 this paradoxical state of t1 and t2 hyperintensity may persist for 1 month or more (fig 7) . 45 ferritin and hemosiderin. after 2 to 6 weeks, modified macrophages (gitter cells) infiltrate from surrounding brain and remove iron from the hematoma (chronic hematoma). this eventually eliminates the remaining protein and inhomogeneous susceptibility effects. the center of the hematoma eventually either collapses or is replaced by cse 45 the iron is deposited at the periphery of the hematoma as hemosiderin and ferritin, which behave paramagnetically. 43 the iron in these storage forms is not accessible to water, so paramagnetic (dipole-dipole) interaction does not occur, but the inhomogeneous susceptibility effect shortens t2. a chronic hematoma has a rim that is hypointense on tl-weighted images and very hypointense on t2-weighted images. the rim becomes thicker as the hematoma resolves. 45 whenever intracramal hemorrhage is identified, it is important to determine if it is associated with an underlying tumor or is caused by a nonneoplastic lesion. the use of a contrast agent often allows identification of an underlying tumor. 4° an important finding in a hemorrhagic tumor is persistent surrounding edema (decreased attenuanon on ct, hyperintensity on t2-weighted images), whereas edema has usually resolved by the chronic stage of a nonneoplastic hematoma. 43 in neoplastic hemorrhage, mri signal intensity patterns are more heterogeneous and the temporal evolution of intensity patterns is often delayed compared with nonneoplastic hematomas, probably because hypoxia within the tumor delays methemoglobin formation. 4°,43 neoplastic hemorrhage often lacks the well-defined hypointense rim characteristic of nonneoplastic hematomas in their subacute and chronic stage. 4°,~3 this is because the persistently altered blood-brain barrier of tumors may allow more efficient removal of ferritm and hemosiderin. 43 in situations in which the diagnosis is still uncertain, repeat scanning is often useful because this allows evaluation of the expected temporal changes. 4° the most common is the arteriovenous malformation (avm). clinical signs are caused by spontaneous hemorrhage and are usually suggestive of a focal cerebral lesion. the onset of signs is usually acute and occurs in middle-aged to older animals. 3>33 the relatively late onset of signs associated with a congenital lesion can be explained by contmued hemodynamic stress and consequent attenuation of the abnormal vessels, which eventually leads to hemorrhage, z° when a cerebrovascular malformation has bled, early ct or mri usually shows intraparenchymal hemorrhage (fig 6) . 30 ct of capillary malformations is often normal, but may show an isodense to slightly hyperdense mass. contrast enhancement is usually minimal. 51 mri is more sensitive, and shows a lace-like region of stippled contrast enhancement with no or subtle abnormality on unenhanced images. 5a a cavernous malformation appears on ct as a focal hyperdense region with variable calcification with mild contrast enhancement. 5152 on mri, a cavernous malformation is seen as a central region of mixed intensity, corresponding to methemoglobin, surrounded by circumferential rings of hypomtense hemosiderin and ferritin)2 53 whenever a spontaneous intraparenchymal hematoma is identified, it is important to look for any associated large vessels to suggest a vascular malformation. however, the failure to identify large vessels does not entirely exclude a vascular malformation, because compression or obliteration of vessels by adjacent hematoma, extremely slow flow, and thrombosis may obscure the abnormal vessels. 51,52 although ct and mri are useful in detecting hemorrhage associated with vascular malformations and depicting the vascular anatomy of some of these lesions, catheter angiography or magnetic resonance angiography (mra) is often necessary for complete assessment of cerebrovascular malformations m human patients. 51,52,~4 (refer to the tidwell & jones article in the may 1999 issue for a discussion of mra.) obstruction of flow in the vessels of the brain can result in a sudden onset of neurological signs caused by infarction. arterial obstruction can be caused by thrombosis or embolism. a thrombus is a blood clot developing within a vessel that causes obstruction at the site of formation. embolism is occlusion of a vessel by a fragment of blood clot or other substance that has flowed to the site of obstruction from a distant location. because of abundant venous anastomoses, venous infarction is less common than arterial thromboembolism. 55 in human patients, cerebrovascular thrombosis or embolism is often secondary to atherosclerosis. 56 atherosclerosis also occurs in dogs, especially older dogs, dogs with hypothyroidism, and miniature schnauzers with idiopathic hyperlipoproteinemia. 29,57,5s atherosclerosis associated with these conditions can lead to cerebral thromboembohsm and neurological dysfunction. 33 57-59 other diseases associated with cerebral thromboembolism in dogs include sepsis, coagulopathy, neoplasia, and heartworm infection. 3>6° the hallmark of brain infarction is an acute onset of focal bram dysfunction. neurological deficits depend on the site of the lesion and are typically asymmetric. involvement of the forebrain, such as with thromboembolism of the middle cerebral artery, usually results in contralateral hemiparesis with decreased postural reactions. seizures are very common in dogs and cats with infarction affecting the forebrain. there may be contralateral bhndness with normal pupillary light reflexes, and the patient may circle or turn toward the side of the lesion. lesions in the cerebellum may cause ataxia, hypermetria, vestibular dysfunction, or opisthotonos. brainstem involvement is characterized by gait deficits ranging from ipsilateral hemlparesis to tetraplegia, cranial nerve deficits, and abnormal levels of consciousness, including coma. 30, 31, 33, 36, 38, 55, 61 nonhemorrhagic infarction computed tomography. changes may be detected on ct as early as 3 to 6 hours after onset of signs and consist of a slight decrease in attenuation and subtle mass effect. 62 63 these changes are related to edema and reach a maximum at 3 to 5 days (fig 8) and resolve by 2 to 3 weeks after infarction. 62 the location and shape of the lesion correspond with the distribunon of the involved vessel(s), most commonly the middle cerebral artery. 62 abnormal contrast enhancement may be seen as early as 24 hours but often does not become evident until about 1 week after infarction. 62,63 enhancement is most apparent at the periphery of the lesion and reflects growth of new capillaries without a normal blood-brain barrier. 62 enhancement of hypodense infarcts can result in isodense lesions, thereby masking their presence. 62 for this reason, unenhanced as well as enhanced ct should be performed. in the chronic phase (3 to 4 weeks), the hypodense region becomes more sharply marginated as necrotic tissue is resorbed. 62 ultimately there is a loss of parenchymal volume with attendant dilatation of adjacent sulci and ventricles. 62 contrast enhancement does not usually occur in the chronic stage because the integrity of the blood-brain barrier is restored. 62 magnetic resonance imaging. early changes on mri consist of a subtle increase in intensity on t2-weighted and proton density-weighted images. these changes reflect edema and can be detected as early as 1 hour after vascular occlusion. 64 mri is thus more sensitive than ct in early infarction. also, mri is more sensitive in detecting small infarcts and those involving the brainstem. 65 in acute infarction, tl-weighted mri is less sensitive than t2-weighed and proton density-weighted images and may be normal. ~6 parenchymal enhancement with gadolinium is uncommon within the first 24 hours. 66,67 the use of functional mri techniques such as diffusion and perfusion studies for the detection of acute infarction is currently being investigated and is discussed in the arucle by tidwell &jones in the may 1999 issue. after the first 24 hours, the hyperintensity on t2-welghted and proton density-weighted images becomes more obvious (fig 9) .64'66'68 during this time, tl-weighted images may show decreased signal. 64,66 68 gyral swelling and mass effect are more prominent, becoming maximal 2 to 4 days after onset of occlusion. 6466 parenchymal enhancement becomes evident within 4 to 7 days and may persist for 3 to 4 weeks. 66 after several weeks, the signal changes seen on earlier scans become smaller and better defined. there is focal atrophy with dilatanon of nearby sulci and ventricles. there may be a collection of fluid with the s~gnal characteristics of cerebrospinal fluid. 69 contrast enhancement does not usually occur. 66 the above description applies to nonhemorrhagic or "bland" infarcts. however, many infarcts will have attendant bleeding caused by reperfusion of damaged blood vessels. 66 this results in a hemorrhagic infarct. petechial hemorrhage into an infarct may result in an isodense lesion on unenhanced ct because of the combined effects of the brain edema, which decreases attenuation, and acute hemorrhage, which increases attenuation. mass effect caused by the edema usually provides a clue to the presence of a lesion. if hemorrhage is a large component of an acute infarct, unenhanced ct shows a hyperdense hematoma surrounded by hypodense edema. these changes are often confined to a vascular territory, a feature that is helpful in differentiating hemorrhagic infarctions from other causes of hemorrhage. 4° in the subacute phase, the region around the infarct may enhance, increasing the possibility of hemorrhage within a tumor. mass effect from a hemorrhagic infarct is usually minimal or decreasing at the time of maximal contrast enhancement (2 to 4 weeks), which is useful in differentiating between hemorrhagic infarct and tumor. 5~ the mri features of hemorrhagic infarction are similar to those of lntraparenchymal hemorrhage. 4° compared with other causes of lntraparenchymal hemorrhage, hemorrhagic infarcts tend to have a higher p02 because of earlier revascularization and collateral perfusion. the higher p02 decreases the amount of deoxyhemoglobin, and therefore minimizes the acute t2 relaxation effect. 51 uncontrolled seizures or status epileptmus may result in neuronal degeneration progressing to necrosis. the distribution of lesions varies somewhat among individual patients, but the hippocampus, basal nuclei, and frontal and pyriform lobes of the cerebrum are most severely affected. f° lesions are usually bilateral, but may affect one side more severely. f° these changes are thought to be the consequence of ischemia secondary to accumulation of cytotoxic agents and a mismatch between brain metabolism and blood flow during prolonged seizures. 71 persistent or reversible mr lesions have been described in human patients after seizures, and similar abnormalities have there is also a smaller wedge-shaped region of hyperintensity in the vascular territory of the right middle cerebral artery. se, 1500/105, 0.064 t. been described in dogs, [72] [73] [74] the most consistent finding is unilateral or bilateral lesions of the hippocampus, pyriform lobe, and frontal lobe. the lesions are hypointense on t1weighted images and hyperintense on proton densityweighted and t2-weighted images. there is minimal mass effect and no or moderate contrast enhancement tm (fig 10) . these lesions may persist or disappear on subsequent scans. head trauma results from a variety of causes, including motor vehicle accidents, bites, kicks, and gunshot wounds. previously, imaging of head trauma was limited to skull radiography for the detection of fractures. plain radiography, however, cannot identify many traumatic brain lesions, such as hemorrhage, and therefore provides only limited diagnostic information. the development of ct provided a sensitive means for detecting and localizing intracranial hemorrhage, permitting expeditious surgical treatment, and improving outcome. 75 mri has been shown to be similar in sensitivity for detection of hemorrhagic lesions, but is much more sensitive than ct for detecting nonhemorrhagic lesions, such as shearing injuries of white matter. 75 neuroimaging should be considered early in the management of animals with head injury and marked impairment of consciousness or neurological deficits that progressively worsen despite initial medmal therapy. the most critical issue is to detect potential hematomas that may be treatable with sur-gery. 76 in general, ct is the diagnostic study of choice for initial evaluation, because it can be completed quickly and is sensitive to acute hemorrhage, r6 ct also provides fine anatomic detail of bone when viewed at a wide window width allowing accurate characterization of any skull fractures 77 (fig 1 1) . although mri is slightly more sensitive than ct for detection of hematomas, those that are not seen on ct are usually small and typically managed conservatively. 75 a disadvantage of mri is the longer examination ume and the difficulty in monitoring unstable patients in the mri environment. therefore, if ct is available, mr examination is usually delayed until the patient is stabilized. in patients with severe head trauma, mri should be considered in the first 2 weeks after injury, because most parenchymal lesions are more easily detectable during this period. 75 epidural hematomas accumulate in the potential space between the inner surface of the skull and the dura mater. they are usually found in the temporoparleteal region, and have been caused by laceration or tearing of the middle meningeal artery by skull fracture. the arterial force of the bleeding dissects the dura away from the bone, often resulting in a rapidly expanding mass. clinical signs may consist of rapidly progressing focal neurological deficits and deterioration in consciousness. however, patients with associated parenchymal brain injury may have severely impaired consciousness at the time of trauma. 75 on ct, an acute epidural hematoma is typically a well-defined, biconvex lesion between the inner table of the skull and the underlying depressed dura and brain. tm the attenuation of the hematoma is initially greater than brain parenchyma and increases further during the first few hours with coagulation and clot retraction. with time, the attenuation decreases with clot retraction, erythrocyte lysis, and hemoglobin degradation. the hematoma becomes smaller and fades to isodense and then hypodense within 1 or 2 weeks. the inner surface of an epidural hematoma is dura mater, which normally enhances with intravenous contrast material, but this enhancing margin becomes even more prominent as a neovascular membrane develops over time. 78 on mri, the intensity varies depending on the age of the hematoma, as described in the section on intracranlal hemorrhage and summarized in table 1 . occasionally, an epidural hematoma may not have the classic lenticular shape or associated skull fracture, making it difficult to differentiate it from a subdural hematoma. mri can be helpful in this regard, because the medially &splaced dura mater is usually directly visualized as a thin line of low signal separating the hematoma from the underlying compressed brain parenchyma. 75 mri is also superior in the detection of subacute and chronic hematomas, which may be isodense on ct77 78 subdural hematomas accumulate within the potential space between the pia-arachnmd and dura mater. they are usually caused by tearing of veins that traverse the subdural space. subdural hematomas appear to be less common m dogs and cats with head injury, compared with human patients. clinical signs may consist of progressive asymmetrical neurological deficits and decreased levels of consciousness. an acute subdural hematoma appears on ct as a hyperdense, crescentshaped collection conforming to the inner surface of the skull. tm mass effect is evident and may be compounded by contusion and edema of the underlying brain parenchyma. acute hyperdense hematomas may not be vislble on a narrow window width (soft tissue window), appearing only as an apparent thickening of the skull, since the bone and hematoma may have the same pixel brightness, r5 78 mass effect is usually present, however, providing a clue in their detectlon. widening the window width may be necessary to distinguish an acute subdural hematoma from the dense skull, rs as with other intracranial hematomas, the density of a subdural hematoma decreases over time, becoming isodense to gray matter by 1 to 2 weeks. accordingly, subacute subdural hematomas may be difficult to detect on ct but are readily detectable on mri. 75 a chronic subdural hematoma (2 to 3 weeks old or older) is hypodense compared with normal brain on ct and is surrounded by a well-defined capsule (fig 12) . this results in a more focal collection of blood with a straighter medial edge compared with crescent-shaped acute subdural hematomas. 78 the capsule of a chronic subdural hematoma enhances with intravenous contrast material and may calcify. tm subarachnoid hemorrhage is bleeding into the csf-filled subarachnoid space. posttraumatic subarachnoid hemorrhage is relatively common and often associated with cortical contusions. acute subarachnoid hemorrhage is evident on ct as increased attenuation of the sulcl, fissures, or basal cisterns, with the degree of increased attenuation being related to the amount of blood in the subarachnoid space. 78 with time the attenuation decreases and subarachnoid hemorrhage may not be detectable after the first week, unless rebleeding has occurred. 51 acute subarachnoid hemorrhage is usually not detectable on mri, probably because the po2 of the subarachnoid csf is too high for the conversion of oxyhemoglobin to deoxyhemoglobin and methemoglobin. 51 however, mri is excellent at detecting subarachnoid hemorrhage in the subacute or chronic stage. 5i brain contusions are common after head trauma and consist of heterogeneous regions of hemorrhage, edema, and necrosis, often located in the superficial gray matter. in human patients contusions tend to be multiple and bilateral and are much less likely to be associated with severe initial impairment of consciousness compared with diffuse axonal injur~ 75 initial ct findings are often limited to faint, ill-defined hypodense areas mixed with tiny regions of hyperdense hemorrhage. 75 contusions in which edema and necrosis predominate may not be visible imtially on ct but often become apparent several days later as regions of decreased attenuation and mass effect caused by edema. 7s mri, because of its greater sensitivity in detecting edema, is better at detecting early contusions, which appear hypointense on tl-weighted images and hyperintense on t2-weighted images. 75 head trauma that involves rapid angular acceleration may result in diffuse axonal injury. these shearing injuries result from differences in elastic and inertial properties between different but adjacent brain tissues/s,79 in human patients, these injuries are characterized pathologically by disruption of axons, especially at the junction of gray and white matter of the cerebrum, and at the corpus callosum, basal nuclei, and cranial aspect of the brainstem. there is subsequent axonal swelling and infiltration with macrophages. these patients present with severe impairment of consciousness starting from the moment of injury. 76"77,s0 mri is much more sensitive than ct in detecting diffuse axonal injury in human patients, although even mri findings usually underestimate the true extent of these injuries. 75 ct is often normal, but may show scattered hemorrhages. the mri appearance reflects the prolonged t1 and t2 values of increased tissue fluid (edema). there are multiple, small elliptical lesions in the white matter. these lesions are hypomtense on tl-weighted images and hyperintense on t2-welghted images. 75, 78 infectious and inflammatory diseases inflammatory diseases are important diagnostic considerations for patients with brain disease. infectious agents, such as viruses, protozoa, and fungi, cause many inflammatory diseases, but for others the etiology is unknown. despite the numerous causes of mflammatory brain disease, the affected tissue can respond only in a limited number of ways. thus, many of these diseases appear similar on imaging studies and differentiating the potential etiologies based on imaging features alone may be impossible. furthermore, it may be difficult to &scriminate between inflammatory diseases and other categories of disease, such as neoplasia and vascular disorders. accordingly, results of imaging studies must be interpreted in context with clinical features and results of other laboratory tests, especially analysis of cse 8i all inflammatory brain diseases share a common pathological feature--an influx of leukocytes into the brain (cerebritis or encephalitis) or meninges (meningitis). because of the close anatomical association of these structures, more than one area of the nervous system can be involved in the inflammatory process. for example, inflammation of the meninges and brain is called meningoencephalitis, s2 the most common cause of meningitis in dogs is steroidresponsive meningitis-arteritis, a nonseptic suppurative meningitis of unknown etiology that responds to immunosuppressive dosages of corticosteroids, s3 infectious causes of memngitis are less common in small animals and include bacteria, viruses, fungi, and protozoa s4 pathologically. acute leptomeningitls results in congestion and hyperemia of the pia-arachnoid and distension of the subarachnoid space by an exudate containing leukocytes. clinically, affected patients show fever, spinal pain, cervmal rigidity, and stiff gait. several complications can occur m the ensuing days to weeks. there may be extension of the infection to the neural parenchyma, resulting in focal or diffuse encephalitis, myelitis, or abscess. inflammatory exudate may obstruct csf pathways, producing hydrocephalus. endogenous host inflammatory mediators can result in disruption of the blood-brain barrier, cerebral edema, and increased intracranial pressure. 85 definitive &agnosis of meningitis is based on analysis of cse neuroimaging is useful in detectmg some of the complications associated with meningitis and when the differential diagnosis includes other diseases. neuroimaging features of experimental bacterial menmgitls in dogs is comparable with naturally occurring bacterial meningitis in human patients. 868r in human patients with uncomplicated early bacterial or viral meningitis, unenhanced ct and mri are often unremarkable or show mild dilatation of the ventricles or subarachnoid space (fig 13) . 88 with more severe involvement, there may be diffuse or patchy brain edema. 88 postcontrast ct or mri may show abnormal enhancement of the leptomeninges. 89 in experimental studies in dogs, tl-weighted mr images with gadolinium showed abnormal leptomeningeal enhancement better than ct. 8r mri also identifies complications such as encephahtis more effectively than ct. 87 encephalitis generally refers to nonpurulent inflammation of the brain and is distinguished pathologically from suppurauve inflammation of the brain (cerebritis) assooated with bacterial infection. 88 viral encephalitides of small animals include canine distemper and feline infectious peritonitis. other causes of encephalitis include rocky mountain fever, canine ehrlichiosis, toxoplasmosis, and neosporosls. finally, there are encephahtides of unknown etiology, such as pug dog encephalitis and granulomatous meningoencephalitis. distemper encephalitis. the two most common clinical forms of distemper encephalitis are acute encephalitis in young dogs and chronic encephalitis in mature dogs. immature dogs with distemper encephalitis typmafly suffer a rapid onset of systemic illness characterized by conjunctivitis, nasal discharge, cough, vomiting, and diarrhea. neurological dysfunction can occur during or after the systemic illness and includes seizures, abnormal behavior, blindness, and paresis. mature dogs are more likely to develop chromc, multifocal encephalitis with a predilection for the white matter of the brainstem. many of these dogs have an adequate vaccination history, and signs of systemm illness are often absent or transient. 9°-92 these patients often have slowly progressive gait deficits or vestibular dysfunction. 9°, 91 ct of dogs with chromc distemper encephalitis may be normal or show focal or multifocal hypoattenuating lesions with a predilection for the white matter. 93 these lesions may have uniform or ring-like enhancement 93 (fig 14) . some lesions may be associated wlth hypoattenuating edema and mass effect. 93 lesions are typically hypointense or poorly defined on tl-weighted mri, hyperintense on t2-weighted images, and enhance with contrast agent. 94 feline infectious peritonitis. feline infectious peritonitis (fip) is a systemic disease of cats caused by an immune response to a corona virus. neurological signs are generally associated with the parenchymatous (dry) form of fie neurological deficits referable to brainstem involvement predominate and include ataxia, paresis, and vestibular dysfunction. [95] [96] [97] imaging features of fip reflect the pathological changes, which consist of pyogranulomatous inflammation of the leptomeninges, choroid plexus, ependyma, and brain parenchyma. hydrocephalus is common and is probably secondary to obstruction by ependymltis. 95-98 the brainstem and fourth ventricle are consistently involved, but other regions of the central nervous system can be affected. 95 96 the inflammatory process primarily affects the inner and outer surfaces of the brain with only secondary extension into the parenchyma. recognition of this surface-related pattern can be helpful in differentiating fip from other bram diseases in the cat. 98 ct may be normal or show hydrocephalus. 95 mri may show ependymitis, choroiditis, and memngitis. this is evident as hyperintensity of the ventricular lining, choroid plexus, and meninges, respectively, on t2-weighted mri and abnormal enhancement with gadolinium-based contrast agent 97,99 (fig 15) . fungal infections. many fungal agents can sporadically infect the nervous system, causing meningitis or granulomas. cryptococcus neoformans is the most common fungal infectmn to involve the nervous system of dogs and cats. in cats, this organism generally induces a mild, nonsuppurative meningitis or encephalitis, whereas affected dogs typically develop a granulomatous reaction in the brain and meninges. 98 neurological deficits can be acute or chronic and include seizures, lethargy, ataxia, and vestibular dysfunction. 1°° 101 on ct, mass lesions (cryptococcomas) appear as single or multiple isodense or hypodense masses with ring or solid enhancement and surrounding edema. 81,89,93i°2 leptomeningeal enhancement may also be apparent if the meninges are involved. 81,i°2 hydrocephalus may occur secondary to meningitis or obstruction of csf pathways by the mass. 81,89 in human patients, mri is more sensitive than ct and may show clustered foci of signal abnormahties that are lsointense to csf on all sequences. these lesions represent small granulomas or dilated virchow-robin spaces filled with fungal organisms and mucoid. these are often bilaterally symmetrical, and are located in the basal nuclei and midbraln. these lesions do not enhance with gadolinium and are not associated with mass effect or edema. 89 similar changes have been reported on mri of canine cryptococcosis.i°3 cryptococcomas appear as masses that are hypointense on tl-weighted images, hyperintense on t2-welghted images, and enhance with gadolinium. 8<1°3 other fungal organisms sporadically infect the central nervous system, including blastomyces dermatldis, histoplasmosis capsulatum, aspergfllus spp, coccidiodes immitis, and phaeohyphomycosis (fig 16) . l°4qn necrotizing encephalitis (pug dog encephalitis). a necrotizing form of encephalitis has been recognized in pug dogs, maltese terriers, and yorkshire terriers between 6 months and 10 years of age. n2-n6 signs include progressive seizures, abnormal behavior, blindness, ataxia, and walking in circles. pathological changes consist of multifocal necrosis and nonsuppuratlve inflammation, with a striking predilection for the white matter of the cerebrum. lesions are often bilateral but asymmetrical, n2-114,n6 enlargement of the lateral ventricles secondary to shrinkage and cavitation of the cerebral hemispheres (hydrocephalus ex vacuo) is common n2,i14 n6 (fig 2) . in yorkshire terriers, the brainstem may be preferentially revolved. 113 the etiology is unknown. in acute forms of the disease, ct may show one or more focal hypodense lesions, most commonly affecting the cerebral hemisphere. the lesions may or may not enhance with contrast agent. mri shows the early edematous changes as increased signal intensity on proton density-weighted and t2-weighted images and decreased signal intensity on tl-weighted images. in acute cases, there is often substantial mass effect and minimal if any abnormal enhancement with contrast medium (fig 17) . differentials include neoplasia, other inflammatory lesions, and acute infarction. in more chronic cases, necrosis and cystic changes usually predominate. the centers of the lesions appears similar to csf; that is, hypodense on ct, very hypointense on tl-weighted images, and very hyperintense on proton density-weighted and t2-weighted images 114-116 (fig 18) . lesions are usually located in the white matter of the cerebral hemisphere, often in the area lateral to the ventricles. ii3 typically there is no mass effect or even a reverse mass effect (shift of surrounding tissue toward the lesion). lesions usually do not enhance, but may have a ring pattern of enhancement. 93 114, 115 asymmetric enlargement of the lateral ventricles is common. 93 the primary differential is chronic infarction. however, in necrotizing encephalitis the lesions are not confined to a specific vascular territory as is typical of infarction caused by thromboembolism. the onset of signs (sudden in infarction versus slow onset in chronic necrotizing encephalitis) ts also helpful. granulomatous meningoencephalomyelitis. granulomatous meningoencephalomyelitis (gme) is an inflammatory disease of the canine central nervous system characterized pathologically by an accumulation of mononuclear cells in the parenchyma and meninges of the brain and spinal cord. 117 lesions may be disseminated or focal. in the disseminated form, lesions are distributed throughout the central nervous system, with a predilection for the white matter of the cerebrum, cerebellum, caudal aspect of the brainstem, and cervical spinal segments. the focal form is manifested as a single granulomatous mass, most commonly located in the cerebrum, with smaller disseminated lesions, ns-ln the cause is not known. adult dogs of any breed can be affected, although females and toy and terrier breeds are at increased risk. 118122 dogs with disseminated gme usually have rapidly progressive signs including neck pain, vestibular dysfunction, paralysis, and seizures. the focal form is manifested as chronic, gradually progressive signs, with seizures being the most common. iis-123 the clinical presentation of focal gme often mimics that of a tumor. on ct, disseminated gme is seen as multiple foci of ill-defined contrast enhancement involving the parenchyma and meninges. some lesions may be associated with hypoattenuating edema and mass effect. 124 other inflammatory diseases are the primary differentials. focal gme appears on noncontrast ct as an isodense or hyperdense mass, most commonly located within the cerebrum or at the cerebellomedullary junction (fig 19) . 93 '125 on mri, focal gme is usually isointense or slightly hypointense on tl-weighted images and hyperintense on proton density-weighted and t2-weighted images (fig 20) . 126 enhancement is variable, including no enhancement, ring-pattern enhancement, or moderate homogenous enhancement. 93,t24,i25 there may be edema in the white matter surrounding the mass. 93, 125, 126 asymmetric enlargement of the lateral ventricles has also been reported. 93 the primary differentials are neoplasia and other inflammatory lesions. biopsy is often necessary for definitive diagnosis. 125 focal infection of the brain with pyogenic organisms is uncommon in small animals. bacteria may gain access to the brain through penetrating wounds; secondary to direct extension from infections in the eye, ear, nasal passages, or meninges; or via hematogenous spread from extracranial sources, i27128 with hematogenous spread, lesions often arise at the graywhite matter junction of the cerebrum, s8 clinical signs reflect a progressively worsening focal brain lesion. 127 i28 several reports detail the pathological and ct features of experimental cerebritis/abscess in dogs. 129,13° these imaging features are similar to those reported in human patients with spontaneous brain abscess. tm pathologically, serial changes occur over 2 to 3 weeks, starting as cerebritis and culminating in abscess. focal but poorly localized areas of scattered necrosis, edema, vascular congestion, and perivascular inflammatory infiltrates characterize cerebritis. at this stage, unenhanced ct shows only an irregular, poorly circumscribed region of low attenuation, t29-132 scans obtained immediately after contrast administration show ring-like enhancement. on delayed scans, contrast diffuses into the center of the lesion, starting peripherally, until the center of the lesion may be completely filled with contrast by 30 minutes after admmistra-tlonj 29-13t the inherent sensitivity of proton density-weighted and t2-welghted mri to aheradons in tissue water enables earlier detection of cerebritis compared with ct. 89,132 these images show increased signal intensity indistinguishable from or slightly hypointense to surrounding edema, sg,j32 on t1weighted images, cerebritis is isointense to slightly hypomtense to adjacent brain parenchyma, with associated mass effect.89,132 over a period of 1 to 2 weeks, untreated cerebritis may progress to abscess formation when the central zone of necrosis becomes liquefied, better defined, and encircled by a collagen capsule, which is formed by fibroblast migration from the surrounding vessels. because of relatively poor vascularization of white matter, the medial aspect of the capsule may be somewhat thinner. this predisposes to expansion of the abscess into white matter, the formation of daughter abscesses medially, or rupture into the lateral ventricles, lz9,13°,132 the capsule is visible on unenhanced ct as an isodense rim that is visible because it is bordered medially by a hypodense liquid center and surrounded by hypodense edema (fig 21) . 131 the edema may be greater in volume than the abscess itself, causing much of the mass effect. 131 on contrast-enhanced ct, the rim is usually smooth and brightly enhancing. there is no diffusion of contrast into the necrotic center on delayed scans as there is with cerebritis, n9-131 on tl-weighted mri there is mild peripheral hypointensity representing edema and a more markedly hypointense liquid center. the capsule is a discrete rim that is isointense to slightly hyperintense. on t2-weighted images the abscess center is isointense to mildly hyperintense to gray matter. the capsule is seen as a dramatic hypointense rim, possibly caused by paramagnetic free radicals within phagocytic macrophages. 89 the ring pattern of enhancement parallels the enhancement seen on contrast-enhanced ct. the differential diagnosis for a ring-enhancing lesion includes primary brain tumor, metastasis, infarction, granuloma, and resolving hematoma. 42.89 helpful clues that may identify abscesses include the time course, location, temporal pattern of enhancement, and predisposition for the abscess rim to be thinner medially, s9 cuterebra. dogs and more commonly cats can suffer brain disease caused by aberrant migration of cuterebra larvae. [133] [134] [135] fly larvae attach to the host and burrow into the subcutaneous tissue. the developing larva (1 to 10 mm long) may migrate under the skin and enter the brain, most likely through the nasal passages and cribiform plate. 134,135 pathological changes in the brain consist of multifocal meningoencephalitis with malacia and hemorrhage •133-135 affected animals typically have access to outdoors and develop signs from june to october, coinciding with the larval migration portion of the cuterebra life cycle. a recent history of upper respiratory disease is common, likely reflecting migration of the larvae through nasal passages. 135 there is an acute onset of neurological dysfunction, most commonly referable to a focal forebrain lesion. seizures, abnormal mentation, circhng, hemlparesis, and unilateral blindness are common. 134,135 clinical and pathological features in cats are similar to what has been previously reported as feline ischemic encephalopathy.i35 a mottled appearance to the brain has been reported on ct of an affected cat} 35 based on my observations, ct may show focal or muhifocal regions of decreased attenuation with minimal mass effect. the may be one or more tract-like regions of contrast enhancement (fig 22) . lesions may be hypointense on tl-weighted mri and hypermtense on t2-weighted mri. edema is fairly minimal, and there may be small regions of hemorrhage. contrast enhancement is s~milar to that described for ct. an enhancing tract (arrows) extends from the cribiform plate through the right cerebral hemisphere. this cat, which lived outside, presented in october, 5 days after suffering a sudden onset of sneezing followed by seizures, left hemiparesis, depression, and circling to the right. clinical signs gradually resolved over the next 2 weeks, so the diagnosis was not confirmed by necropsy, but the clinical features are consistent with intracranial migration of a cuterebra larva. and does not enhance. i36 the intraparenchymal location is helpful in differentiating these lesions from arachnoid cysts, which are associated with the subarachnoid space. 25,~-6 ct and mri are helpful in identifying and characterizing many nonneoplastic brain disorders in dogs and cats. the imaging features of some of these disorders are unique, permitting definitive diagnosis based on imaging results and clinical features. however, the imaging findings for other brain disorders are nonspecific. in these instances, ct and mri often allow detection and localization of abnormalities, but definitive diagnosis may require other laboratory tests or surgical biopsy. as the use of ct and mr] becomes more widespread in veterinary medicine, further research in the imaging findings associated with various nonneoplastic disorders will improve our ability to diagnose and manage these conditions. imaging the brain (first of two parts) canine hydrocephalus. comp contln ed pract vet computed tomography in evaluation of hydrocephalus diagnosis and management of an atypical case of hydrocephalus, using computed tomography, ventrlculoperitoneal shunting, and nuclear sclntigraphy ultrasonographic diagnosis of canine hydrocephalus. vet radio131 ultrasonographlc imaging of the lateral cerebral ventricles in the dog diagnosis of cerebral ventrlculomegaly in normal adult beagles using quantitative mri normal variation in size of the lateral ventricles of the labrador retriever dog as assessed by magnetic resonance imaging developmental change of lateral ventricle volume and ratio in beagle-type dogs up to 7 months of age magnetic resonance imaging of silastlc-lnduced canine hydrocephalus pathogenesis of chlari malformation: a morphometric study of the posterior cranial fossa pathophysiology of synngomyelia associated with chiarl i malformation of the cerebellar tonsils hydromyelia in the dog occipital dysplasia and associated cranial spinal cord abnormalitles in two dogs magnetic resonance imaging of the brain and spine common 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cases cerebellar infarction caused by artenal thrombosis in a dog cerebral infarction ~n a dog. prog vet neurol computed tomography of an acute hemorrhagic cerebral infarct ~n a dog vet rad~ol ultrasound 35 magnetic resonance ~maging appearance of intracranfal hemorrhage secondary to cerebral vascular malformation ~n a dog magnetjc resonance imaging of brain infarction in seven dogs cerebral hemorrhage in an old dog intracranlal hemorrhage natural history of experimental intracerebral hemorrhage, sonography, computed tomography and neuropathology intracranlal ring enhancing lesions jn dogs: a correlatwe ct scanning and neuropathologlc study vet radlol ultrasound 36 magnetic resonance imaging of the brain and spree biochemical bas~s of the mr appearance of cerebral hemorrhage mr imaging of cerebral hematomas at different field strengths: theory and applications magnetic resonance imaging of intracranial hemorrhage deo-narjne '4. et al. mr imaging of acute intracran~al hemorrhage: 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cuterebra larvae myiasis of the central nervous system cerebral coenurosls in a cat labuc ret al: cerebral coenunasis in a domestic cat magnetic resonance imaging cerebrovascular disease key: cord-321549-r7bmtloy authors: jendrny, paula; schulz, claudia; twele, friederike; meller, sebastian; von köckritz-blickwede, maren; osterhaus, albertus dominicus marcellinus erasmus; ebbers, janek; pilchová, veronika; pink, isabell; welte, tobias; manns, michael peter; fathi, anahita; ernst, christiane; addo, marylyn martina; schalke, esther; volk, holger andreas title: scent dog identification of samples from covid-19 patients – a pilot study date: 2020-07-23 journal: bmc infect dis doi: 10.1186/s12879-020-05281-3 sha: doc_id: 321549 cord_uid: r7bmtloy background: as the covid-19 pandemic continues to spread, early, ideally real-time, identification of sars-cov-2 infected individuals is pivotal in interrupting infection chains. volatile organic compounds produced during respiratory infections can cause specific scent imprints, which can be detected by trained dogs with a high rate of precision. methods: eight detection dogs were trained for 1 week to detect saliva or tracheobronchial secretions of sars-cov-2 infected patients in a randomised, double-blinded and controlled study. results: the dogs were able to discriminate between samples of infected (positive) and non-infected (negative) individuals with average diagnostic sensitivity of 82.63% (95% confidence interval [ci]: 82.02–83.24%) and specificity of 96.35% (95% ci: 96.31–96.39%). during the presentation of 1012 randomised samples, the dogs achieved an overall average detection rate of 94% (±3.4%) with 157 correct indications of positive, 792 correct rejections of negative, 33 incorrect indications of negative or incorrect rejections of 30 positive sample presentations. conclusions: these preliminary findings indicate that trained detection dogs can identify respiratory secretion samples from hospitalised and clinically diseased sars-cov-2 infected individuals by discriminating between samples from sars-cov-2 infected patients and negative controls. this data may form the basis for the reliable screening method of sars-cov-2 infected people. the ongoing covid-19 pandemic highlights the importance of fast and reliable testing for accurate identification of symptomatic and asymptomatic carriers to reduce spread of infection effectively [1] . current testing regimens usually require nasopharyngeal swabs applied by a trained person and a reverse transcription polymerase chain reaction test (rt-pcr) for pathogen identification. obtaining rt-pcr results is time consuming and can be cost-prohibitive, especially for developing countries, and is therefore currently often used in a targeted fashion, testing predominantly patients with covid-19 specific symptoms [1] . there is therefore a need for an additional faster, reliable, noninvasive, and versatile screening tool, especially to identify asymptomatic and pre-symptomatic individuals. several studies have proven the canines' extraordinary olfactory acuity to detect persons with infectious and non-infectious diseases like different types of cancer [2] , malaria [3] , bacterial, and viral infections [4] [5] [6] , with usually high rates of sensitivity and specificity [7] . a pathogen-specific odour that can be detected by dogs may be composed of specific patterns of volatile organic compounds (vocs). compared to bacteria, viruses have no own metabolism, and therefore vocs are released by infected body cells as a result of metabolic host processes [8] . different technical approaches have used the detection of vocs to discriminate infectious diseases successfully, but none is being used routinely in clinical practice [9] . as dogs can be trained quickly, the aim of the present study was to test the concept of using dogs reliably and in real-time to discriminate between samples of sars-cov-2 infected patients and non-infected controls. this method could be employed in public areas such as airports, sport events, borders or other mass gatherings as an alternative or addition to laboratory testing, thus helping to prevent further spreading of the virus or further outbreaks. saliva samples and tracheobronchial secretion samples were collected from hospitalised covid-19 patients that showed clinical symptoms and were diagnosed as sars-cov-2 positive using nasopharyngeal swab samples. negative control samples were obtained from sars-cov-2 rt-pcr negative people with no previous history of covid-19, nor had the individuals any history of a recent cold or infection. none of the samples were screened for different human coronaviruses like beta coronavirus hcov-oc43 or alpha coronavirus hcov-229e. after the sample acquisition, the anonymised samples were transported to the university of veterinary medicine hannover. all collected samples were confirmed as positive or negative using the rt-pcr sars-cov-2-ip4 assay from institut pasteur (recommended by the world health organization [10, 11] , including an internal control system and protocol as described [12, 13] . samples from covid-19 patients (irrespective of the final rt-pcr result) were further subjected to virus quantification (end point dilution assay) and virus isolation analysis using vero e6 cells under biosafety level 3 conditions. the cell layers were assessed for cytopathic effects and final results were obtained 7 days after cell infection. since dogs are susceptible to sars-cov-2 [14] all samples from covid-19 patients were inactivated using beta propiolactone (bpl) in order to protect the dogs and their handlers from infection during training. briefly, samples and reagents were kept at 4°c, 20 μl/ml nahco 3 (7.5%) was added, and samples were incubated for 10 min at 4°c. after addition of 10 μl/ml of 10% bpl, samples were incubated at 4°c for 70 to 72 h. hydrolysis of bpl was conducted at 37°c for 1 to 2 h. samples that showed a cytopathic effect before bpl inactivation using virus isolation or end point dilution assay were tested again after bpl inactivation and were confirmed to be inactivated. only bpl inactivated samples from covid-19 patients were used for the dog training. furthermore, detection dogs were provided both negative control samples with and without previous bpl treatment to exclude hydrolysed bpl as a potential distracting reagent. for the dog training, a volume of 100 μl per sample was pipetted onto a cotton pad, which was placed into a 4 ml glass tube. the presentation of the samples to the dogs was conducted via a device called detection dog training system (ddts; kynoscience ug, germany), which can present samples in a randomised automated manner without trainer interference. for a short video sequence, see additional file 1. ddts was utilised for training and testing. the device is composed of seven scent holes. behind each hole two tubes are leading to two metal containers. in the study, the first container enclosed the target sample and the second one carried the control sample. only one container is presented in each sniffing hole at any given time as the pairs of containers are situated on movable slides inside the device. the metal containers were covered with grids, which allowed the odour to escape and reach the sniffing hole. each tube extension was identical and lshaped, which prevented dogs from physical contact with the samples and excluded any visual cues that may have enabled further detection capabilities. for each trial run, only one hole presented a sars-cov-2 positive sample at a time while the other six holes presented negative samples. after the indication of the hole with the positive sample, the dog was automatically rewarded by the device with food or ball. the indication time was changed during successful training from 1 s to 2 s. while the reward was eaten, the device's software randomly and automatically assigned new positions to the slides for the following session with again only one hole presenting the positive odour sample. the dog, its handler and a person observing the study were blinded during the double-blinded study. all personnel stood behind the dog during the test runs to avoid distraction. the device recorded automatically the number and time length of each nose dip into the scent holes and the location of the positive and negative samples. this was verified by manual time-stamped video analysis. after a 2 weeks habituation process to the ddts, the eight dogs needed 5 days of training in total until the detection rate was above chance. an additional spreadsheet provides background information of the dogs used in the study (see additional file 2). the controlled doubleblinded detection study was then conducted after 7 days of training and in total 10,388 sample presentations ( table 1) . on each training day, unknown and known positive samples and negative control samples were presented to the canines. the response to the new sample was used in order to evaluate if the generalisation process has been achieved. while the dogs had only achieved an average detection rate of 50% on the second day of training, the values increased to 70% on day five and even 81% on day seven indicating a successful generalisation process. after completion of the training process, the detection accuracy of the eight trained dogs was evaluated in a randomised, double-blinded, and controlled study ( table 2) . samples from seven infected and seven healthy individuals were used in this study. two of the positive samples were tracheobronchial secretion, the other samples consisted of saliva. within randomised and automated 1012 sample presentations, dogs achieved an overall average detection rate of 94% (±3.4%) with 157 correct indications of positive, 792 correct rejections of negative, 33 false positive and 30 false negative indications. the canines discriminated between infected and non-infected individuals with an overall diagnostic sensitivity of 82.63% (95% confidence interval [ci]: 82.02-83.24%) and specificity of 96.35% (95% ci: 96.31-96.39%). sensitivity ranged from 67.9 to 95.2% and specificity from 92.4 to 98.9% (fig. 1) . there was no notable difference in detection ability between saliva and tracheal secretion (average hit rates 85.1 and 87.7%, respectively). timely and accurate detection of sars-cov-2 infected individuals is of uttermost importance for a society to control the pandemic. our data indicate that detection dogs can be trained in just about a week to discriminate between samples of people infected and non-infected by sars-cov-2. the average detection rate was 94%. analysis for accuracy and precision revealed a diagnostic sensitivity of 82.63% (95% ci: 82.02-83.24%) and a high diagnostic specificity of 96.35% (95% ci: 96.31-96.39%) for all dogs. all dogs had a high diagnostic specificity with a small range in variation, which could be important for population screening to avoid false positive results. however, there was quite a range in variation of sensitivity for the individual dog and inbetween dogs. this can in part be explained with the dogs' variable training background (see additional file 2), signalment, personality traits and short training period of 7 days. to avoid a bias concerning hospital specific smells, positive samples were obtained from two different hospitals to include a variation in a covariate factor and this appears to have not influenced the current results. understanding better why there is this range in sensitivity and how to potentially improve it would be important prior to considering the use of detection dogs in the field. in comparison, the current gold standard diagnostic rt-pcr test of a nasopharyngeal swab can, in trained hands, have a false detection rate of 25% and a false positive rate of 2.3-6.9% [16] . a new, not yet published study indicated a clear, nearly 100% voc specific pattern of sars-cov-2 infected individuals compared to negative controls and individuals infected by the influenza virus using multicapillary column coupled ion mobility spectrometry of breath [17] . this provides further indications that unique voc imprints exist and can be used for the development of diagnostic procedures. the current study results are promising, although they should be regarded as preliminary and suitability for this detection method in the field can only be acquired after further research has been conducted. our work provides the very first steps of the development of a new sars-cov-2 screening method. our inclusion criteria for the samples collected were rather non-specific and not stratified by severity of symptoms, disease status or virus load. future studies are needed to address this including a higher number of different samples to evaluate the analytical sensitivity (e.g. dilution of samples/detection level, different disease phenotypes and stages) and analytical specificity (differentiation to other lung diseases or pathogens such as cancer or infection with other seasonal respiratory virus infections, e.g. influenza, respiratory syncitial virus, adenovirus, other than sars-cov-2 coronaviruses, rhinovirus). in the current study negative control samples were acquired from healthy individuals without clinical signs of respiratory disease. the individuals were only tested for sars-cov-2 virus and therefore one cannot exclude that a former infection, especially with another human coronavirus like hcov-oc43 resulted in false positive indications of the dogs and that cross detection occurred. on the other hand, samples included in the current study were from severely affected, hospitalised covid-19 patients, but one of the main challenges in controlling the current pandemic is to identify pre-symptomatic covid-19 patients and asymptomatic carriers, which may constitute most covid-19 cases [18] . the sensitivity of detection by dogs may also vary across the course of the disease. future research should therefore focus on the ability of dogs to identify the different covid-19 disease phenotypes and phases of disease expression, such as asymptomatic, pre-symptomatic, mild and severe clinical cases as well as to test samples of the same individuals at different timepoints across the course of the disease. one of the most important requirements regarding handling of infectious samples is infection prevention and control. initially, it was assumed that dogs cannot get infected by sars-cov-2, but recent single cases have been reported showing that dogs can get infected by sars-cov-2 and could potentially play a role in viral spread [14, 19] . there is evidence of human-to-animal transmission with a subsequent infection of dogs. it is still unclear whether dogs can function as spreaders of the virus by infecting other animals or humans [14, 20] . nevertheless, this needs to be considered when using dogs for detection of infected material or people. it is also unclear how an infection in the dog will alter its sense of smell. in the current study we chose to use an inactivation procedure which should not affect vocs. however, this is not practical for testing in the field and we are currently developing new strategies for a secure presentation of non-inactivated samples. this would eliminate potential risks of virus transmission by detection dogs when used in a non-laboratory setting. detection dogs were able to discriminate respiratory secretions of infected sars-cov-2 individuals from those of healthy controls with high rates of sensitivity and specificity. the current pilot study had major limitations which needs to be elucidated in future studies. sars-cov-2 detection dogs may then provide an effective and reliable infection detection technology in various settings like public facilities and function as an alternative or addition to regular rt-pcr screening. in countries with limited access to diagnostic tests, detection dogs could then have the potential to be used for mass detection of infected people. further work is necessary to better understand the potential and limitation of using scent dogs for the detection of viral respiratory diseases. supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-05281-3. additional file 1: additional video. detection dog working with ddts. the video (additional file 1) shows the labrador retriever "seven" can be seen at the bottom of the video. the scent hole with a sample of an sars-cov-2 infected individual is marked in green on the video (please note the green mark was not seen by the dog and was only used in the video as a visualisation tool for the viewer to demonstrate the dog's search and detection behaviour). at each detection trial run only one hole is presenting the target scent with the other six holes presenting saliva samples from sars-cov-2 negative tested individuals. when the dog detects the target scent, the nose will be left within the hole for ≥2 s to indicate the detection. this will be recorded by the device. a beeping sound announces the food or ball reward, which is automatically ejected by the device, distracting the dog for a short time period. in the meantime, the device rearranges the sample presentation in an automatic and random fashion, presenting one other scent hole with a sample of a sars-cov-2 positive tested individual and six control scent holes with negative control samples. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review diagnostic accuracy of canine scent detection in early-and late-stage lung and breast cancers trained dogs identify people with malaria parasites by their odour using dog scent detection as a point-of-care tool to identify toxigenic clostridium difficile in stool canine detection of the volatilome: a review of implications for pathogen and disease detection realtime detection of a virus using detection dogs biomedical scent detection dogs: would they pass as a health technology? the human volatilome: volatile organic compounds (vocs) in exhaled breath, skin emanations, urine, feces and saliva the scent of disease: volatile organic compounds of the human body related to disease and disorder protocol: real-time rt-pcr assays for the detection of sars-cov-2 sensitivity assessment of sars-cov-2 pcr assays developed by who referral laboratories a universal heterologous internal control system for duplex real-time rt-pcr assays used in a detection system for pestiviruses infection of dogs with ars-cov-2 sensitivity, specificity, and predictive values: foundations, pliabilities, and pitfalls in research and practice. front pub health false positives in reverse transcription pcr testing for sars-cov-2. medrxiv rapid detection of sars-cov-2 infection by multicapillary column coupled ion mobility spectrometry (mcc-ims) of breath. a proof of concept study medrxiv community prevalence of sars-cov-2 virus in england during may 2020: react study the risk of sars-cov-2 transmission to pets and other wild and domestic animals strongly mandates a onehealth strategy to control the covid-19 pandemic. one health susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the members of the id-uke-covid-19 study group marylyn m. addo, etienne bartels, thomas t. brehm, christine dahlke, anahita fathi, monika friedrich, svenja hardtke, till koch, ansgar w. lohse, my l. ly, stefan schmiedel, l. marie weskamm, julian schulze zur wiesch at the university medical-center hamburg-eppendorf by helping us with recruitment of patients and sample collection. we further would like to thank stefan hampel, sina knisel and miguel acosta for support at the bundeswehr school of dog, german armed forces, ulmen during dog training and leander buchner from the central institute of medical services, german armed forces in koblenz for the support in getting samples. a special thanks goes to hans ebbers, kynosciences, for providing the ddts free of charge and for the support in dog training. we would like to thank our doctoral student saskia irene peek for her support during sample collection. special thanks go to our "doggy noses" coyote, elli, lotta, donnie, hoss, luigi, jo and seven. heartfelt thanks go to all the people providing us with samples, especially to the sars-cov-2 infected persons and their relatives with the sincere intention to contribute to the containment of covid-19 and to scientific progress. we wish you lots of strength and full recovery during the current pandemic. authors' contributions pj participated in the planning of the study, carried out the main practical work, data analyses and drafted the manuscript. cs participated in the planning of the study and carried out the laboratory work including rt-pcr and virus inactivation, as did vp. ft, sm and hav designed and coordinated the study, drafted the manuscript, conducted and coordinated (ft) the sample acquisition and were responsible for data analyses. mvkb and admeo participated in the planning of the laboratory part of the study and were in charge for the legal permission for sample processing. je programmed the ddts software. ip, tw, mpm, af and mma were in charge for the ethical approval, patient recruitment and sample collection (ip, af) at hannover medical school (ip, tw, mpm) and university medical-center hamburg-eppendorf (af, mma). es participated in the planning of the study, was responsible for the dog training and helped with data analyses. all authors have read and approved the final manuscript. not applicable. the datasets used and/or analysed during the current study are available at jendrny, paula, twele, friederike, schulz, claudia, meller, sebastian, von köckritz-blickwede, maren, volk, holger andreas. (2020). sars-cov-2 detection dogs -a pilot study [data set]. zenodo. https://doi.org/10.5281/zenodo. 3950074ethics approval and consent to participate the study was conducted according to the ethical requirements established by the declaration of helsinki. the local ethics committee of hannover medical school (mhh) and hamburg medical association at the university medical-center hamburg-eppendorf (uke) approved the study (ethic consent number 9042_bo_k_2020 and pv7298, respectively). written consent from all people were collected before sample collection. not applicable. the authors declare that they have no competing interests. key: cord-002560-pue5q5wp authors: moreno, paloma s.; wagner, josef; mansfield, caroline s.; stevens, matthew; gilkerson, james r.; kirkwood, carl d. title: characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics date: 2017-06-01 journal: plos one doi: 10.1371/journal.pone.0178433 sha: doc_id: 2560 cord_uid: pue5q5wp the virome has been increasingly investigated in numerous animal species and in different sites of the body, facilitating the identification and discovery of a variety of viruses. in spite of this, the faecal virome of healthy dogs has not been investigated. in this study we describe the faecal virome of healthy dogs and dogs with acute diarrhoea in australia, using a shotgun metagenomic approach. viral sequences from a range of different virus families, including both rna and dna families, and known pathogens implicated in enteric disease were documented. twelve viral families were identified, of which four were bacteriophages. eight eukaryotic viral families were detected: astroviridae, coronaviridae, reoviridae, picornaviridae, caliciviridae, parvoviridae, adenoviridae and papillomaviridae. families astroviridae, picornaviridae and caliciviridae were found only in dogs with acute diarrhoea, with astroviridae being the most common family identified in this group. due to its prevalence, characterisation the complete genome of a canine astrovirus was performed. these studies indicate that metagenomic analyses are useful for the investigation of viral populations in the faeces of dogs. further studies to elucidate the epidemiological and biological relevance of these findings are warranted. interest in the virome, or the entire population of viruses present in a biological sample, has increased recently due to improved availability of high throughput sequencing or next generation sequencing (ngs) technologies, and improved metagenomic analytical methods [1, 2] . a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the virome comprises all types of viruses, including those that infect prokaryotic and eukaryotic organisms, dna or rna viruses, and viruses that cause acute or chronic infections. many of these viruses are difficult or impossible to propagate in cell culture, and molecular detection is difficult as no common gene such as the ribosomal 16s gene that is present in bacterial species exists in viruses. these limitations have hindered the identification and characterisation of uncultured viruses [3, 4] . recently, due to the advent of molecular enrichment protocols, high throughput sequencing and new metagenomic analytical methods we are now able to explore, identify and characterise viruses from different biological and environmental samples with a greater capacity [2, [5] [6] [7] [8] [9] [10] [11] in studies of human faeces, the virome has been shown to include viruses that infect eukaryotic organisms and viruses that infect prokaryotes (bacteriophages) [2, 5, [12] [13] [14] [15] [16] [17] [18] . bacteriophages have been reported in many studies to be the most frequently detected viral constituent in the gut of humans [1, 2, 5, 8, 16, 19, 20] . the faecal virome has been characterised for several animal species including pigs, bats, cats, pigeons, horses and ferrets [2, 6, 7, [9] [10] [11] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . in dogs, the presence of enteric viral pathogens such as canine parvovirus, coronavirus, rotavirus and distemper virus (paramyxoviridae) have been identified only through targeted studies [32] [33] [34] [35] . to date, only one published study has used high throughput sequencing to investigate the faecal viral population in diarrhoeic dogs [6] . these investigators analysed faeces from dogs with acute diarrhoea and detected two new virus species, canine sapovirus and canine kobuvirus; known canine enteric viruses such as canine coronavirus, canine parvovirus, canine rotavirus as well as plant and insect viruses were also reported [6] . the aim of this study was to describe the faecal virome of samples collected from healthy dogs, and compare these findings to the faecal virome of dogs with acute diarrhoea in australia, using an illumina miseq shotgun metagenomic sequencing approach. a total of 16 faecal samples (8 from healthy and 8 from diarrhoeic dogs) were subjected to viral nucleic acid extraction, followed by nucleic acid enrichment, reverse transcription, random amplification and the creation of two libraries for each sample (dna and cdna), before being sequenced by illumina miseq platform (table 1) . after sequencing, a total of 93,744,624 raw sequences were generated. all raw sequences are available in ncbi, (bioproject id: prjna380672). after trimming by quality 80,414,313 high quality reads (hqrs) were available. all sequences corresponding to dog and cellular organisms (383,785 and 27,825,631 respectively) were removed and the resultant reads were de novo assembled (fig 1) generating in total 1,672,615 contigs and singletons (reads). from these contigs/singletons 1,285,171 (76.8%) had no hits in the database (s1 table) . further analysis of contigs/singletons with no hits confirmed most sequences had no hits, while a limited number matched bacterial, human or animal sequences with a very low coverage. in addition to the contigs/singletons with no hit in the database, some contigs/singletons matching to cellular organisms and some with low complexity were identified, however, were not analysed any further (s1 fig) . sequences similar to twelve viral families were identified in faecal samples from healthy and diarrhoeic dogs after analyses with two different bioinformatic pipelines and comparison against viral and ncbi databases (fig 1) . eight of these viral families infect eukaryotic organisms, and the remaining four infect prokaryotes. despite the known bias of sispa in the resultant sequences after de novo assembly [36], we report the number of contigs/singletons matching viral families and the subsequent analysis of alignments with the lowest common ancestor according to megan v5.2.1 [37]. faecal samples were collected from eight healthy dogs (table 1) . genetic analyses identified 659,696 contigs/singletons with no hits and 3968 contigs/singletons were classified as viral, matching to five viral families that infect eukaryotes and four that infect prokaryotes. 75.9% (3012 contigs/singletons) of the total number of viral contigs/singletons were classified as bacteriophages in the healthy canine faecal virome. bacteriophages were detected in the faeces of all dogs in this group and belonged to caudovirales order and microviridae family. viral contigs/singletons from five eukaryotic virus families were identified in faecal samples from 4 of the 8 healthy dogs (table 1) . three out of five viral families detected were dna viruses. adenoviridae and papillomaviridae were detected in a single sample containing only (table 1) , however, these were all detected in one sample. after analysis, only 76 contigs/singletons matched the reference sequence of alphacoronavirus 1 (feline infectious peritonitis virus, nc_002306.3) and covered only 0.5% of the complete genome (minimum match: 75% and minimum overlap: 50), which represented 3.2% of fipv_gp02 (receptor binding molecule) region. contigs/singletons belonging to the reoviridae family were found in only one sample. genetic analysis revealed they covered between 11%-35.5% of vp1, vp2, vp3 and vp4 genes of reference sequences of rotavirus a (nc_011506-nc_011510). another eukaryotic viral family found in one healthy dog sample was parvoviridae, genetic analysis of the 3 contigs/singletons showed a coverage of approximately 3.5% of the complete genome of canine parvovirus reference sequence (nc_001539), or 9.3% of the polyprotetin ns1-ns2. in eight faecal samples from dogs with acute diarrhoea, a total of 625,475 contigs/singletons had no hits and 17,242 were identified as viral contigs/singletons comprising 6 eukaryotic and 4 prokaryotic viral families (table 1) . bacteriophages comprised 98.19% of the total of viral contigs/singletons and they were present in all individuals and were identified as belonging to order caudovirales and microviridae family. eukaryotic families found in this group were coronaviridae, parvoviridae, reoviridae, caliciviridae, astroviridae, and picornaviridae ( table 1) . the most common eukaryotic viruses identified were rna viruses (5/6 viral families). interestingly, all 8 samples in this group contained at least one eukaryotic family each. co-infection was identified in 6 individual dog samples from this group. from the 8 samples from dogs with acute diarrhoea, 2 different eukaryotic virus families were detected in five samples and 3 eukaryotic families were detected in one sample ( table 1 ). the most prevalent family identified was astroviridae, present in 5 dogs followed by reoviridae present in 4 of 8 dogs with acute diarrhoea (table 1) . astroviridae contigs/singletons from 5 dogs were compared with reference sequence of canine astrovirus (nc_026814.1), the lowest common ancestor according to megan, and they covered between 2.4% and 5% of compete genome and between 6.3% and 12.9% of the orf2. the sample with the most contigs/singletons was later characterised. reoviridae contigs/singletons found in 4 dogs were compared with reference sequence of rotavirus a (nc_011503.2) and 3 of them covered between 10.5% and 23.4% of the vp4 gene and the other sample covered 23.6% of the vp7 gene. furthermore, contigs/singletons matching to canine parvovirus, were found in 2 dog samples with acute diarrhoea. one of the samples covered approximately 5.8% of the complete genome of canine parvovirus reference sequence (nc_001539), corresponding to 17.6% of vp2. the other sample contained 41 contigs/singletons that matched to this same reference sequence and in total they cover 100% of vp1, 66.2% of polyprotein ns1 and ns2 (cpvgp1) and 87.9% of vp2 genes. in this group were also found contigs/singletons matching to the coronaviridae family, covering between 0.6% and 1.7% of the complete genome of reference sequence alphacoronavirus 1 (fipv, nc_002306.3). one dog with acute diarrhoea contained contigs/singletons similar to a canine norovirus (jf930689.1), covering approximately 7.9% of the complete genome. other dog sample had contigs/singletons similar to a canine kobuvirus (jn387133.1), covering 2.2% of complete genome. to further explore the high abundance of contigs/singletons from astroviridae family in dogs with acute diarrhoea and their absence in healthy dogs, a near complete full genome of a representative canine astrovirus was generated through sanger sequencing (dd1, table 1 ). the genome encoded the complete three open reading frames (orfs): orf1a, orf1b and orf2. the total length was 6513 nucleotides, excluding the 3' poly (a) tail and the nucleotide composition was 28% a, 22% g, 26% t, 23% c. the g/c composition was 45%. the genbank accession number for the canine astrovirus sequence is kx756441. a phylogenetic tree was constructed using the protein alignment from the conserved region of the capsid (orf2) of the astrovirus characterised in this study (dd1) and other canine astrovirus orf2, together with mamastrovirus sequences from different mammalian species, including a chicken astrovirus as an outgroup. the phylogenetic analysis grouped our canine astrovirus within the canine astrovirus clade. the closest canine astroviruses to our australian sample were from uk and china with an identity between 98.82%-99.41% (fig 2) . using next generation sequencing and metagenomics analysis, the virome in faecal samples from 8 healthy dogs and 8 dogs with acute diarrhoea is described. only a single previous shotgun metagenomic study investigating the faecal virome of dogs with diarrhoea has been reported. in that study, mammalian viruses were found in 15 samples and two new virus species were described [6] . our study analysed 16 faecal samples from dogs (8 healthy and 8 diarrhoeic), and identified eukaryotic viruses in 12 samples, including all diarrhoeic samples and 50% of the healthy samples. thus, 70% of canine faeces contained eukaryotic viruses, suggesting that mammalian viruses are a common component of the enteric microbial population in dogs. our results must be interpreted with caution, due to bias created by sispa. areas of exaggerated depth appear when the sispa method is used, creating artefacts during de novo assembly. this results in regions of repetitive sequences [36] . in order to overcome this bias, all contigs/singletons were analysed at family level and only for viral eukaryotic families were the results further analysed to evaluate what percentage they covered to some specific viral species. the most common viral contigs/singletons identified in both groups were bacteriophages, similar to previous findings from human and other animal faecal virome studies [2, 5, [11] . bacteriophages modify diversity of bacterial populations due to their lytic life cycle and also promote different characteristics in the bacterial population due to their lysogenic life cycle transferring genes such as encoding toxins or resistance to antibiotic [38] . this life-cycle may lead to bacteriophages conferring advantage to some bacterial species in the environmental niche [39]. therefore, it is possible that the greater amount of contigs/singletons corresponding to bacteriophages identified in the group of dogs with acute diarrhoea, when compared to healthy dogs, means a higher amount of bacteriophages. if so, bacteriophages could have generated a change in the normal balance of bacterial population resulting in dysbiosis, and ultimately causing diarrhoea. conversely, it could be that an initial change in the bacterial population in these dogs resulted from the acute diarrhoea [40, 41] is the cause of the variation in the bacteriophage population. in our sample population, the latter explanation is most likely, because in a shelter environment a higher number of circulating pathogens, changes in diet and a stressful environment could contribute to the dysbiosis associated with acute diarrhoea [42] . the bacterial microbiome and the analysis of contigs/singletons matching specific bacteriophages were not assessed in this study, therefore further microbiome/virome cross analysis is necessary to elucidate the association between bacteria and bacteriophages in dogs. however, even this analysis would be unlikely to determine the cause or effect relationship between bacteriophages and dysbiosis at a single point in time. the analysis of the lowest common ancestor of eukaryotic viral families, according to megan, identified eight eukaryotic virus species (table 1) . however, each of these results require validation by targeted pcr, or whole genome characterisation of each species, as the ngs results after sispa amplification may be biased and not accurate depiction at a species level [36] . sequences matching those of human viruses (adenovirus and papillomavirus) were found in one sample from a healthy dog. only one contig from each virus covering a very small percentage of the genome in both cases. this finding could suggest contamination during collection or processing. known enteric pathogenic families parvoviridae and coronaviridae were identified in samples from both healthy dogs and dogs with acute diarrhoea. interestingly, almost all positive samples were from puppies (between 4-8 months) that had been vaccinated less than one month prior to sampling. the lowest common ancestry analysis in megan of the contigs/singletons matching parvoviridae family, suggested they were canine parvovirus (cpv), but as cpv positive dogs (as tested by faecal antigen tests) were not included in this study it is highly likely these results represent vaccine derived sequences not detected by the cpv antigen detection kit, or represent a virus load below the level of detection. previous studies have demonstrated that modified live vaccine virus can be detected in faecal samples for extended periods of time after vaccination [43] . further genome characterisation of these canine parvovirus is warranted to confirm this hypothesis. three individual samples contained coronaviridae contigs/singletons, two of which were from puppies [nd10 and dd8] ( table 1 ) and one from an adult dog [dd1] ( table 1 ). our results are consistent with li et al 2011, who also reported the highest number of coronaviridae reads in one sample collected from a puppy [6] . canine coronavirus can be shed in faeces in high numbers for up to 156 days [44, 45] . these findings validate the affinity of the coronaviridae viral family to infect young individuals [46] , and present as a common enteric pathogen in a shelter environment [42, 45, 47] . the uncommon viruses, canine kobuvirus and canine norovirus, were identified only in samples from dogs with acute diarrhoea. previous studies have suggested these viruses may have some association with enteric disease in dogs, however, both viral species have been detected in both healthy dogs and dogs with diarrhoea [6, 48] our shotgun metagenomic sequence data indicated that the most frequent rna viral family in dog samples with acute diarrhoea was astroviridae, being identified in more than half of the diarrheal samples. [49] . in dogs, astrovirus has been previously detected mainly in puppies with diarrhoea, but has also been occasionally reported in healthy dogs [50] [51] [52] [53] [54] . the only previous report of a possible canine astrovirus in australia was described in canine faeces in the 1984, where astrovirus-like particles were detected using electronic microscopy in healthy dogs [55] . to date, canine astrovirus has been reported in usa [56] , china [51] , italy [50, 57, 58] , uk [52] , france [53] , brazil [59] , korea [60] and japan [54] . the first description of the complete genome of two canine astroviruses was reported by a group of researchers from the uk in 2015 [52] . the current study contributes the first description of the complete genome of a canine astrovirus identified in australia. in our study, using sanger sequencing a near complete genome of a canine astrovirus was assembled from one dog with acute diarrhoea. a phylogenetic tree, analysing the capsid region (orf2) of this australian canine astrovirus and other astrovirus sequences present in genbank, determined that it belonged to the canine astrovirus clade, very closely related to the canine astrovirus strains from the uk and china (fig 2) . it is interesting to note that all canine astrovirus positive samples, were collected from the same shelter and obtained within a short period of time (sept-nov 2012). we could infer that this virus was endemic at that time in that shelter, and or maybe could represent an outbreak of diarrhoea in the shelter within that period of time. a more sensitive test (i.e.: quantitative pcr) in a larger number of samples from cases and controls may be useful to better understand the potential role of astroviruses as an aetiological agent in acute diarrhoea of dogs. in this study we analysed the faecal virome in healthy dogs and compared these findings with the faecal virome of dogs with acute diarrhoea. known dna and rna viruses were found, together with different proportions of bacteriophages in each group. in addition, we described and characterised the first complete genome of a canine astrovirus in australia. future longitudinal studies analysing viruses, bacteria and other potential pathogens should be performed to assess the aetiology of diarrhoea in dogs and further elucidate the pathological importance of viruses found in dog intestines. faecal samples from a total of 16 dogs were obtained between september 2012 and march 2013. all dogs were aged between 2.5 months and 7 years; and comprised 5 females and 11 males of various breeds ( table 1) . all faecal samples were collected from a single shelter in melbourne (lost dogs home), australia. all samples were maintained at 4ë�c for up to 24 hrs, then were transported on dry ice before storing up to five aliquots of 500 mg of faeces each at -80ë�c until further analyses. information about age, sex, breed, diet, vaccination and deworming status was recorded for each dog (university of melbourne animal ethics committee approval ids 1413272.2 and 1112035.1). animals were determined to be healthy based on physical examination by a veterinarian and absence of any clinical signs of disease. faecal consistency was considered normal as per published criteria (faecal scoring chart, purina), and all dogs had been treated with deworming drugs for prophylaxis (ilium pyraquantal, troy or milbemax, novartis). all samples were lifted from the floor, first thing in the morning before cleaning, during november 2012. faecal samples from 8 dogs with an acute onset of diarrhoea (less than 3 days of duration), were collected by a veterinarian from within the animal's enclosure. all dogs with acute diarrhoea were tested for the presence of canine parvovirus antigen in faeces using the anigen rapid cpv/ccv ag test kit, (bionote). positive samples were excluded from the study. none of the dogs had been treated with antimicrobial drugs within the previous 8 weeks of sample collection. the majority of healthy dogs were receiving commercial dry food and some of the dogs with diarrhoea were being fed a high-fibre prescription veterinary diet (hill's i/d diet). faecal samples were processed as described previously [6, 12] . briefly, aliquots of 500 mg of faecal sample were thawed and re-suspended in saline buffer (0.01m tris solution (ph7.5), 0.15m nacl, 0.01m cacl 2 ) at 3:1 ratio of solid mass. one mm zirconia/silica beads were added to the stool solution, filling around 150î¼l of an eppendorf tube, and vortexed vigorously for 3 minutes. the samples were then centrifuged at 17900 x g for 5 min, collecting the supernatant and repeating this step three more times. to reduce solid faecal matter and bacterial contamination, 500 î¼l of this solution was filtered through a 0.45 î¼m tube filter (corning costar spin x) by centrifugation at 3800 x g for 5 minutes, then the filtrate was transferred to 2 ml tubes. to enrich for viral dna and rna, a dnase/rnase step was incorporated using a modified protocol described previously [6, 12] . each sample was treated with a cocktail of dnases (turbo dnase, from ambion, baseline-zero from epicentre, benzonase from novagen and dnase i from roche) and rnase a (qiagen). this mixture was incubated in a water bath at 37ë�c for 3 hours. to stop the enzymatic activity, edta (amresco) was added in a final concentration of 15 mm to each sample and incubated at 75ë�c for 10 min. viral dna/rna protected from digestion within viral capsids were extracted using qiaamp viral rna mini kit (qiagen), according to manufacturer's recommendations. a second dnase/rnase step was performed on the extracted viral rna for elimination of genomic dna, using dnase i recombinant, rnase free (10u/î¼l) (roche) and protector rnase inhibitor (40 u/î¼l) (roche). after digestion of the dna, the viral rna was transcribed with sensiscript reverse transcriptase kit (qiagen; sensiscript rt kit) to generate cdna, according to manufacturer's instructions with minor modifications. briefly, for a more sensitive detection in the subsequent pcr, a mixture of oligo-dt primers (oligo (dt)15 primer, promega) and random primers (random hexamers, taqman reverse transcription reagents, roche, applied biosystems) were used and a rna denaturation step (95ë�for 3 minutes) was added. viral cdna and genomic dna were randomly amplified using a modified sispa protocol [61, 62] . briefly, a second strand synthesis was performed with large (klenow) fragment (new england biolabs) and random hexamers (roche, biosystems, 50î¼m) followed by digestion of the second strand product with the restriction enzyme cviqi (csp6.1), (new england biolabs). then a csp11/nbam24 adaptor was ligated to the digested dna using t4 dna ligase (invitrogen) followed by pcr amplification of the adaptor-ligated product with nbam24 pcr primers. an aliquot of the pcr product was validated on a 1% agarose tbe gel, where a positive smear with multiple bands confirmed the random sispa amplification of nucleic acid products. the amplified pcr products were cleaned up using wizard sv gel and pcr clean-up system (promega) following manufacturer's recommendations and two libraries with dual indexing for each sample were generated (dna and cdna) with illumina nextera xt dna sample preparation kit, according to manufacturer indications. after visualise it with agilent 2200 tape station system (agilent technologies), the libraries were submitted to the australian genome research facility (agrf) for a 250 bases paired-end sequencing on the miseq illumina platform. all raw sequences were deposited under bioproject id: prjna380672 at ncbi database. raw sequences were trimmed by quality score with prinseq software (v0.20.3) [63] , filtering for low quality reads from both ends using the dust score [64] with a threshold of 7. poly a/t tails in both ends (ten nucleotides of each end) and sispa primers sequences were also removed using this software. the mothur software v.1.31.2 [65] was applied and the sequences were trimmed again, eliminating homopolymers, ambiguous bases and sequences less than 100bp. after these trimming steps, high quality reads (hqr) were obtained and all bad quality reads were removed from the group file (fig 1) . subsequently, these dog free sequences were compared against a bacterial database (cam-era prokaryotic nucleotide database 10572.v7, nov 2012; http://camera.calit2.net/) [66] to eliminate bacterial sequences, using the blastn (blast 2.2.29+ standalone) algorithm with an 80% identity cut off. to extract cellular organism sequences from the group file, megan v5.2.1 and mothur software were used as described above (fig 1) . the host and bacteria free sequence reads, were de novo assembled with metavelvet (velvet 1.2.08, kmer51) [67] using kmer size 51 and contigs and singletons were created. these singletons were clustered with a 98% similarity using cd-hit-est (version.4.5.4 2011) [68] (fig 1) . all contigs and singletons clusters were analysed through two pipelines. (1) contigs and singletons clusters were compared against the camera viral nucleotide sequence database 10570.v9, using tblastx search with an e-value cut off 10 â��5 ; (2) contigs and singletons clusters were compared against the ncbi nucleotide database (2012) using blastn search with an e value cut off 1. all blast searches were performed in blast 2.2.29+ standalone. these files were then analysed by megan v5.2.1 [37] and the lowest common ancestor of known viral sequences were identified (fig 1) . finally, all viral contigs and singletons of eukaryotic organisms present in both analyses were aligned and compared with the ncbi reference sequence of the lowest common ancestor given by megan v5.2.1. all alignments were made using sequencher version 5.0.1 sequence analysis software (gene codes corporation, ann arbor, mi usa) with minimum match percentage of 70%-80% and minimum overlap 50 as assembly parameters, evaluating the percentage of coverage of the genome. all contigs and singletons with no hits were re-evaluated using online blastn with an e value cut off 10 and visualised with megan v5.2.1 to evaluate the alignment with its lowest common ancestor. in order to acquire the complete genome of canine astrovirus, multiple sets of primers were selected from the literature or designed based on sequences obtained from illumina reads (s2 table) . nucleic acids from a single faecal sample from a dog with acute diarrhoea (dd1), which had 18 contigs/singletons of canine astrovirus (after tblastx analysis) was used to determine the complete genome sequence. rna was extracted directly from the centrifuged sample after faecal extraction, previous to enrichment of viral nucleic acids as outlined before. rt-pcr was performed with superscript iii one-step rt-pcr system with platinum taq (invitrogenâ�¢). pcr conditions used were: 45ë�c for 60 min and 95ë�c for 5 min, 35 cycle of 94ë�c for 40 sec, 55ë�c for 1min and 72ë�c for 5 min, and a final elongation step of 72ë�c for 5 min, followed by final hold at 4ë�c. pcr products were run on a 1.2% agarose tbe gel stained with redsafeâ�¢ nucleic acid staining solution (intron biotechnology). all pcr products were excised and cleaned up with wizard sv gel and pcr clean-up system (promega) following manufacturer's protocol and sequenced using sanger sequencing at the agrf. the near complete genome of the canine astrovirus was assembled using sequencher version 5.0.1 sequence analysis software (gene codes corporation, ann arbor, mi usa) with minimum match percentage 80 and minimum overlap 50 as assembly parameters. phylogenetic analysis of this canine astrovirus was performed aligning protein sequences of the 172 conserved amino acids of the capsid region (orf2) from different species (s2 fig), using clustal w, from mega version 6.0 [69] with default settings. a phylogenetic tree with 1000 bootstrap was generated using the maximum likelihood method based on the jtt matrix-based model [70] , using mega version 6.0. the percentage of identity was calculated with clustalo 1.2.4 [71] supporting information s1 fig. megan viruses in the faecal microbiota of monozygotic twins and their mothers metagenomic analyses of an uncultured viral community from human feces viral metagenomics. reviews in medical virology viral metagenomics as an emerging and powerful tool in veterinary medicine. veterinary quarterly viral diversity and dynamics in an infant gut viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses the human gut virome: inter-individual variation and dynamic response to diet the fecal virome of pigs on a high-density farm feline fecal virome reveals novel and prevalent enteric viruses viral metagenomic analysis of feces of wild small carnivores metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis rna viral community in human feces: prevalence of plant pathogenic viruses metagenomic analysis of human diarrhea: viral detection and discovery study of the viral and microbial communities associated with crohn's disease: a metagenomic approach characterization of microbial dysbiosis and metabolomic changes in dogs with acute diarrhea common and emerging infectious diseases in the animal shelter. veterinary pathology long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination m gene evolution of canine coronavirus in naturally infected dogs. the veterinary record an update on canine coronaviruses: viral evolution and pathobiology canine coronavirus-associated puppy mortality without evidence of concurrent canine parvovirus infection infectious diseases of the dog and cat novel norovirus in dogs with diarrhea fields virology,. 1. 6th ed. philadelphia detection and characterization of canine astroviruses isolation and characterization of canine astrovirus in china complete genome sequence of canine astrovirus with molecular and epidemiological characterisation of uk strains prevalence and risk factors of astrovirus infection in puppies from french breeding kennels detection of canine astrovirus in dogs with diarrhea in japan viruses and virus-like particles in the faeces of dogs with and without diarrhoea astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea enteric disease in dogs naturally infected by a novel canine astrovirus molecular characterisation of calicivirus and astrovirus in puppies with enteritis phylogenetic analysis of astrovirus and kobuvirus in korean dogs a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species sequence-independent, single-primer amplification (sispa) of complex dna populations quality control and preprocessing of metagenomic datasets a fast and symmetric dust implementation to mask low-complexity dna sequences introducing mothur: opensource, platform-independent, community-supported software for describing and comparing microbial communities community cyberinfrastructure for advanced microbial ecology research and analysis: the camera resource metavelvet: an extension of velvet assembler to de novo metagenome assembly from short sequence reads cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences mega6: molecular evolutionary genetics analysis version 6.0 the rapid generation of mutation data matrices from protein sequences. computer applications in the biosciences fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega the authors gratefully acknowledge the helpful assistance of all staff at lost dogs home and university of melbourne u-vet werribee animal hospital with the faecal sample collection. also to dr celeste donato for her valuable scientific input and her assistance with the construction of the astrovirus phylogenetic tree. minot key: cord-011669-hkkpw2bl authors: rodríguez-sánchez, diego noé; pinto, giovana boff araujo; thomé, edval fernando; machado, vânia maria de vasconcelos; amorim, rogério martins title: lissencephaly in shih tzu dogs date: 2020-06-20 journal: acta vet scand doi: 10.1186/s13028-020-00528-0 sha: doc_id: 11669 cord_uid: hkkpw2bl background: lissencephaly is a brain malformation characterized by smooth and thickened cerebral surface, which may result in structural epilepsy. lissencephaly is not common in veterinary medicine. here, we characterize the first cases of lissencephaly in four shih tzu dogs, including clinical presentations and findings of magnetic resonance imaging of lissencephaly and several concomitant brain malformations. case presentation: early-onset acute signs of forebrain abnormalities were observed in all dogs, which were mainly cluster seizures and behavioral alterations. based on neurological examination, the findings were consistent with symmetrical and bilateral forebrain lesions. metabolic disorders and inflammatory diseases were excluded. magnetic resonance imaging for three dogs showed diffuse neocortical agyria and thickened gray matter while one dog had mixed agyria and pachygyria. other features, such as internal hydrocephalus, supracollicular fluid accumulation, and corpus callosum hypoplasia, were detected concomitantly. antiepileptic drugs effectively controlled cluster seizures, however, sporadic isolated seizures and signs of forebrain abnormalities, such as behavioral alterations, central blindness, and strabismus persisted. conclusions: lissencephaly should be considered an important differential diagnosis in shih tzu dogs presenting with early-onset signs of forebrain abnormalities, including cluster seizures and behavioral alterations. magnetic resonance imaging was appropriate for ante-mortem diagnosis of lissencephaly and associated cerebral anomalies. lissencephaly in mammals occurs due to the failure of neuroblasts to migrate to the cerebral cortex, during development [1, 2] . it is characterized by smooth cortical appearance and by the absence of surface folds (agyria) or abnormally broad folds (pachygyria). histopathology demonstrates thickening of the cerebral cortex, altered gray-to-white matter ratio and replacement of a normal 6-layered cortex with a 4-layered disorganized cortex [2, 3] . two types of lissencephaly can be distinguished in humans: classical lissencephaly (or type i), characterized by thickened brain surface with agyria or pachygyria that results from neuronal migration arrest [4, 5] and cobblestone lissencephaly (or type ii), characterized by thin and nodular brain surface, resulting from glial and neuronal overmigration [4, 5] . muscular dystrophy, ocular alterations, obstructive hydrocephalus, and malformation of the brainstem and cerebellum are often associated with cobblestone lissencephaly [4] [5] [6] . lissencephaly has been described in lhasa apso [7, 8] , pekingese [9] , australian kelpie [10] , wire-haired fox terrier [11] , irish setter [11] , and mixed-breed dogs [12] . in humans, lissencephaly is associated with gene mutations related to brain development or cerebral metabolism [1, 5, 13] . in addition, nongenetic causes, such as intrauterine viral infections, vascular events (hypoxia or hypoperfusion), and maternal metabolic disorders that interrupt cortical formation, have been described [2, 3, 13] . the neurological signs in dogs commonly begin with an early-onset of seizures and behavioral alterations, leading to disability [7, 8, 10] . clinical findings and magnetic resonance imaging (mri) features of lissencephaly in shih tzu dogs have not been reported previously, and reports of concomitant brain malformations are scarce. four apparently unrelated shih tzu dogs were presented with lissencephaly between 2011 and 2018 at the veterinary neurology service of são paulo state university (unesp), brazil. details regarding clinical features, neurolocalization, ancillary diagnostics and antiepileptic treatment are shown in table 1 . mri was performed using a 0.25 tesla scanner (vet-mr grande, esaote, italy) in all dogs to obtain t1-weighted, t2-weighted, fluid-attenuated inversion recovery (flair) and postcontrast t1 sequences. in addition, gradient echo (gre) sequences were obtained in three dogs, and hybrid contrast enhancement (3d hyce) sequences were obtained in two dogs. all mris were evaluated and interpreted by two researchers (ra and vm). a summary of the mri findings, equipment, positioning, sequences, imaging parameters, and the contrast medium are detailed in additional file 1. the first case was an 8-month-old spayed female referred in 2011 due to seizures of suspected idiopathic origin that were poorly controlled with phenobarbital (gardenal ® , 2.5 mg/kg, orally q12h; safoni, brazil). the dog was evaluated by the referring veterinarian 2 months after the onset of tonic-clonic seizures, which had progressed over the last 2 weeks to 2-3 seizures per week. in our service, the owner reported that the dog experienced cluster seizures that occurred over 48 h and were treated with diazepam and thiopental. on presentation, neurological examination was performed 1 week after cluster seizures and was unremarkable. based on a history of seizures, the lesion was localized to the forebrain. physical examination findings, biochemical profile, and complete blood count (cbc) were normal; polymerase chain reaction (pcr) for canine distemper virus in urine and indirect immunofluorescent antibody tests (ifat) for antibodies against toxoplasma gondii and neospora caninum were negative. mri showed mixed parieto-occipital agyria and pachygyria of the frontal and parietal lobes (fig. 1 ). temporal and occipital regions lacked gyri and sulci. mri diagnosis indicated lissencephaly and supracollicular fluid accumulation (sfa) (fig. 1) . treatment with phenobarbital (gardenal ® , 3 mg/kg, orally q12h; safoni, brazil) and levetiracetam as an adjunct (keppra ® , 20 mg/kg, orally q8h; ucb biopharma, brazil) effectively controlled cluster seizures after presentation. levetiracetam was discontinued after 4 weeks. the neurological signs were nonprogressive and this dog experienced only isolated episodes (interictal period of 2-3 months) over a period of 24 months after diagnosis of lissencephaly (> 50% reduction in the frequency of seizures). it was not possible to obtain information regarding survival for this dog. the second case involved a 43-month-old castrated male which was referred in 2014 due to the occurrence of cluster seizures. the dog was diagnosed by the referring veterinarian with presumed idiopathic epilepsy at 24 months of age and treatment with phenobarbital was then initiated (gardenal ® , 2.5 mg/kg, orally q12h; safoni, brazil). during the first 16 months after diagnosis of presumed idiopathic epilepsy and the onset of treatment, the dog experienced both isolated seizures and cluster seizures, with an interictal period less than 30 days. relatively low levels of phenobarbital (< 25 µg/ ml: therapeutic window 25-35 µg/ml) were detected in serum; therefore, the dose of phenobarbital was gradually increased (gardenal ® , 4 mg/kg, orally q12h; safoni, brazil). however, despite the increase in serum phenobarbital concentration (35 µg/ml), the dog continued to have repeated tonic-clonic seizures. therefore, potassium bromide (kbr) (30 mg/kg orally, q24h) was also prescribed 1 month before referral to unesp. on presentation in our service, in addition to cluster seizures the owner reported behavioral changes between seizures, such as difficulties in learning basic commands, changes in sleep cycle and compulsive pacing. aggression was noted in our service during manipulation for physical examination. during anamnesis, the owner reported polyuria, polydipsia and polyphagia. biochemical profile showed increased levels of alkaline phosphatase (216: reference interval 20-156 u/l). these alterations were presumed to be associated with phenobarbital treatment. neurological examination revealed no abnormalities except for the presence of bilateral central blindness and bilateral ventromedial strabismus during cranial nerve examination. physical and ophthalmological examinations were normal. the anatomical neurolocalization was compatible with a forebrain lesion. pcr in urine for canine distemper virus and ifat in the serum for t. gondii and n. caninum were negative. mri showed absence of sulci and gyri with superficial undulations in the frontal and temporal lobes. the main gyri, including the marginal, ectomarginal, suprasylvian, and ectosylvian gyri, were absent. a rudimentary lateral rhinal sulcus was present, while the cingulate gyrus was not apparent. the internal capsule was abnormally small (fig. 1) . a diagnosis of lissencephaly, internal this dog remained stable and neurological signs were nonprogressive for 36 months after diagnosis of lissencephaly (interictal interval of 3-4 months with > 50% reduction in the frequency of seizures) using combined polytherapy involving both phenobarbital (gardenal ® , 4 mg/kg, orally q12h; safoni, brazil) and kbr (30 mg/ kg, orally, q24h). levetiracetam (keppra ® , 20 mg/kg, orally q8h for 4-6 weeks, ucb biopharma, brazil) was initially included as an adjunct treatment modality. no more cluster seizures were reported after presentation. a carbonic anhydrase inhibitor (acetazolamide, diamox ® , 10 mg/kg, orally q8h, genom, brazil) and a proton-pump inhibitor (omeprazole, gaviz ® , 10 mg/dog, orally q24h, agener, brazil) were used for supportive treatment of hydrocephalus. however, the difficulties in learning basic commands, changes in sleep cycle, compulsive pacing, strabismus and aggression were persistent despite treatment. phone conversation with the owner revealed that the dog was alive 6 years after diagnosis. the third case involved an 18-month-old intact male which was referred in 2014 due to the occurrence of cluster seizures starting 12 days prior to referral. the dog was previously treated with phenobarbital (gardenal ® , 6 mg/kg, orally q12h; safoni, brazil); however, due to poor response to treatment, adjunctive therapy with kbr (40 mg/kg, orally, q24h) was initiated. on presentation, anamnesis revealed that 6 months prior to referral, the dog had experienced over 14 isolated seizures within 45 days and subsequent episodes monthly. at evaluation, the dog experienced two tonic-clonic seizures, and emergency treatment was provided using diazepam (1 mg/kg, per rectum and repeated iv bolus 3x). during the 48-h postictal re-evaluation, neurological examination revealed central blindness, and the owner reported that the dog demonstrated abnormal vocalizations. in addition, aggressiveness during the interictal period was noted, mainly during dog handling. anatomical neurolocalization was consistent with a forebrain lesion. physical and ophthalmological examination was unremarkable. laboratory data, including pcr in urine for canine distemper virus and ifat in the serum for t. gondii and n. caninum were negative. mri of the third dog showed presence of some sulci in the temporal lobe, including the caudal sylvian gyri and lateral rhinal sulci. however, the main gyri, including the marginal, ectomarginal, suprasylvian gyri, and suprasylvian sulcus (division between the parietal and temporal lobe), were absent (fig. 2) . the internal capsule was abnormally small. diagnosis was consistent with lissencephaly, asymmetrical internal hydrocephalus, and corpus callosum hypoplasia. this dog showed progressive reduction in isolated seizures throughout the year following diagnosis (> 50% reduction in seizure frequency) with an interictal interval of 2 months, maintaining combined polytherapy involving phenobarbital (gardenal ® , 6 mg/kg, orally q12h; safoni, brazil) and kbr (30 mg/kg, orally, q24h). serum concentration of phenobarbital was not tested due to financial constraints. no more cluster seizures were observed with combined polytherapy. a carbonic anhydrase inhibitor (acetazolamide, diamox ® , 10 mg/kg, orally q8h, genom, brazil) and a proton-pump inhibitor (omeprazole, gaviz ® , 10 mg/dog, orally q24h, agener, brazil) were used for supportive treatment of hydrocephalus. behavioral changes and central blindness persisted despite treatment. overall survival was 6 years after diagnosis, confirmed by the owners after our phone call. the fourth case was an 18-month-old spayed female which was referred with seizures in 2018. the referring veterinarian suspected meningoencephalitis of unknown etiology and reported poor control of seizures with phenobarbital (gardenal ® , 2 mg/kg, orally q12h; safoni, brazil). prednisolone treatment (predsim ® , 0.5 mg/kg, orally q12h; medley, brazil) was added empirically 1 week prior to referral by the referring veterinarian. in our service, the owner reported the first isolated tonic-clonic seizure when the dog was 9 months old. after the interictal period of 9 months, the owner reported that these seizures began again 3 weeks prior to examination. over the 3-week period before examination, during the interictal periods, subtle behavioral changes such as aggressiveness (mainly after handling), compulsive pacing, abnormal vocalizations, and licking at things were observed. the dog presented with cluster seizures in our service and was treated with diazepam. after stabilization, the postictal state in the following 24 h was characterized by ataxia, paresis, and behavioral signs. upon neurological examination after postictal stage, consciousness, posture, gait, and postural reactions were normal. cranial nerve examination showed bilateral central blindness and bilateral ventromedial strabismus. the findings were consistent with a forebrain lesion. physical and ophthalmological examination findings, biochemical serum profile and cbc test results were unremarkable. thorax radiography and abdominal ultrasound did not show alterations. pcr in urine for canine distemper virus and , h) in the third and fourth cases. mri of the third dog showed a few sulci in the temporal lobe, including the caudal sylvian gyri and lateral rhinal sulci (arrow). the main gyri (including the marginal, ectomarginal and suprasylvian) and the suprasylvian sulcus were absent. the internal capsule was abnormally small, and internal hydrocephalus was visualized (a-d). mri of the fourth dog showed generalized agyria with an absence of sulci and thickened gray matter with smooth appearance (arrow) (e, f). rhinal sulci in the temporal lobe were not apparent and cingulate gyrus was absent (arrowhead). internal hydrocephalus and supracollicular fluid accumulation associated with dorsocaudal outpocketing of the third ventricle (type sfa-iii) were observed (g, h) ifat in the serum for t. gondii and n. caninum were negative. mri showed generalized absence of sulci and gyri and thickened gray matter. the main sulci and gyri, including the marginal, ectomarginal, suprasylvian, ectosylvian, and lateral rhinal sulci, were absent. the cingulate gyrus was not recognizable, and the subcortical internal capsule was abnormally small (fig. 2) . the diagnosis was consistent with lissencephaly with sfa, internal hydrocephalus and corpus callosum hypoplasia. prednisolone was then discontinued due to lack of indication. clinical presentation and mri did not support a diagnosis of meningoencephalitis. after dose readjustment with phenobarbital (gardenal ® , 2.7 mg/kg, orally q12h; safoni, brazil) and levetiracetam as an adjunct (keppra ® , 20 mg/ kg, orally q8h for 4 weeks; ucb biopharma, brazil), seizure frequency was reduced for the first 3 months (> 50% frequency reduction of seizures). adjunct treatment with levetiracetam was discontinued after approximately 4 weeks. at the 12-month follow-up examination, seizure reduction with an interictal interval over 2-3 months was noted and no more cluster seizures were observed. however, the behavioral changes, blindness and strabismus observed were persistent despite treatment. a phone conversation with the owners revealed that the dog was alive at the time of writing this report 2 years after diagnosis. malformations of cortical development have rarely been reported in dogs, but epileptic seizures seem to be a frequent clinical sign in affected dogs [7, 9, 10] . lissencephaly represents an uncommon disorder of the cortical gyration in dogs [7, 9, 10] . lissencephaly without concurrent intracranial malformations was described in lhasa apso dogs, in an australian kelpie dog and in a small mixed-breed dog [7, 10, 12] . this report is the first on lissencephaly in shih tzu dogs with other concurrent intracranial malformations and the first report providing a detailed neuroanatomical description of lissencephaly identified on mri. in humans, malformations of cortical development can lead to lissencephaly type i, characterized by pachygyria or agyria with thickened and smooth brain surface [4, 5] . apart from lissencephaly, cortical development disorders that may cause epilepsy as subcortical band heterotopia, polymicrogyria, and cobblestone malformations have been described [1, 2] . such disorders are rarely reported in dogs [14] . four shih tzu dogs without any known close relationship, were referred because of epileptic seizures. the dogs were not selected among breeds. the onset of seizures was at the age of 6, 9, 12 and 24 months. previous studies have reported the onset of seizures in dogs between the age of approximately 12 months and 5 years in two lhasa apso [7, 8] , one pekingese [9] , one australian kelpie [10] , and one mixed-breed dog [12] . in humans with lissencephaly, approximately 83% of patients experience earlyonset seizures earlier than 7-months-old [15] . neuro-ophthalmological abnormalities have been poorly characterized in dogs with lissencephaly. central blindness was previously reported [7, 9] . we detected central blindness in three out of four dogs. bilateral vision deficits were thought to derive from occipital lobe lesions. bilateral ventromedial strabismus (esotropia) was detected in two out of four dogs. only one other report has described ventromedial strabismus in a dog with lissencephaly, which was associated with an orbital anatomical abnormality or short medial rectum muscle [7] . we assumed that maldevelopment of the primary visual cortex and visual motor control mechanism may lead to esotropia [16] . humans with lissencephaly have visual abnormalities, including no ocular fixation or tracking, poor visual tracking, nystagmus, variable esotropia, oculomotor apraxia, optic nerve and macular hypoplasia/ atrophy, delayed visual maturation, and cortical visual impairment [17] . approximately 67% of human patients with lissencephaly type i have neuro-ophthalmological abnormalities [17] . severe mental retardation may be observed in humans with lissencephaly and is associated with neuronal migration defects in spatial learning areas [3, 5, 18] . motor disability as early hypotonia, spastic tetraplegia and opisthotonos is observed in human cases of lissencephaly due to defects in motor areas [3, 5] . humans with lissencephaly often die before adulthood [1, 6, 17] . in our study, early onset of several behavioral changes was detected in three dogs between 6 and 24 months old. alterations in locomotor behavior (pacing and changes in sleep pattern), aggressiveness (irritability to manipulation) and vocalization in addition to seizures were observed. previous studies observed late-onset behavioral changes in dogs with lissencephaly over 12-months-old [7] [8] [9] [10] 12] , aggressiveness being frequently reported [7, 9, 10] . the different clinical pictures between human and canine lissencephaly could be explained by the fact that the cerebral cortex is less essential in dogs than in humans for motor function [11] . in dogs, motor function may be maintained despite frontoparietal lobe (motor area) and pyramidal system lesions. however, cognitive and learning abilities may be affected [11] . magnetic resonance imaging in dogs with lissencephaly showed thickened cortical gray matter with smooth appearance, an abnormally small internal capsule, and absence of the major gyri and sulci when compared with healthy shih tzu dogs (additional file 2). previous studies did not report detailed neuroanatomical examinations of cortical gyri and sulci in dogs with lissencephaly [7, 9, 10] . in addition, mri was informative for correct ante-mortem diagnosis of multiple congenital anomalies, determination of cerebral morphology, and the degree of lissencephaly in shih tzu dogs. lissencephaly is graded in humans using a 6-point grading system based on the severity and anterior-posterior brain gradient of the abnormalities. grade 1 represents severe lissencephaly with complete agyria, while grade 6 represents mild subcortical band heterotopia [3, 5] . grades 1a to 6a are more severe posteriorly and 1b to 6b anteriorly [3, 5] . using these criteria, the second (fig. 1e) , third (fig. 2a) and fourth (fig. 2e) cases presented here were classified as grade 2a with diffuse agyria and few shallow undulations in the frontal and temporal lobes. the first dog was classified as grade 3a due to mixed parieto-occipital agyria and frontal pachygyria (fig. 1a) . previous studies have reported lissencephaly grade 2a [9] and 2b [10] in dogs. in our study, behavioral alterations and central blindness were noted in dogs with more severe lissencephaly grade, indicating a possible correlation between mri severity and clinical signs. the grade of lissencephaly was not related to the severity of the clinical signs in previous studies [9, 10] . two forms of lissencephaly have been described in humans: classic lissencephaly (or type i), characterized by abnormal thick cortical layers (four layered, with a cell-sparse zone), agyria or pachygyria without malformation of the brainstem and cerebellum [2] . mutations in cytoskeletal genes, such as the platelet activating factor acetylhydrolase 1b regulatory subunit 1, doublecortin, and tubulin a1a genes [1, 2, 5] , can lead to lissencephaly in humans and mice [1, 5] . cobblestone lissencephaly (type ii) is characterized by multiple shallow furrows, a thin cerebral mantle and malformation of the brainstem and the cerebellum [2, 5] . in dogs, genetic mutations for lissencephaly have not yet been described [7, 11] . in our study, muscular and ocular disorders were not observed and mri did not showed nodular brain surface and brainstem or cerebellum malformations associated to cobblestone lissencephaly [4] [5] [6] . therefore, our dogs likely have lissencephaly analogous to type i human lissencephaly [2, 5, 19] . lissencephaly is overrepresented in the lhasa apso dogs and was also reported in the genetically related pekingese breed [7, 9] . considering the genetic relation among lhasa apso, pekingese and shih tzu breeds [20] , it is possible that lissencephaly may be a genetic disease in dogs. hydrocephalus and sfa are intracranial malformations most often reported in young and toy breed dogs [21] [22] [23] . enlarged ventricles (a condition known as ventriculomegaly) comprise a common finding in adult brachycephalic dogs [21, 23] . dogs with only ventriculomegaly do not have increased intraventricular pressure and are considered to be asymptomatic [21, 23] in our study, internal hydrocephalus was confirmed based on specific mri features that indicated increased intraventricular pressure in three dogs [21] . hydrocephalus with concomitant lissencephaly has not been reported in dogs. in dogs, lissencephaly has been diagnosed together with ventriculomegaly rather that with hydrocephalus [7, 10, 12] . in a human study, ventriculomegaly has been observed in 73.7% of patients with lissencephaly type i [15] . supracollicular fluid accumulation without concomitant lissencephaly has been reported in male brachycephalic dogs, and the shih tzu dog breed was most often reported [22] . in dogs, most sfas are associated with dorsocaudal outpocketing of the third ventricle (sfa-iii) [24] . an expansion of both the third ventricle and the quadrigeminal cistern is another type of sfa (sfa iii-qc) [24] . a few cases present with enlargement of the sole quadrigeminal cistern (sfa-qc) [24] . previously, it was hypothesized that type sfa-iii in predisposed breeds can be part of hydrocephalus rather than an anomaly itself [24] . in our study, the first dog presented with sfa-qc and type sfa-iii was detected in the second and fourth dogs. although sfa can be related to the presence of neurological signs in dogs with lissencephaly, the clinical significance of sfa is variable and sfa may be incidental in dogs with other intracranial diseases [22] . there is no reported genetic relationship between sfa and lissencephaly in neither dogs nor humans. in humans, supracollicular fluid accumulation is associated with a defect in leptomeninges development [19] . corpus callosum abnormalities have been sporadically reported and are still poorly understood in dogs, typically being an isolated abnormality or associated with holoprosencephaly and inborn errors of metabolism. the most frequent clinical signs described are hypodipsia/ adipsia, tremors, and seizures [25] . we observed concurrent corpus callosum hypoplasia in the second, third, and fourth dogs, which is not commonly reported in dogs with lissencephaly [11] . epileptic seizures may be related to corpus callosum hypoplasia. in humans, corpus callosum abnormalities are associated with classic lissencephaly (type i), cobblestone lissencephaly (type ii) and polymicrogyria [2, 15] . the antiepileptic drugs used resulted in good control of cluster seizures with mild adverse effects in the long-term follow-up (12-36 months). seizures were persistent, although, compared to pretreatment, a reduction in the frequency (> 50% or more) and severity of seizures as well as cessation of cluster seizures was observed. other signs of forebrain abnormalities, such as behavioral alterations, central blindness, and bilateral strabismus were persistent. several medications, including carbonic anhydrase inhibitor acetazolamide and proton-pump inhibitor omeprazole, with the goal of decreasing cerebrospinal fluid production, have been proposed as medical management in dogs with hydrocephalus [23, 26, 27] . experimental studies in healthy dogs and rabbits reported that cerebrospinal fluid production was reduced after acetazolamide and omeprazole treatment, respectively [26, 28] . due to hydrocephalus, the second and third dogs were medically treated with acetazolamide and omeprazole. nevertheless, aggressiveness, changes in sleep cycle, compulsive pacing, central blindness, and strabismus were persistent during the follow-up period despite treatment with acetazolamide and omeprazole. one previous report has described no significant effects on recovery of the neurological signs or ventricular volume reduction after treatment with acetazolamide in dogs with hydrocephalus [29] . furthermore, chronic oral omeprazole therapy in healthy dogs did not affect cerebrospinal fluid production [30] . the major limitation of this study was the lack of histopathological evaluation, which was not possible because dogs were not euthanized during the follow-up period. although transcranial ultrasonography or computed tomography may aid in diagnosis, these methods are not precise [9, 10] . in our study, the use of low-field mri provided a good resolution for ante-mortem diagnosis of lissencephaly in all dogs. variations in signal intensity, identification of abnormal cortical layers and depth of cortical sulci were observed. all anatomical structures of the cerebral cortex were identified (additional file 2). in agreement, previous studies performed diagnosis in dogs using mri fields between 0.4-and 1.5-tesla [7, 9, 10] . mri is the modality of choice for lissencephaly diagnosis and for the differentiation of other neuronal migration disorders, showing a correlation with histopathological features in dogs and humans [1, 2, 7] . lissencephaly should be considered an important differential diagnosis in shih tzu dogs presenting predominantly with early-onset signs of forebrain abnormalities, including tonic-clonic seizures, behavioral alterations, central blindness, and bilateral ventromedial strabismus. low-field mri may be a useful diagnostic tool to detect cases of lissencephaly. hydrocephalus, sfa, and corpus callosum hypoplasia comorbidities could also be associated with lissencephaly in shih tzu dogs. genetic malformations of cortical development a developmental and genetic classification for 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domestic dog magnetic resonance imaging signs of high intraventricular pressure-comparison of findings in dogs with clinically relevant internal hydrocephalus and asymptomatic dogs with ventriculomegaly clinical and magnetic resonance imaging characteristics of quadrigeminal cysts in dogs congenital hydrocephalus multidetector computed tomographic and low-field magnetic resonance imaging anatomy of the quadrigeminal cistern and characterization of supracollicular fluid accumulations in dogs corpus callosal abnormalities in dogs inhibition of cerebrospinal fluid formation by omeprazole different effects of omeprazole and sch 28080 on canine cerebrospinal fluid production effect of certain drugs on cerebrospinal fluid production in the dog effect of acetazolamide and subsequent ventriculo-peritoneal shunting on clinical signs and ventricular volumes in dogs with internal hydrocephalus evaluation of the effect of oral omeprazole on canine cerebrospinal fluid production: a pilot study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are grateful to heraldo andré catalan rosa for technical support with mri. data have not been published previously. supplementary information accompanies this paper at https ://doi. org/10.1186/s1302 8-020-00528 -0.additional file 1: table s1 . doc. summary of magnetic resonance imaging (mri) findings of lissencephaly and concomitant congenital malformations in shih tzu dogs. details regarding malformation type, mri scan, positioning of the patient, sequence types, imaging parameters and contrast medium are described.additional file 2: figure s1 . doc. brain magnetic resonance imaging (mri) in a healthy shih tzu dog. transverse t1-weighted (a and b) and transverse and sagittal t2-weighted (c and d) imaging. the following structures were identified at the level of the interthalamic adhesion: marginal gyri (a); marginal sulci (b); middle ectomarginal gyri (c); ectomarginal sulci (d); middle suprasylvian gyri (e); middle suprasylvian sulci (f ); middle ectosylvian gyri (g); caudal ectosylvian sulci (h); caudal sylvian gyri (i); pseudosylvian fissure (j); lateral rhinal sulci (k); splenial sulci (l); cingulate gyri (m) and corpus callosum (o) (a). the following structures were identified at the level of the mesencephalic aqueduct: marginal gyri (a); marginal sulci (b); middle ectomarginal gyri (c); ectomarginal sulci (d); caudal suprasylvian gyri (e); caudal suprasylvian sulci (f ); ectosylvian gyri (g); lateral rhinal sulci (h); parahippocampal gyri (i) and caudal composite gyri (j) (b). in transverse and sagittal t2-weighted images, all anatomical structures were normal, including the lateral ventricles, quadrigeminal cistern and corpus callosum (c and d). authors' contributions dnrs and rma conceived and designed the study. dnrs, gbap, eft and vmm performed the diagnostic work-up, clinical assessment, and provided professional discussion regarding the cases. rma and vmm evaluated and interpreted magnetic resonance imaging. dnrs and gbap wrote the manuscript. rma critically revised the manuscript. all authors read and approved the final manuscript. not applicable. mri equipment was acquired through funding (procedure number 2009/54028-8) provided by the são paulo research foundation (fapesp). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. this study was not submitted for ethics committee review because it is a retrospective analysis of medical records of the veterinary neurology service, school of veterinary medicine and animal science form sao paulo state university-unesp -brazil. not applicable. the authors declare that they have no competing interests. key: cord-309587-xc4jaw31 authors: lembo, tiziana; hampson, katie; kaare, magai t.; ernest, eblate; knobel, darryn; kazwala, rudovick r.; haydon, daniel t.; cleaveland, sarah title: the feasibility of canine rabies elimination in africa: dispelling doubts with data date: 2010-02-23 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000626 sha: doc_id: 309587 cord_uid: xc4jaw31 background: canine rabies causes many thousands of human deaths every year in africa, and continues to increase throughout much of the continent. methodology/principal findings: this paper identifies four common reasons given for the lack of effective canine rabies control in africa: (a) a low priority given for disease control as a result of lack of awareness of the rabies burden; (b) epidemiological constraints such as uncertainties about the required levels of vaccination coverage and the possibility of sustained cycles of infection in wildlife; (c) operational constraints including accessibility of dogs for vaccination and insufficient knowledge of dog population sizes for planning of vaccination campaigns; and (d) limited resources for implementation of rabies surveillance and control. we address each of these issues in turn, presenting data from field studies and modelling approaches used in tanzania, including burden of disease evaluations, detailed epidemiological studies, operational data from vaccination campaigns in different demographic and ecological settings, and economic analyses of the cost-effectiveness of dog vaccination for human rabies prevention. conclusions/significance: we conclude that there are no insurmountable problems to canine rabies control in most of africa; that elimination of canine rabies is epidemiologically and practically feasible through mass vaccination of domestic dogs; and that domestic dog vaccination provides a cost-effective approach to the prevention and elimination of human rabies deaths. rabies is a viral zoonosis caused by negative-stranded rna viruses from the lyssavirus genus. genetic variants of the genotype 1 lyssavirus (the cause of classical rabies) are maintained in different parts of the world by different reservoir hosts within 'host-adaptive landscapes' [1] . although rabies can infect and be transmitted by a wide range of mammals, reservoirs comprise only mammalian species within the orders carnivora (e.g. dogs, raccoons, skunks, foxes, jackals) and chiroptera (bats). from the perspective of human rabies, the vast majority of human cases (.90%) result from the bites of rabid domestic dogs [2] and occur in regions where domestic dogs are the principal maintenance host [3] . over the past three decades, there have been marked differences in efforts to control canine rabies. recent successes have been demonstrated in many parts of central and south america, where canine rabies has been brought under control through large-scale, synchronized mass dog vaccination campaigns [4] . as a result, not only has dog rabies declined, but human rabies deaths have also been eliminated, or cases remain highly localized [5] . the contrast with the situation in africa and asia is striking; here, the incidence of dog rabies and human rabies deaths continue to escalate, and new outbreaks have been occurring in areas previously free of the disease (e.g. the islands of flores and bali in indonesia [6] ; http://wwwn.cdc.gov/travel/ contentrabiesbaliindonesia2008.aspx). in this paper, we identify four major reasons commonly given for the lack of effective domestic dog rabies control including (1) low prioritisation, (2) epidemiological constraints, (3) operational constraints and (4) lack of resources (table 1) , focussing on the situation in africa. we address each of these issues in turn, using outputs from modelling approaches and data from field studies to demonstrate that there are no insurmountable logistic, practical, epidemiological, ecological or economic obstacles. as a result, we conclude that the elimination of canine rabies is a feasible objective for much of africa and there should be no reasons for further delay in preventing the unnecessary tragedy of human rabies deaths. this paper compiles previously published data (see references below) and additional analyses of those data, but we present a brief summary of the data collection methods below. hospital records of animal-bite injuries compiled from northwest tanzania were used as primary data sources. these data informed a probability decision tree model for a national disease burden evaluation [7] , which has since been adapted for global estimates of human rabies deaths and disability-adjusted life years (dalys) lost due to rabies [3] , a standardized measure for assessing disease burden [8, 9] . hospital records were also used to initiate contact tracing studies [10] [11] [12] , whereby bite-victims were interviewed to obtain more detail on the source and severity of exposure and actions taken, allowing subsequent interviews with other affected individuals (not documented in hospital records) including owners of implicated animals. statistical techniques applied to these data for estimating epidemiological parameters and inferring transmission links are described elsewhere [10, 12] . rabies monitoring operations including passive and active surveillance involving veterinarians, village livestock field officers, paravets, rangers and scientists were used to collect samples from carcasses (domestic dogs and wildlife whenever found), which were subsequently tested and viral isolates were sequenced [10, [13] [14] [15] [16] , with results being used to inform estimates of rabies-recognition probabilities [7] and for phylogenetic analyses [10, 16] . operational research on domestic dog vaccination strategies was carried out in a variety of settings [14, 17] . household interviews were also used for socio-economic surveys and to evaluate human:domestic elimination of canine rabies has been achieved in some parts of the world, but the disease still kills many thousands of people each year in africa. here we counter common arguments given for the lack of effective canine rabies control in africa presenting detailed data from a range of settings. we conclude that (1) rabies substantially affects public and animal health sectors, hence regional and national priorities for control ought to be higher, (2) for practical purposes domestic dogs are the sole maintenance hosts and main source of infection for humans throughout most of africa and asia and sufficient levels of vaccination coverage in domestic dog populations should lead to elimination of canine rabies in most areas, (3) the vast majority of domestic dog populations across sub-saharan africa are accessible for vaccination with community sensitization being of paramount importance for the success of these programs, (4) improved local capacity in rabies surveillance and diagnostics will help evaluate the impact of control and elimination efforts, and (5) sustainable resources for effective dog vaccination campaigns are likely to be available through the development of intersectoral financing schemes involving both medical and veterinary sectors. dog ratios, levels of vaccination coverage achieved and reasons for not bringing animals to vaccination stations [17, 18] . the study was approved by the tanzania commission for science and technology with ethical review from the national institute for medical research (nimr). this retrospective study involved collection of interview data only, without clinical intervention or sampling, therefore we considered that informed verbal consent was appropriate and this was approved by nimr. permission to conduct interviews was obtained from district officials, village and sub-village leaders in all study locations. at each household visited, the head of the household was informed about the purpose of the study and interviews were conducted with verbal consent from both the head of the household and the bite victim (documented in a spreadsheet). approval for animal work was obtained from the institutional animal care and use committee (iacuc permit #0107a04903). (a) there is not enough evidence to define rabies control as a priority a principal factor contributing to a low prioritization of rabies control has been the lack of information about the burden and impact of the disease [19, 20] . data on human rabies deaths, submitted from ministries of health to the world health organization (who), are published in the annual world surveys of rabies and through the who rabnet site (www.who.int/ rabies/rabnet/en). for the who african region (afro) comprising 37 countries, these surveys report an average of 162 human deaths per year between 1988 and 2006. it is therefore unsurprising that for national and international policy-makers, rabies pails into insignificance in comparison with other major disease problems. this perceived lack of significance of human rabies is reflected in the absence of any mention of rabies in either of the two published global burden of disease surveys [21, 22] , which assessed more than 100 major diseases. these surveys adopted the metric of the daly which is widely used as the principal tool for providing consistent, comparative information on disease burden for policy-making. until recently no estimates of the daly burden were available for rabies. official data on human rabies deaths submitted to who from africa are widely recognized to greatly under-estimate the true incidence of disease. the reasons for this are manifold: (1) rabies victims are often too ill to travel to hospital or die before arrival, (2) families recognize the futility of medical treatment for rabies, (3) patients are considered to be the victims of bewitchment rather than disease, (4) clinically recognized cases at hospitals may go unreported to central authorities, and (5) misdiagnosis is not uncommon. the problems of misdiagnosis were highlighted by a study of childhood encephalitis in malawi, in which 3/26 (11.5%) cases initially diagnosed as cerebral malaria were confirmed as rabies through post-mortem tests [23] . several recent studies have contributed information that consistently demonstrates that the burden of canine rabies is not insubstantial. human rabies deaths. estimates of human rabies cases from modeling approaches, using the incidence of dog-bite injuries and availability of rabies post-exposure prophylaxis (pep), indicate that incidence in africa is about 100 times higher than officially reported, with ,24,000 deaths in africa each year [3, 7] . consistent figures have subsequently been generated from detailed contact-tracing data: in rural tanzanian communities with sporadic availability of pep (a typical scenario in developing countries), human rabies deaths occur at an incidence of ,1-5 cases/100,000/year (equivalent to 380-1,900 deaths per year for tanzania) [11] . similarly, a multi-centric study from india reported 18,500 human rabies deaths per year [24] , consistent with model outputs of 19,700 deaths for india [3] . a crude comparison of annual human deaths for a range of zoonotic diseases is shown in figure 1 (top). while diseases such as severe acute respiratory syndrome (sars), rift valley fever and highly pathogenic avian influenza cause major concerns as a result of pandemic potential and economic losses, these figures provide a salutary reminder of the recurrent annual mortality of rabies and other neglected zoonoses, such as leishmaniasis and human african trypanosomiasis (hat). decision-tree models applied to data from east africa and globally indicate that the daly burden for rabies exceeds that of most other neglected zoonotic diseases (figure 1 -bottom) [3, 25, 26] . human animal-bite injuries and morbidity. most of the rabies daly burden is attributed to deaths, rather than morbidity because of the short duration of clinical disease. the daly burden for rabies is particularly high, because most deaths occur in children and therefore a greater number of years of life are lost [25, 27] . daly estimates incorporate non-rabies mortality and morbidity in terms of adverse reactions to nerve-tissue vaccines (ntvs) [3] , which are still widely used in some developing countries such as ethiopia, however rabies also causes substantial 'morbidity' as a direct result of injuries inflicted by rabid animals, and this is not included in daly estimates. contact-tracing studies suggest an incidence as high as 140/ 100,000 bites by suspected rabid animals in rural communities of tanzania [11] . thus, for every human rabies death there are typically more than ten other rabid animal-bite victims who do not develop signs of rabies, because they obtain pep (figure 1 bottom) or are simply fortunate to remain healthy. the severity of wounds has not yet been quantified, but case-history interviews suggest that injuries often involve multiple, penetrating wounds that require medical treatment. economic burden. the major component of the economic burden of rabies relates to high costs of pep, which impacts both government and household budgets. with the phasing out of ntvs, many countries spend millions of dollars importing supplies of tissue-culture vaccine (,$196 million usd pa [3] ). at the household level, costs of pep arise directly from anti-rabies vaccines and from high indirect (patient-borne) costs associated with travel (particularly given the requirement of multiple hospital visits), medical fees and income loss [3, 28] . indirect losses, represent .50% of total costs ( figure 2 ). total costs have been estimated conservatively at $40 us per treatment in africa and $49 us in asia accounting respectively for 5.8% and 3.9% of annual per capita gross national income [3] . poor households face difficulties raising funds which results in considerable financial hardship and substantial delays in pep delivery [11, 28] . shortages of pep, which are frequent in much of africa, further increase costs as bite victims are forced to travel to multiple centres to obtain treatment, also resulting in risky delays [11] . additional economic losses relate to livestock losses derived from an incidence of 5 deaths/100,000 cattle estimated to cost $12.3 million annually in africa and asia [3] . however, substantially higher incidence has been recorded in tanzania, with 12-25 cases/100,000 cattle reported annually in rural communities (hampson, unpublished) . canine rabies introduced from sympatric domestic dog populations is also recognized as a major threat to endangered african wild dogs (lycaon pictus) and ethiopian wolves (canis simensis) [29] [30] [31] [32] . potential losses of tourism revenue may be substantial; african wild dogs are a major attraction in south africa national parks with the value of a single pack estimated at $9,000 per year [33] and ethiopian wolves are a flagship species for the bale mountains national park. psychological impact. an important, but often underappreciated component of disease burden is the psychological impact on bite-victims and their families. in rural tanzania, .87% of households with dog bite victims feared a bite from a suspected rabid animal more than malaria [28] because malaria can be treated whereas clinical rabies is invariably fatal and malaria treatment is generally affordable and available locally in comparison to pep. when human rabies cases occur, the horrifying symptoms and invariably fatal outcome result in substantial trauma for families, communities and health care workers [34] . increasing incidence of rabies in africa has prompted concerns that the epidemiology of the disease may be more complex, involving abundant wildlife carnivores that may sustain infection cycles [13, [35] [36] [37] [38] . there is also uncertainty about the level of vaccination coverage needed to control rabies particularly in rapidly growing domestic dog populations [39, 40] . to eliminate infection, disease control efforts need to be targeted at the maintenance population [41] . this is clearly demonstrated for fox rabies in western europe, whereby control of rabies in foxes (through mass oral vaccination) has led to the disappearance of rabies from all other 'spill-over' hosts [42] . despite the predominance of domestic dog rabies in africa, the role of wildlife as independent maintenance hosts has been debated, and many perceive the abundance of wildlife as a barrier to elimination of canine rabies on the continent. it has also been argued that the predominance of dog rabies is an artefact of poor surveillance and under-reporting in wildlife populations [43] . in the wildlife-rich serengeti ecosystem in tanzania, evidence suggests that domestic dogs are the only population essential for maintenance [10, 13, 16] : (1) phylogenetic data showed only a single southern africa canid-associated variant (africa 1b) circulating among different hosts [16] ; (2) transmission networks suggested that, for wildlife hosts, within-species transmission cannot be sustained [16] ; and (3) statistical inference indicated that cross-species transmission events from domestic dogs resulted in only relatively short-lived chains of transmission in wildlife with no evidence for persistence [10] . the conclusion that domestic dogs are the only maintenance population in such a species-rich community suggests that elimination of canine rabies through domestic dog vaccination is a realistic possibility, and provides grounds for optimism for wider-scale elimination efforts in africa. in other parts of central and west africa, transmission of rabies appears to be driven by domestic dogs [44] . an outstanding question relates to southern africa. earlier and recent evidence indicate that jackal species (canis mesomelas and c. adustus) and bat-eared foxes (otocyon megalotis) may maintain the canid variant in specific geographic loci in south africa and zimbabwe [2, [36] [37] [38] [45] [46] [47] [48] [49] [50] , but it is still not clear whether these cycles can be sustained over large spatial and temporal scales in the absence of dog rabies [13, 51, 52] . independent wildlife cycles may preclude continent-wide elimination of this variant through dog vaccination alone and wildlife rabies control strategies, in conjunction with dog vaccination, may need to be considered in specific locations [38] . a critical proportion of the population must be protected (p crit ) to eliminate infection and this threshold can be calculated from the basic reproductive number (r 0 , defined as the average number of secondary infections caused by an infected individual in a susceptible population) [53] . vaccinating a large enough proportion of the population to exceed p crit will not only protect the vaccinated individuals but will reduce transmission such that, on average, less than one secondary infection will result from each primary case (effective reproductive number, r e ,1), which can ultimately lead to elimination. vaccination has eliminated canine rabies in many countries demonstrating the success of this concept [54] . however, theory suggests that r 0 increases with population density [39] and thus higher coverage will be needed in higher density populations. however, evaluation of historical outbreak data from around the world and recent data from tanzania indicate that r 0 in domestic dog populations is consistently low (between 1.0 and 2.0) [12] , confirming the feasibility of rabies elimination through vaccination in african domestic dog populations. an important conclusion of this study was that in populations with rapid turnover (such as those in many african countries) at least 60% of the population must be vaccinated during annual campaigns to prevent coverage falling below p crit between campaigns. data from africa clearly show that very few control efforts have reached these levels of coverage [ table 2 ], which is why rabies remains a persistent problem [12] . although emergence of new variants maintained in wildlife also remains a possibility, as shown in the usa, where wildlife rabies now dominates since elimination of canine rabies [55] . for africa, these questions are likely only to be resolved with large-scale intervention involving mass vaccination of dogs. several arguments are given for why mass vaccination campaigns have failed to achieve the high levels of coverage that are necessary to interrupt rabies transmission. we counter these arguments below: a perception of many inaccessible stray/ownerless dogs. a common claim is that the majority of dogs in africa are unowned 'stray' animals, and therefore inaccessible for parenteral vaccination. it is not hard to see why this perception has arisen -unrestrained dogs, without any apparent evidence of ownership, are commonly observed. further investigation, however, usually reveals that the vast majority are owned, and at least one household claims some responsibility, including presentation for vaccination. published studies in africa, which quantify the proportion of unowned dogs, are admittedly sparse, but all support this observation [56] [57] [58] . capture-mark-recapture methodologies and household questionnaires used in african settings have all found consistently low estimates (tunisia ,7% [57] , 1%, 8% and 11% in three sites in n'djamena, chad [56] , and 1% in a peri-urban site in tanzania [58] ). notably, the tanzanian site was selected specifically on the basis of reports of many unowned dogs. while mark-recapture methods yield reliable estimates of unowned dog numbers, their implementation and analysis is not trivial and efforts are underway to develop simpler, yet robust methodologies [59] . certainly in traditional africa, i.e. most of sub-saharan africa, the issue of roaming dogs seems not to be one of a lack of ownership, but rather an inability or unwillingness by owners to confine their dogs. unwillingness/inability to bring dogs for vaccination. published studies tend to refute the idea that owners are often unable or unwilling to restrain their dogs for parenteral vaccination. a multi-country who-commissioned study (tunisia, sri lanka and ecuador) concluded that ''dogs which are not catchable by at least one person are rare and represent generally less than 15% of the dog population'' [57] . similarly a study from nepal found that 86-97% of dogs were accessible to parenteral vaccination [60] . although an early study in turkey concluded that 48% of all free-roaming owned dogs could not be captured by their owners [61] , more recent surveys found that most unvaccinated dogs could be handled (only 16% could not) and that a much larger proportion (56%) resulted from a lack of information about the campaign -a much easier problem to remedy (unpublished data). in africa, very similar figures were obtained in a multi-site study in urban and rural tanzania, where only 15% of vaccination failures were due to a reported inability by the owner to handle the dog, while 53% of cases were due to poor information dissemination [17] . however, there may be settings in transitional africa (e.g. parts of southern africa including kwazulu natal [2] ) where handling of dogs is more difficult due to a break-down in traditional animal husbandry and other social factors, and more intensive efforts may be required for these special cases. given that most dogs are accessible for parenteral vaccination, high coverage can be achieved with well-planned vaccination campaigns. during pilot programmes in urban and rural africa which have not charged owners for vaccination, coverages obtained have exceeded 60% [14, 17, 56] . pastoral communities pose particular challenges due to remote locations and seminomadic lifestyles, but .80% coverages can still be achieved through house-to-house delivery strategies or community-based animal health workers [17] . young pups usually make up a large proportion (.30%) of african dog populations [62] and there is a widespread perception among veterinary authorities and dog owners that they should not be vaccinated, which leads to insufficient coverage [17] . however, rabies vaccines can safely be administered to pups ,3 months of age [63] , and in village campaigns in tanzania, vaccines consistently induced high levels (.0.5 iu/ml) of rabies virus neutralizing antibody [64] . the issue of inclusion of pups can effectively be addressed through appropriate advertising before campaigns. cost-recovery, through charging dog owners for rabies vaccination, is widely promoted for sustainable programmes and to encourage responsible dog ownership. however, charging for a vaccination that represents a public rather than a private good, can be counterproductive, resulting in low turnouts and coverage (,30%) with little or no impact [65] . charging for vaccination may indeed be the principal reason why owners are unwilling to bring dogs for vaccination. ineffective campaigns that achieve ,30% coverage are a waste of resources and can be highly demoralising for veterinary staff and communities. when resources are spread thinly, such that only low coverage is achieved or only small pockets are well vaccinated, then large-scale failure is inevitable. a more epidemiologically sensible strategy is to focus resources into a single (preferably well-bounded) area where high coverage can be consistently achieved. uncertainty about dog population sizes and ecology for effective design and planning of vaccination campaigns. official figures used for planning frequently underestimate true population sizes. for example, gsell [58] found that the owned dog population in a municipality in tanzania was six times larger than official records. although standard survey methodologies for estimating dogs/household or dog:human ratios [57, [66] [67] [68] are not without problems (for example, double ownership of dogs), a rough estimate of owned dog populations can be derived from national (human) population censuses, and can be corrected for different demographic and ecological settings [18, 69] . more detailed studies can be conducted to identify key household determinants of dog ownership (for example, religion, age and sex of household heads, household size, socio-economic level, and livestock presence/absence [18, 28, 70, 71] . such determinants have been used to generate a 'dog density' map of tanzania, for assistance in planning national rabies vaccination campaigns ( figure 3 ). the above factors are all generally described as obstacles that ultimately lead to a lack of investment into rabies control and surveillance. we suggest that investment would actually reap multiple benefits including economic ones, if appropriate strategies are implemented overcoming the constraints described. a lack of surveillance and diagnostic capacity for rabies detection. poor surveillance and diagnosis capacity means that (1) data is insufficient to demonstrate disease burden and motivate policy-makers, and (2) impacts of control efforts cannot be evaluated. considerable progress has been made in the development of simple and inexpensive techniques for sample preservation and rapid post-mortem diagnosis suitable for laboratories with limited storage and/or diagnostic resources with potential to increase incountry capabilities for surveillance. a new direct rapid immunohistochemical test (drit) requires only light microscopes [72] , which are widely available. the test is simple and can be performed by a range of operators if appropriate training is provided. field evaluation studies in africa demonstrated that this assay has characteristics equivalent to those of the direct fluorescent antibody (dfa) test, the global standard for rabies diagnosis, including excellent performance on glycerolated field brain material [15, 73] , the preservative of choice under field conditions [74, 75] . other simple field-diagnostics that allow rapid screening, including enzyme immunoassays [76] , dot blot enzyme immunoassays [77] and lateral-flow immunodiagnostic test kits [78, 79] are being evaluated. these tools offer hope of extending diagnostic capacity in resource-limited settings. animal-bite injury data from hospitals are an easily accessible source of epidemiological information and have been verified as reliable indicators of animal rabies incidence and human exposures [11, 14] . furthermore, increasing availability of communication infrastructure through mobile phone network access in remote areas could enhance surveillance by allowing real-time reporting. costs of effective dog vaccination campaigns are beyond the budget of veterinary services. veterinary services in africa usually report very limited budgets and often have to divert resources during outbreaks of other diseases [80, 81] . this is clearly the most significant constraint to effective rabies control. however, with increasing human and dog populations, dog rabies incidence, human exposures to rabies and the costs required to prevent human rabies deaths through pep will invariably continue to rise unless rabies can be controlled at the source, i.e. in domestic dog populations [82] . many countries in asia, such as thailand, vietnam and sri lanka have greatly reduced human rabies deaths through increased pep use, but at a very high cost [83] . in vietnam, for example, deaths fell from 285 in 1996 to 82 in 2006 with administration of .600,000 pep courses per year at an estimated cost of ,$27 million/year [84] . although domestic dog populations need to be targeted for the effective control of rabies, this is usually deemed to be the responsibility of veterinary services even though many of the benefits accrue to the medical sector. in rural tanzania, dog vaccination campaigns led to a rapid and dramatic decline in demand for costly human pep [14] . in pastoral communities, vaccination not only reduced rabies incidence, but has now resulted in a complete absence of exposures reported in local hospitals for over two years (figure 4) . large-scale campaigns can therefore translate into human lives and economic savings through reduced demand for pep. costs per dog vaccinated are generally estimated to be low (rural tanzania ,$1.73 [17] , philippines ,$1.19-4.27 [85] , tunisia ,$1.3 [86] , thailand ,$1.3 [86] and urban chad ,$1.8 [87] ) and preliminary studies suggest that including dog vaccination in human rabies prevention strategies would be a highly cost-effective intervention at ,us $25/daly averted (s. cleaveland, unpublished data; see also 82) . developing joint financing schemes for rabies prevention and control across medical and veterinary sectors would provide a mechanism to use savings in human pep to sustain rabies control programs in domestic dogs. although conceptually simple, the integration of budgets across different ministries is likely to pose political and administrative challenges. however, given sufficient political will and commitment, developing sustained programmes of dog vaccination that result in canine rabies elimination should be possible. in conclusion, here we show that a substantial body of epidemiological data have now been gathered through multiple studies demonstrating that: (1) rabies is an important disease that exerts a substantial burden on human and animal health, local and national economies and wildlife conservation, (2) domestic dogs are the sole population responsible for rabies maintenance and main source of infection for humans throughout most of africa and asia and therefore control of dog rabies should eliminate the disease, (3) elimination of rabies through domestic dog vaccination is epidemiologically feasible, (4) the vast majority of domestic dog populations across sub-saharan africa are accessible for vaccination and the few remaining factors compromising coverage can be addressed by engaging communities through education and awareness programs, (5) new diagnostic and surveillance approaches will help evaluate the impact of interventions and focus efforts towards elimination, and (6) dog rabies control is affordable, but is likely to require intersectoral approaches for sustainable programmes that will be needed to establish rabies-free areas. appendix s1 appendix with additional references. can rabies be eradicated? emerging epidemic dog rabies in coastal south africa: a molecular epidemiological analysis re-evaluating the burden of rabies in africa and asia overview of rabies in the americas current status of human rabies transmitted by dogs in latin america rabies on flores island, indonesia: is eradication possible in the near future? estimating human rabies mortality in the united republic of tanzania from dog bite injuries quantifying the burden of disease: the technical basis for disability-adjusted life years estimating the ''avoidable'' burden of disease by disability adjusted life years (dalys) exploring reservoirs dynamics: a case study of rabies in the serengeti ecosystem rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies transmission dynamics and prospects for the elimination of canine rabies maintenance of a microparasite infecting several host species: rabies in the serengeti a dog rabies vaccination campaign in rural africa: impact on the incidence of dog rabies and human dog-bite injuries evaluation of a direct, rapid immunohistochemical test for rabies diagnosis molecular epidemiology identifies only a single rabies virus variant circulating in complex carnivore communities of the serengeti rabies control in rural africa: evaluating strategies for effective domestic dog vaccination a cross-sectional study of factors associated with dog ownership in tanzania strategies for the control and elimination of rabies in asia canine and human rabies in cameroon the global burden of disease: a comprehensive assessment of mortality and disability from diseases, injuries and risk factors in 1990 and projected to 2020 the world health report 2002: reducing risks, promoting healthy life rabies encephalitis in malaria-endemic area assessing the burden of human rabies in india: results of a national multi-center epidemiological survey estimating the public health impact of rabies the epidemiology of animal bite injuries in uganda and projections of the burden of rabies an important public health problem: rabies suspected bites and post-exposure prophylaxis in a health district in turkey rabies control in rural tanzania: optimising the design and implementation of domestic dog mass vaccination programmes rabies in african wild dogs (lycaon pictus) in the serengeti region rabies and mortality in ethiopian wolves (canis simensis) integrating epidemiology into population viability analysis: managing the risk posed by rabies and canine distemper to the ethiopian wolf the cost efficiency of wild dog (lycaon pictus) conservation in south africa rabies and other lyssavirus diseases infectious diseases of livestock with special reference to southern africa canine rabies ecology in southern africa molecular epidemiology of rabies in bat-eared foxes (otocyon megalotis) in south africa molecular epidemiology of rabies: focus on domestic dogs (canis familiaris) and black-backed jackals (canis mesomelas) from northern south africa immunization coverage required to prevent outbreaks of dog rabies comparison of vaccination strategies for the control of dog rabies in machakos district identifying reservoirs of infection: a conceptual and practical challenge rabies in europe -epidemiological cycles and impact of oral vaccination of foxes rabies in southern africa dog population structure and cases of rabies among dog bite victims in urban and rural areas of borno state molecular epidemiology of rabies virus in south africa rabies in bat-eared foxes in south-africa molecular epidemiology of rabies virus in south africa: evidence for two distinct virus groups molecular epidemiology of canid rabies in zimbabwe and south africa mongoose rabies in southern africa: a re-evaluation based on molecular epidemiology the epidemiology of rabies in zimbabwe. 2. rabies in jackals (canis adustus and canis mesomelas) rabies in zimbabwe: reservoir dogs and the implications for disease control the epidemiology of rabies in zimbabwe. 1. rabies in dogs (canis familiaris) infectious diseases of humans: dynamics and control synchronous cycles of domestic dog rabies in sub-saharan africa and the impact of control efforts enzootic rabies elimination from dogs and reemergence in wild terrestrial carnivores, united states coverage of pilot parenteral vaccination campaign against canine rabies in n'djamã©na report of a who consultation on dog ecology studies related to rabies control. geneva: world health organization (who/rab demographic, spatial and behavioural heterogeneities in an urban dog (canis familiaris) population, relevant in planning rabies control in iringa surveying roaming dog populations: guidelines on methodology accessibility of dog populations for rabies control in kathmandu valley report of the 3 rd consultation on oral immunization of dogs against rabies. geneva: world health organization (who/rab dog rabies and its control is it possible to vaccinate young canids against rabies and to protect them? proceedings of the sixth southern and eastern african rabies group/world health organization meeting welfare and health assessment of tanzanian dogs and its relationship to immunological response to rabies vaccination owner valuation of rabies vaccination of dogs a dog ecology study in urban and semi-rural area of zambia the epidemiology and control of domestic dog rabies ecological and epidemiological data requirements for the planning of dog rabies control colombo humane dog population and rabies management project aspects of dog ownership and canine rabies control in africa and asia dog ecology and demography information to support the planning of rabies control in machakos district standard operating procedure for the direct rapid immunohistochemistry test for the detection of rabies virus antigen. national laboratory training network course. atlanta: us department of health and human services rabies diagnosis for developing countries simplified technique for the collection, storage and shipment of brain specimens for rabies diagnosis. who/rab.res/88 simple technique for the collection and shipment of brain specimens for rabies diagnosis development and evaluation of an enzyme immunoassay for rapid diagnosis of rabies in humans and animals rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay evaluation of a rapid immunodiagnostic test kit for rabies virus a simple and rapid immunochromatographic test kit for rabies diagnosis proceedings of the sixth southern and eastern african rabies group/world health organization meeting proceedings of the seventh southern and eastern african rabies group/world health organization meeting transmission dynamics and economics of rabies control in dogs and humans in an african city preventing the incurable: asian rabies experts advocate rabies control report to the world health organization. dog rabies elimination demonstration project rabies control in the republic of the philippines: benefits and costs of elimination economics of human and canine rabies eliminationguidelines for programme orientation costdescription of a pilot parenteral vaccination campaign against rabies in dogs in n'djamã©na, chad we are indebted to the ministries of livestock development and fisheries and of public health and social welfare, tanzania national parks, tanzania wildlife research institute, ngorongoro conservation area authority, tanzania commission for science and technology, and national institute for medical research for permission and collaboration; the frankfurt zoological society and the mwanza and arusha veterinary key: cord-021772-5v4gor2v authors: levine, gwendolyn j.; cook, jennifer r. title: cerebrospinal fluid and central nervous system cytology date: 2019-05-31 journal: cowell and tyler's diagnostic cytology and hematology of the dog and cat doi: 10.1016/b978-0-323-53314-0.00014-6 sha: doc_id: 21772 cord_uid: 5v4gor2v nan analysis of csf is an important adjunctive diagnostic tool in the workup of patients with cns disease and must be interpreted within the context of the patient's history, clinical signs, clinicopathological data, imaging studies, and other ancillary diagnostics. rarely is csf solely used to provide an etiological diagnosis (exceptions include cytological visualization of infectious agents or overtly neoplastic cells), but analysis may significantly narrow the field of pathophysiological differentials, guiding further diagnostic and therapeutic options. csf analysis is most sensitive in detecting inflammatory disease. 3 positive findings in csf tend to be more diagnostically helpful compared with negative findings but are often nonspecific because many different diseases may cause a common csf pathology (e.g., neutrophilic pleocytosis). 4 occasionally, the magnitude of change within the csf may be as instructive as the character of the change (e.g., a marked increase in protein concentration raising diagnostic concern for feline infectious peritonitis, marked neutrophilic pleocytosis raising diagnostic concern for steroid-responsive meningitis-arteritis in a young dog in pain). 4 more frequently, however, specific disease etiologies will present with csf changes of variable character and magnitude. csf that falls within laboratory reference intervals should never be used to rule out a differential diagnosis because negative findings may represent early or mild disease, disease suppressed or masked by therapeutic intervention, or a disease process that does not present within the particular area of the extracellular space being sampled. csf analysis may or may not correlate with imaging studies; a retrospective study of 92 cats receiving magnetic resonance imaging (mri) for spinal signs showed that abnormal csf was not a predictor for abnormal mri. 5 in another study, approximately 25% of dogs with intracranial signs and inflammatory csf had normal brain mri results. 6 the conventionally accepted theory of csf secretion and transport is based on the concept of active transport of ions within the ventricular ependymal cells and choroid plexi, subsequent passive flow of fluid, and circulation and drainage of csf into dural venous sinuses. these ideas have recently come under scrutiny as potentially simplistic and inconsistent with the past 100 years of experimental evidence. 7 analysis of past experiments, coupled with new data, supports a "global production" hypothesis-that instead of exclusive formation within the ventricles, csf is continually created and reabsorbed diffusely by cerebral capillaries that have slight variances in hydrostatic and osmotic pressure. canine studies have documented csf production within the ventricular system and the sas. 2 a study of 158 dogs with focal, noninflammatory disease showed that in cases of spinal lesions, csf was more likely to be abnormal if collected from the lumbar cistern, that is, caudal to the lesion. 12 this observation may be explained by presupposing cranial to caudal flow of csf, but the traditionally held theory of csf flow has recently been contested. 7 in canines, csf collected from the cerebromedullary cistern generally has lower microprotein concentrations compared with samples collected from the lumbar cistern. 13 blood contamination may be more pronounced in lumbar collection, as the desired subarachnoid space is more difficult to enter and yields a smaller volume of fluid that tends to flow more slowly. 9, 10 moreover, hemodilution may contribute to increased measured protein concentration. 13 rare instances of csf contamination with hematopoietic precursors have only been reported from lumbar sites. 14 a low, but potentially catastrophic, risk for puncturing the cervical spinal cord or caudal brainstem exists during cerebromedullary collection. because the spinal cord length is variable, spinal cord puncture is a possibility during lumbar collection, but it is associated with less severe adverse effects compared with injury following cisternal puncture. in a case series of four accidental cisternal parenchymal punctures (documented by using mri), three of the four patients suffered neurological decompensation and subsequently had to be euthanized. 15 the following equipment should be assembled: anesthesia and monitoring equipment, clippers, aseptic preparation materials for the skin, sterile gloves, and a spinal needle with stylet. for cerebromedullary cistern collection in dogs weighing less than 25 kg and for cats, a 22-gauge, 1.5-inch spinal needle is usually adequate, and a 22-gauge, 2.5-inch spinal needle is recommended for dogs weighing greater than 25 kg. for lumbar puncture, a 22-gauge spinal needle up to 6 inches long may be required for obese or extremely large patients. if available, fluoroscopic equipment may aid in the acquisition of cisternal or lumbar csf. at the authors' institution, fluoroscopy is often used before cisternal csf acquisition in toy-breed dogs to exclude the possibility of subclinical atlantoaxial subluxation. the anesthetized patient is placed in lateral recumbency (it is generally easier for a right-handed clinician to have the patient in right-lateral recumbency, and vice versa), with the neck and back flush to the edge of a sturdy table. for collection from the cerebromedullary cistern, the neck is flexed such that the dorsum of the muzzle is 90 degrees to the long axis of the body (if needed, stabilizing the endotracheal tube to prevent kinking and deflating the cuff to prevent tracheal trauma), and the snout is propped up slightly, if necessary, to keep it parallel with the table and not angulated from the sagittal plane. 10 a wide area (3-5 cm) around the atlanto-occipital joint (beyond atlas wings and axis spinous process and to the external occipital protuberance) is shaved and aseptically prepared, and landmarks are palpated with a gloved, nondominant hand. 9 the needle is inserted at the intersection of two imaginary perpendicular lines that run (1) along the dorsal midline (dividing the patient sagittally) from the occipital protuberance to the cranial spinous process of the axis (c2) and (2) across the craniolateral aspects of the wings of the atlas (c1) (dividing the patient craniocaudally). for lumbar collection, the pelvic limbs are brought forward into full flexion, and the needle is inserted cranial and parallel to the dorsal spinous process of l6 for dogs and l7 for cats, advancing the needle until the ventral aspect of the vertebral canal is encountered; the needle is then retracted slightly and csf is collected from the ventral sas. 10, 16 the pelvic limbs may be kicked or may twitch slightly during collection because of irritation of the cauda equina or spinal cord parenchyma. for either location, once landmarks are palpated, the needle is held stably with the dominant hand and very slowly advanced, stylet in place. the heel of the dominant hand may be supported against the table. for cisternal collection, it is important to advance the needle toward the point of the nose without angulation. the stylet is removed with the nondominant hand every 2 to 3 mm to check for fluid within the needle hub, waiting a few seconds. it is common to feel a decrease in resistance to forward needle movement once the thecal space is entered. if bone is hit or frank hemorrhage is observed from the needle, it should be withdrawn slowly and collection reattempted. 9 if clear or slightly blood-tinged fluid is observed, advancement of the needle is stopped, and open tubes are placed directly under the needle hub to collect freely falling drops. csf is collected passively and should not be aspirated. there are no significant objective data regarding the maximal amount of csf that may be collected in dogs. several authors claim that it is safe to collect 0.2 milliliters (ml) of csf per kilogram of body weight (1 ml/5 kg); in other species much higher volumes of csf per body weight are acquired standardly. 17 in general, 0.5 to 1 ml of csf is adequate for routine diagnostic tests, including cell counts, protein concentration, and cytological analysis. larger volumes are necessary for additional diagnostics (cultures, titers, polymerase chain reaction [pcr], flow cytometry, protein electrophoresis, etc.). two sets of tubes should be readied and ideally handled by an assistant. an ethylenediaminetetraacetic acid (edta)-treated (purple-top) tube is used for cell counts, flow cytometry, and pcr testing for organisms, and plain (red-top) tubes are used for protein concentration, culture, or immunologic assays. 10 some sources indicate that plain tubes are recommended, as edta could increase protein concentration. if csf analysis will occur rapidly (within 1 hour), collection into a plain tube is adequate, whereas preservation of cells may be improved with collection into edta if analysis will be delayed. if low volume is present, priority is given to the edta tube. if csf appears red, then iatrogenic hemorrhage (puncture of a dural vessel) or actual cns hemorrhage has occurred. in this instance, the first few drops are allowed to collect into the first set of tubes, and the second set of tubes are reserved for the latter portion of the sample, as iatrogenic hemorrhage tends to clear over time. if the hemorrhage does clear, a decision may be made about discarding the first set of tubes or keeping them for ancillary testing not affected by the hemorrhage. after collection, the needle is withdrawn without the stylet, and the csf within the needle is allowed to drip into one of the tubes or is placed in an additional plain tube and saved for culture. as with other clinicopathological and cytological samples, evaluation of a fresh specimen is preferred to minimize cellular degradation, to which csf is particularly vulnerable because of its relatively low protein concentration. sample degradation will affect cell differential count to a greater extent than the total nucleated cell count or the protein concentration. 18 a study of 30 canine csf samples with pleocytosis concluded that delay of analysis up to 8 hours was unlikely to alter interpretation, especially in samples with protein concentrations above 50 milligrams per deciliter (mg/dl). 18 preservative should be added to low protein samples unless analysis is to be completed within 60 minutes (see next section), and a dilutional effect must then be factored into cell counts. 18 samples to be shipped to a reference laboratory overnight should be kept at refrigeration temperature and shipped with ice packs for analysis within 48 hours. 9, 16 the reference laboratory should be prenotified to ensure prompt analysis. if analysis is likely to be delayed by more than 1 hour and the csf sample has a protein concentration less than 50 mg/dl, one of the following may be added as a protein source to maintain cellular integrity: (1) hetastarch (add 1:1 volume), (2) fetal calf serum (3.7 g/dl protein; add 20% by volume), or (3) autologous plasma or serum (fresh or frozen; 11% by volume ≡ one drop from 25-gauge needle (approximately 0.03 ml) mixed into 0.25 ml csf). 10, 19, 20 the sample should be labeled with the protein source and amount added to the sample. one study demonstrated better preservation of mononuclear cells in canine samples when fetal calf serum was used instead of hetastarch. 18 all samples should be refrigerated at 4°c to minimize cellular degradation. a hemocytometer may be employed in practice to count nucleated cells and erythrocytes. both sides of the cover-slipped hemocytometer are loaded with unstained csf, which is then placed in a humidified container for 10 to 15 minutes to allow cells to settle on the glass. because the fluid is unstained, the microscope condenser is lowered to improve contrast. erythrocytes and nucleated cells are differentiated by size, refraction, granularity, and smoothness of plasma membrane. 21 some laboratories stain csf samples with new methylene blue (nmb), as leukocytes will take up stain, whereas erythrocytes remain unstained, making differentiation of leukocytes (specifically small lymphocytes) and erythrocytes easier (fig. 14.1) . 22 a small volume of csf is drawn into a capillary tube coated with nmb or a tube that has a small volume of nmb followed by an air pocket. 22 the tube containing nmb and csf is gently rocked back and forth, allowing the cells to take up some stain without diluting the csf with a volume of nmb. 22 the hemocytometer is then loaded, and each population is counted and totals are calculated, as follows: neubauer chamber: (1) both areas of large nine squares are counted, and the average of the number of leukocytes and erythrocytes is found; (2) the average is multiplied by 9 to get the cells per microliter (cells/μl). 10 the advia 120 (siemens medical solution, fernwald, germany) hematology instrument has been validated for analyzing canine csf samples and shows excellent correlation with manual methods used in dogs with increased total cell counts (pleocytosis), but the instrument may overestimate the cell count in samples without pleocytoses and has not been validated for the identification of eosinophils. 23 the automated differential count is also more accurate at higher cell numbers and thus should be compared with a traditional manual differential. the advia 2120 hematology analyzer displayed satisfactory agreement with the standard hemocytometer method. 24 validation experiments using 67 canine samples showed a sensitivity of 100% and specificity of 89% for accurately identifying samples with pleocytosis when manual counting was considered the gold standard (>5 cells/μl). 24 the instrument tended to be less accurate at lower (within reference interval) nucleated cell counts. 24 erythrocytes may be a source of interference, as a red blood cell (rbc) count of 250 cells/μl was shown to elevate the nucleated cell count. 24 with regard to differential cell count, the instrument performed better in the presence of pleocytosis, whereas monocytes were overcounted at lower nucleated cell counts. 24 automated cell counts thus should not replace a manual differential but may be used as another level of quality control. automated instruments cannot recognize altered cell types, such as atypical neoplastic cells. measurement of csf specific gravity is not considered to be helpful because of low sensitivity for detecting abnormalities. 12 csf microprotein may be semiquantitatively measured by using urine dipsticks that detect albumin. this assay has a lower detection limit of 100 mg/ dl; therefore, it has low sensitivity for mild to moderate csf protein concentration elevations (30 mg/dl to 100 mg/dl). false-positive or false-negative reactions may occur if the dipstick reads at trace or 1+, but this method is useful if other techniques are not available. 11 reference laboratories apply a similar but more sensitive methodology to measurement of csf microprotein as that of serum protein, using the trichloroacetic acid method, the ponceau s red dye-binding method, or the coomassie brilliant blue method. 22 csf globulin production is typically screened for with the pandy reaction. in this test, a few drops of csf are added to 1 ml of 10% carbolic acid solution, and the resulting turbidity is graded 0 to 4+. any pandy score above zero is considered elevated. globulin concentration below 50 mg/dl will be undetectable with either test. 10, 21 protein electrophoresis and immunoelectrophoresis may be performed on csf and serum for maximum fractionation. 25 the utility of protein electrophoresis or immunoelectrophoresis of csf lies in discriminating altered blood-brain barrier (bbb) permeability from increased localized production of immunoglobulin, which may be suggestive of (but not specific for) a disease entity for which an electrophoretic pattern has been established. cytological analysis is a critical component of csf evaluation because the differential count (percentages) of cells may be abnormal, even if the total nucleated count is within reference interval. cytology also enables examination for neoplastic cells, infectious agents, and evidence of prior hemorrhage. it may also serve as a quality control point, allowing for correlation between observed cellularity and the total count generated by a hemocytometer or an automated analyzer. because of its low cellularity, csf must be concentrated before cytological smear preparation. use of an in-house sedimentation chamber (sörnäs procedure) may be very useful and preserves cell-free fluid for ancillary testing. 10 this technique will recover approximately 60% of total cells, which is sufficient for analysis. 16 a syringe barrel (with the tip and needle aseptically removed with a scalpel blade) is turned upside down and the smooth, top side is placed in warm petroleum jelly and then onto a clean slide. once a seal has formed, fresh csf (at least 0.5 ml) is placed in the syringe and allowed to sit for 30 minutes. 16, 21 then, the supernatant is aspirated carefully with a pipette so as not to disturb the bottom layer contacting the slide. the syringe barrel is removed, and any excess csf is carefully absorbed with a small piece of filter paper or paper towel. the slide is completely and rapidly air-dried without heat (inadequate drying results in cellular distortion), excess petroleum jelly removed with a scalpel blade, and the slide is stained with routine romanowsky stains (e.g., diff-quik). if csf is sent to a reference laboratory, a cytological slide will likely be prepared using cytocentrifugation (500-1000 revolutions per minute [rpm] for 5-10 minutes, either onto a slide coated with albumin or with the addition of 0.05 ml of 30% albumin for improved cell capture) for maximal concentration of nucleated cells onto one slide. 16 cytocentrifuged cytology may show excellent cellular detail, but the preparation may enlarge cells slightly and create an artifactual foamy or vacuolated appearance. 16 slides are air-dried and stained with conventional romanowsky stains. multiple cytospin preparations may be made to yield 200 intact nucleated cells for classification. as it is rare for etiologic agents to localize only within the cns, all cases of suspected infection may be aided diagnostically by fine-needle aspiration (fna) cytology, biopsy with histopathology, culture of nonneural lesions, or all of these. 21 bacterial culture and sensitivity testing of csf is recommended for most cases of neutrophilic pleocytosis, given the appropriate clinical index of suspicion for a septic lesion. even when organisms are visualized on csf cytology, speciation and susceptibility testing may help guide prognostic and treatment decisions. alternatively, bacterial or fungal culture may be negative regardless of cytological observation of organisms. 10, 20 it must be remembered that bacterial cns infection is highly uncommon in dogs and cats compared with other domestic animal species. 26 advanced techniques for neurological disease diagnosis are expanding rapidly. enzyme-linked immunosorbent assay (elisa)-based assays for antibody detection and pcr-based assays for nucleic acid detection of several medically important microbes have been developed for use on csf and may be instructive in the diagnosis of viral, rickettsial, protozoal, or fungal diseases. 20 a large canine study that included a subset of 16 dogs with neoplastic or inflammatory disease showed that csf titer provided diagnosis in 25% of cases. 3 antibody assays should be interpreted cautiously because the presence of antibody may indicate prior exposure or vaccination rather than active infection. moreover, compromise to the bbb in states of inflammation may translate to the presence of antibodies within the csf without local production. occasionally cross-reactive antibodies may be present that do not represent presence of the disease agent under assessment. similarly, specimens for pcr should be submitted to a laboratory with strict quality control to minimize false-negative and false-positive results. poor collection technique may result in false-positive results, especially for bacterial species that are ubiquitous in the environment. 27 as with other aspects of csf analysis, a negative pcr result does not definitively rule out the presence of a pathogen because of the sampling limitation of a small portion of the extracellular space. 20 csf contains glucose, electrolytes, neurotransmitters, and enzymes, but these substances are not measured routinely, although this measurement represents a rapidly expanding area of research in the effort to give clinicians better tools for diagnosing patients and determining prognoses. csf enzymes originate from the bloodstream, the cns, or cells within csf. 10 one study of 34 cats with noninflammatory cns disease showed that measurement of csf activities of lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase (ck) were not diagnostically sensitive but may be useful in detection of acute injury. 28 multiple studies have correlated elevations in csf ck activity with poor prognosis in dogs with neurological disease or spinal cord injury. 29, 30 immunoassays for vascular endothelial growth factor (vegf) and s-100 calcium-binding protein have shown elevations of both molecules in the csf of experimentally induced hypothyroid dogs, suggesting endothelial and glial contribution to increased bbb permeability in this population. 31 myelin basic protein (mbp) has been found to be elevated in lumbar csf in dogs with degenerative myelopathy, supporting the conclusion that it is a demyelinating lesion. 32 mbp concentration is elevated in the csf of dogs affected by intervertebral disk herniation (ivdh) and has been found to be an independent predictor of poor prognosis. 33 beta-2-microglobulin, a major histocompatibility complex i (mhc-i)-associated molecule, has been assayed by using elisa and found to be elevated in the csf of dogs with ivdh and inflammatory disease and also positively correlated with normal total nucleated cell count (tncc). 34 the amino acids tryptophan and glutamine have been found to be elevated in the csf of dogs with portosystemic shunts because of abnormal ammonia metabolism. 35 one study found increased oxytocin in the csf of dogs with spinal cord compression, where it is believed to have an analgesic effect. 36 gamma-aminobutyric acid (gaba) and glutamate neurotransmitter concentrations have been measured in dogs with epilepsy. 37 normal csf is clear and colorless, with few cellular elements and a protein concentration approximately 200 to 300 times less than that of plasma or serum. red or yellowish coloration indicates prior lesional hemorrhage or iatrogenic hemorrhage during collection. in the latter case, a pellet of rbcs will be present after centrifugation. true xanthochromia (yellowish color of hemoglobin breakdown products) that does not clear on centrifugation, cytological evidence of erythrophagia, or both indicate prior hemorrhage into the subarachnoid space. 20 increased bilirubin leakage into the sas or high concentrations of csf protein (>100-150 mg/dl) may cause xanthrochromia. 21 increased turbidity of the sample may be caused by increased number of cells present (>400 rbcs/μl or >200 nucleated cells/μl) but is usually not affected by mild changes. 10, 11 cell counts tncc is fewer than 5 cells/μl in the dog and fewer than 8 cells/μl in the cat, and elevation above this range is termed pleocytosis. 10 grading of pleocytosis is somewhat subjective: in one reference, "mild" was defined as 6 to 50 cells/μl; "moderate" as 51 to 1000 cells/μl; and "marked" as more than 1000 cells/μl. 4 depending on laboratory-specific reference intervals, normal protein concentration is usually less than 25 to 30 mg/dl for cisternal csf and less than 45 mg/dl for lumbar csf. 10, 20 approximately 80% to 95% of csf protein is albumin, and 5% to 12% of csf total protein comprises gammaglobulins. 2 eighty percent of csf protein is transferred from plasma, with the remainder produced within the cns. the latter population includes molecules also produced by other organs and proteins unique to the csf that may potentially be used as markers of cns tissue damage. experimental evidence and earlier literature support a gradient of increasing protein concentration from cranial to caudal within the subarachnoid space, which has been attributed to slower flow and greater blood-csf permeability caudally. 12 normal csf is acellular or contains small numbers of small lymphocytes (figs. 14.2 and 14.3) and large mononuclear cells (macrophages, ependymal lining cells, meningothelial lining cells, choroid plexus cells) (figs. 14.4 and 14.5). large mononuclear cells may be vacuolated and contain phagocytized material ( fig. 14.6) . a low frequency of nondegenerate neutrophils (<25%), which are usually indicative of blood contamination during collection, may be present. 38 a study of 359 samples of canine csf found a 7.5% incidence of meningeal, choroid plexus, ependymal, endothelial cells, or all of these. 39 no correlation existed between the presence of these cells and the presence of pleocytosis, elevated protein concentration, or the primary disease etiology. 39 thus it is postulated that the presence of these cells is an artifact of collection and should not be overinterpreted. the authors recommended the term "surface epithelial cells" for the combined grouping (which cannot be distinguished cytologically), although not all of these cells (meningeal, endothelial) are of epithelial origin. 39 occasionally, anucleate superficial squamous epithelial cells may be seen; these may be caused by contamination from the skin (fig. 14.7 ). occasionally, small amounts of granular, foamy extracellular material are present and are consistent with myelin or myelin-like material, which will stain positively with luxol fast blue stain. this material may consist of myelin fragments, which are generated from demyelination, or may consist of myelin figures (a nonspecific term for layered phospholipids exfoliated from damaged cells). 40 the two cannot be distinguished with light microscopy. the significance of this material remains unclear because it may be observed in samples from patients with no discernible cause. a study of 98 canine cerebromedullary and lumbar csf samples showed 20% incidence of myelin-like material, with a higher percentage in samples from the lumbar cistern or from small dogs (<10 kg). 41 the presence of the material was not correlated with case outcome. 41 similarly, in a study of 61 cavalier king charles spaniels with chiari-like malformations, myelinlike material was observed in 57% of lumbar csf collections and 12% of cerebromedullary collections. 42 thus myelin-like material may be a procedural artifact or may be consistent with a demyelinating (e.g., canine distemper virus, degenerative myelopathy) or potentially necrotizing disorder (e.g., ivdh, other spinal trauma, or a necrotic neoplasm). 40, 41 normal csf should not contain erythrocytes, but hemodilution is a common occurrence. varying reports on the effect of blood contamination on tncc, leukocyte differential, and protein concentration have been published. [43] [44] [45] [46] deciding whether increased tncc or protein concentration is the result of hemodilution alone or a significant change concurrent with hemodilution necessarily remains, to an extent, a subjective assessment and must be critically evaluated in light of the magnitude of csf findings along with the other pertinent facts of the case. correction formulas for csf parameters in the face of hemodilution (e.g., adding 1 nucleated cell/μl per 100 or 500 rbcs/μl) are unreliable. 45, 46 in a recent study of 106 canine csf samples without pleocytosis (tncc <5/μl) but containing at least 500 rbcs/μl, the mean percentage of neutrophils (45.2% versus 5.7%), percentage of samples with eosinophils present (36.8% versus 6.8%), and mean protein concentration (40 mg/dl versus 26 mg/dl) were found to be significantly increased in the samples with blood contamination when compared with controls. 47 significant rbc contamination warrants repeat sampling, if possible. marked hemorrhage or evidence of prior hemorrhage (erythrophagocytosis, xanthochromia, hemosiderin-laden macrophages) may be useful in the diagnosis of cns trauma, which may be accompanied by neutrophilic to mixed cell pleocytosis and mild increase in protein concentration. 4 elevated protein concentration in csf (>30 mg/dl) may occur with or without pleocytosis, and in the absence of pleocytosis is termed albuminocytological dissociation (acd). high protein concentration may be the result of leakage of plasma or cellular proteins across the bbb, localized production of immunoglobulin, localized tissue damage or necrosis, decreased clearance of protein into the venous sinuses, obstruction of csf circulation, or all of the above. as such, it is a nonspecific change that indicates cns damage or hyperproteinemic disease and is consistent with disease of any etiology (e.g., trauma, metabolic, infectious, inflammatory, degenerative, or neoplastic). caution should be exercised when diagnosing acd if the sample is hemodiluted (>500 rbcs/μl). 47 as is true for pleocytosis, inflammation of the meninges and superficial regions of parenchyma will result in greater csf protein elevations than for lesions that are more remote from the sas. occasionally, an abnormal leukocyte differential (shifted from mononuclear predominance to neutrophil predominance) without pleocytosis occurs. this may only be detected if cytological analysis (after sedimentation or cytocentrifugation of csf) is performed. increased percentages of neutrophils may occur in early or mild inflammatory disease, noninflammatory cns disease, disease that is remote from the sas or sampling site, or in cases of hemodilution. an increased proportion of neutrophils is present when neutrophils comprise greater than 25% of all nucleated cells, and increased percentage of eosinophils occurs when eosinophils comprise greater than 1% of the differential. 10 when present (with or without increased tncc), neutrophils should be evaluated for toxic change, degenerative change, and intracellular organisms or other inclusions ( fig. 14.8 ). increased percentage of neutrophils without pleocytosis has been associated with healthy dogs, blood contamination, degenerative disk disease, neoplasia, cerebrovascular accident, fracture, cns aspergillosis, and fibrocartilaginous embolism (fce). 10, 42, 48 a study of 61 cavalier king charles spaniels with chiari-like malformation documented that those with syringomyelia were more likely to have an increased percentage of neutrophils, but it was not reported whether this subpopulation also had a concurrent pleocytosis. 42 in another study, cats with cns neoplasia had increased percentage of neutrophils or lymphocytes without a pleocytosis. 28 although not a classic pattern, infectious or inflammatory disease should not be ruled out if increased neutrophils are visualized without pleocytosis. increased percentage of eosinophils has been reported in parasitic and protozoal diseases, such as neospora caninum infection. 38 one cat with eosinophilic meningoencephalitis (eme) of unknown etiology had an increased percentage of eosinophils and lymphocytes without pleocytosis. 49 the specific diseases mentioned in the next section on various categories of pleocytosis are a survey of the current literature and meant to be a helpful starting point in the generation of particular differential diagnoses. thus disease entities are listed in the section under which they are most commonly present, but it is important to note that for all disease entities, variability in the nature and the magnitude of pleocytosis may emerge in a particular patient at a particular point in time. wherever possible, other categories of pleocytosis that have been reported for a disease have been mentioned. generally, pleocytoses are defined by the cell type that comprises 70% or more of the nucleated cell population. if all cell types are 50% or less, the pleocytosis is classified as a mixed cell pleocytosis. and if, for example, lymphocytes are greater than 50% but less than 70%, some pathologists will classify the pleocytosis as mixed cell, lymphocyte predominant. a pleocytosis will be classified as eosinophilic if eosinophils compose at least 10% to 20% of the nucleated cell population. 11 bacterial meningoencephalomyelitis. bacterial infections of the cns are unusual and represent a small portion of neutrophilic pleocytoses. typically, this pleocytosis is severe (could be over 1000 cells/μl), neutrophilic, and accompanied by significantly elevated protein concentration, but the cell population may change to mononuclear during the course of treatment. 10, 20, 50 rare instances of brain abscessation secondary to sepsis (which may be a sequela of iatrogenic immunosuppression) may result in marked neutrophilic pleocytosis, markedly elevated protein concentration, visualization of bacterial organisms (see fig. 14.8) , and abnormal mri findings. 51 staphylococcus intermedius was cultured from the csf of a dog presenting with a retrobulbar abscess and neurological signs. 52 the csf showed a moderate neutrophilic pleocytosis (75 cells/μl) and borderline elevation in protein concentration (30 mg/dl). 52 local extension of severe otitis interna resulting in meningoencephalitis and ventriculitis in a dog has been reported. 53 this patient exhibited a severe neutrophilic pleocytosis (3672 cells/μl) and protein elevation (>400 mg/dl). 53 pasteurella multocida meningoencephalomyelitis in a kitten was characterized by marked neutrophilic pleocytosis (981 cells/μl) with mild protein elevation (31 mg/dl) and rare extracellular and intracellular bacterial rods. 54 bacterial culture and susceptibility testing are recommended but may yield false-negative results if organisms are not circulating in the extracellular space or if prior antibiotic therapy had been given. serology and csf-pcr (using organism-specific or universal bacterial [ub] pcr) are recommended. 27, 54 cryptococcosis in dogs. cryptococcus spp. are a large genus of systemic dimorphic fungi with a predilection for cns tissue, which is infected hematogenously or via direct penetration of the cribriform plate. only two species at this time are medically important: (1) cryptococcus neoformans (var. neoformans and var. grubii) and (2) cryptococcus gattii. in a recent study of 31 dogs with cryptococcosis, 68% had cns infection, with neurological signs being the most common reason for presentation. 55 dogs and cats with cryptococcosis typically have pleocytoses and elevated protein concentrations, but pleocytoses may be variably neutrophilic, eosinophilic, mononuclear, or mixed. in a recent study of 15 dogs with cns cryptococcosis, organisms were found in 11 of 15 csf samples (figs. 14.9 and 14.10). 56 all affected dogs had pleocytoses that were mixed to mononuclear, whereas cats tended to have neutrophilic pleocytoses. 56 of the samples, 11 of 12 also had increased protein concentrations (mean 494 mg/dl), which were significantly higher than in cats in the same study (mean 45 mg/ dl). 56 capsular antigen latex agglutination testing on serum or csf is highly sensitive and specific and is recommended if cryptococcosis is suspected but organisms are not visualized cytologically. 57 this test may yield negative results if disease is present but localized (i.e., within the respiratory tract), so appropriate clinical signs should guide testing. culture of csf may also be helpful and may distinguish c. neoformans from c. gattii with the use of selective media. the finding of inflammatory foci on mri may be supportive of the presence of fungal disease; cryptococcosis may result in mass lesions, meningitis, or pseudocyst formation. cryptococcosis is the most common systemic fungal disease of cats and is believed to infect the cns less frequently than in the dog. a recent study found that 42% of 62 cats with cryptococcosis had cns infection, but respiratory signs were still a more common reason for presentation. 55 mild to marked neutrophilic or mononuclear pleocytosis may occur, with variable and occasionally normal protein concentrations. 4 a study of cats with cns cryptococcosis showed organisms in 9 of 11 of the csf samples, and a majority of cases (9 of 10) had neutrophilic pleocytosis and increased protein concentration (8 of 10). 56 eosinophilic pleocytosis may also occur. capsular antigen latex agglutination testing on serum or csf is recommended for confirmation of cryptococcus spp. infection, with rare false-negative reactions if disease is highly localized. fungus that has been visualized in canine csf and may be extracellular or within leukocytes. 58 ehrlichiosis. neutrophilic pleocytosis has been reported in cases of granulocytic ehrlichia spp. in dogs (fig. 14.11 ). 61 neurological signs are uncommon in this disease, and affected dogs may display features ranging from ataxia to seizures. (fip) has been traditionally linked to marked csf changes, but the current literature paints a somewhat more varied picture. one study of natural fip infection showed neutrophilic pleocytosis (as defined by >50% neutrophils) in the majority (7 of 11) of cases, with fewer cases of mononuclear (3 of 11; as defined by >80% mononuclear cells) and mixed cell (1 of 11) pleocytosis, all of variable severity. 4 most cases (7 of 9) also had differing degrees of elevated protein concentrations. 4 diagnosis was confirmed by histopathology or suggested by elevated feline coronavirus antibody titers and reduced albumin-to-globulin ratios in both serum and body cavity effusions. 4 a slightly older study of 16 csf samples (natural and experimental infections) showed pleocytosis in 2 of 16 cases (neutrophilic and lymphocytic) and elevated protein concentration in 4 of 16 cases. 62 in a larger study of 67 cats with fip or non-fip disease, incidence of pleocytosis was highest in the neurological fip group, but 20% of these patients did not have a pleocytosis. 63 additionally, protein concentrations were variably elevated and not statistically different in fip compared with non-fip neurological disease. 63 another study of 12 cats with cns fip showed 8 of 12 with unspecified pleocytosis and 3 of 12 with elevated protein concentration. 64 in cats with cns disease, sensitivity of feline coronavirus (fecov) immunoglobulin g (igg) in csf for the diagnosis of fip was 60%, and specificity was 93%, with a positive predictive value of 75% and a negative predictive value of 87% (fip prevalence in this population was 25.6%). 63 definitive diagnosis of this disease remains challenging, with virus identification (pcr or immunohistochemistry) accompanied by pyogranulomatous inflammation in tissues being the gold standard. hypergammaglobulinemia, elevated serum α 1 -acid glycoprotein (agp), mri abnormalities (typically involving the ventricular lining and meninges), and positive feline coronavirus igg titer or pcr from serum, tissue, or csf are supportive but not specifically diagnostic, and negative findings do not rule out disease. 20, 63, 65 toxoplasmosis in cats. cats are the definitive hosts for toxoplasma gondii and may be subclinically infected; thus, diagnostics should only be performed on patients with appropriate clinical signs. cats typically present with mild neutrophilic or mononuclear pleocytosis and normal to mildly elevated protein concentration, but marked protein elevation may occur. 4 mild lymphocytic pleocytosis is also reported. 65 diagnosis may be confirmed by direct visualization of organisms in csf, aspirates of other inflammatory foci, histopathology of affected tissues, or fecal examination. serology must be interpreted cautiously because igg may remain elevated for up to 6 years after exposure. therefore, paired serum igm-igg titers, indicating acute exposure, or documentation of rising serum igg titers are more useful, but the latter is difficult to document in the advanced state of disease. 65, 66 spinal epidural empyema in dogs. epidural empyema is an uncommon disease in dogs, resulting from pyogenic infection in the epidural space. one study showed 4 of 5 dogs with neutrophilic pleocytosis of variable magnitude (11-342 cells/μl). 67 no organisms were visualized on any of the samples. 67 except for one case with a lumbar csf protein concentration of 726 mg/dl, protein elevations were modest. 67 three csf samples were cultured with no growth, and two dogs for which follow-up csf was obtained showed resolution of pleocytosis. 67 these results are not surprising, as the dura likely provides a barrier to prevent infection extending from the epidural space to the subarachnoid space. reported in a young cat with a marked neutrophilic pleocytosis with intracellular and extracellular merozoites observed on csf cytology. 68 diagnosis was confirmed with decreasing paired serologic titers, and speciation to the level of sarcocystis dasypi or sarcocystis neurona was conducted with pcr from blood. 68 a case of systemic acanthamoeba spp. infection in a young boxer, diagnosed post mortem, had antemortem csf with marked neutrophilic pleocytosis (4956 cells/μl), marked increase in protein concentration (259 mg/ dl), and subnormal csf iga concentration (33 mg/dl; reference interval 35-270 mg/dl). 69 postmortem pcr for the organism was positive on extraneural tissue but not on csf or spinal cord. 69 the patient had been deliberately immunosuppressed on the basis of a preponderance of evidence of steroid-responsive meningitis arteritis at initial presentation and thus may have been infected either before or opportunistically after treatment. 69 another case report of canine cerebellar balamuthia mandrillaris infection (diagnosed post mortem with immunohistochemistry) displayed a marked neutrophilic pleocytosis (234 cells/μl), but other cases with lymphocytic pleocytosis have been reported. 70 because of tissue encystment, it is suggested that extraneural tissue be used for immunohistochemistry or pcr for antemortem confirmation of amoebic infection; pcr of csf may be diagnostic but is not widely available. 69, 70 two dogs with aberrant spinal migration of spirocirca lupi nematodes had moderate to marked neutrophilic to mixed or eosinophilic pleocytoses (800 cells/μl with 91% neutrophils; 180 cells/μl with 60% neutrophils, 30% eosinophils). 71 steroid-responsive meningitis arteritis. steroid-responsive meningitis arteritis (srma) is presumptively an immune-mediated disease of mainly young, medium-and large-breed dogs: beagles, boxers, bernese mountain dogs, weimaraners, and nova scotia duck tolling retrievers are overrepresented. 20 csf analysis is important in diagnosis and typically features a moderate to marked neutrophilic pleocytosis (a left shift may be present) and markedly elevated protein concentration. chronically, pleocytosis may change to a more mononuclear or mixed population (fig. 14.12 ) and may become mild or even fall into reference intervals. 72 a study of 20 affected dogs showed neutrophilic pleocytosis in 12 of 20 cases and mononuclear pleocytosis in 8 of 20 cases. 72 concurrent elevations of serum and csf iga titers (elevated igg and igm fractions may be present), serum concentration of cross-reactive protein (crp), or serum α 2 -macroglobulin is diagnostically supportive but not specific. 20, 50 increases in iga have been linked to a t-helper 2 (th2)-dominated immune response driven by elevated interleukin-4 (il-4) and decreased il-2 and interferon-gamma (ifn-γ). 73 serum amyloid a (saa), serum agp, and serum haptoglobin may also be elevated. 74 another study of 36 dogs with srma reported statistically significant elevations of csf and serum crp, but not serum α 2 -macroglobulin, in dogs with srma compared with other neurological diseases. 75 in a study of 20 dogs, serum crp was positively correlated with csf tncc. 72 additionally, serum haptoglobin and serum and csf iga remained increased throughout successful treatment, indicating that these parameters are more useful for diagnosis than for monitoring therapy. 72 serum and csf concentrations of crp and saa have been documented to fall significantly during treatment, and repeat measurement of serum crp or saa may be used to guide therapy and predict relapse, which is less invasive and more sensitive than repeat csf sampling. 72, 74, 75 rare cases have been documented in cats with marked mononuclear or mixed pleocytosis and mild to moderate protein concentration elevations. 4 intervertebral disk herniation. csf from patients with ivdh may be extremely variable; data indicate that csf findings correlate with location of sampling, disk herniation location, chronicity of the lesion, and severity of spinal cord injury. bearing this in mind, it is no surprise that some reports in the literature state that neutrophilic, lymphocytic, mixed, and mononuclear pleocytoses are most common in dogs with ivdh. 12, 30, 76 a study of 423 cases of ivdh showed 51% with pleocytosis, of which 31% were neutrophilic, 41% were lymphocytic, 20% were mixed, and 7.4% were mononuclear. 76 of all cases, 71% had elevated protein concentrations. 76 interestingly, a larger number of cases of lymphocytic pleocytosis were observed in the samples analyzed more than 7 days after onset of clinical signs. 76 the magnitude of pleocytosis, in general, was also shown to decrease with increasing time between clinical onset and sampling, and this observation has been corroborated by other studies. 12, 76 prior treatment with corticosteroids was observed to reduce the number of observed lymphocytes in csf. 76 the authors also found a higher incidence of pleocytosis in thoracolumbar disease (61%) compared with cervical disease (23%), but this may have been caused by exclusive sampling of lumbar csf closer to the lesion. 76 ivdh is rare in cats and has been reported to feature mild mixed cell pleocytosis and elevated protein concentration. 4 patients typically present with nonpainful, progressive, asymmetrical neurological signs. as only histopathology is confirmat ory, it is a multimodal diagnosis of exclusion. a study of 32 dogs with presumptive fce, based on history, clinical signs, imaging, and outcome, showed 53% with normal csf, 25% with acd, and 19% with mild to moderate pleocytosis (7-84 cells/μl; median 12/μl). 77 pleocytoses were neutrophilic or mixed. 77 one study of 36 confirmed cases in dogs showed that 64% had normal csf and the remainder displayed mild changes. 78 another study looking at five dogs suggested that pleocytosis may be marked, up to 529 cells/μl. 3 fce is much less common in cats. in general, the disease process and clinical signs are similar to those in dogs, with the exception that the disease presents in cats in middle or older age, usually with cervical spinal cord signs. a case series of five cats showed csf ranging from normal to marked neutrophilic pleocytosis with moderately elevated protein concentration and variable correlation to clinical outcome. 79 the case with the most severe csf changes had extensive myelomalacia at necropsy. 79 it was suggested in this study that csf is more likely to be abnormal if collected closer to the lesion and that mri is helpful for localization and in supporting the diagnosis. 77, 79 thiamine deficiency in cats. thiamine deficiency is a rare nutritional disorder of patients fed noncommercial, misformulated commercial, or irradiated diets. two case reports showed increased percentage of neutrophils or mild neutrophilic pleocytosis, presumptively from cerebrocortical necrosis. 4 diagnosis is based on history, response to treatment, mri features compatible with the disease (cortical and brainstem hyperintensities), or histopathology. 80 spaniels with chiari-like malformation showed that 40% of dogs with concurrent syringomyelia and cisternal csf sampling had mild (up to 15 cells/μl) pleocytoses and increased percentages of neutrophils compared with the subpopulation without syringomyelia, but it was not specifically documented whether pleocytoses were, in fact, neutrophilic or mixed with an increased percentage of neutrophils. 42 a positive correlation was also seen to exist between tncc and syrinx size. 42 neoplasia. it is important to perform csf in neurology patients with suspected neoplasia, as definitive diagnosis may be achieved if neoplastic cells are directly observed via cytology. inflammatory pleocytoses or elevated protein concentrations are common in patients with cancer, tend to be mild to moderate in magnitude, and may represent paraneoplastic inflammation, compromise of the bbb, lesional necrosis, or all of these. 28 normal csf is also a common finding in cases of neoplasia. moreover, in the absence of overtly neoplastic cells, no defined patterns connect specific tumors with specific types of inflammatory pleocytoses. neutrophilic pleocytosis of unspecified magnitude was found in the csf of 2 of 11 cats with spinal lymphoma and in 3 of 7 cats with nonlymphoma spinal neoplasia (astrocytoma or osteosarcoma). 81 additionally, the remaining four cats with nonlymphoma spinal tumors (meningioma, peripheral nerve sheath tumor, plasma cell tumor) had either normal csf or acd of unspecified magnitude. 81 metastatic tumors to the cns should also be considered in a patient with neurological signs. eosinophilic meningoencephalitis of dogs. eme is an idiopathic diagnosis of exclusion that is typically steroid responsive and is postulated to be triggered by an underlying hypersensitivity, allergy, or self-limiting infection. the disease may be overrepresented in rottweilers and golden retrievers. 82 a study of 23 dogs with eosinophilic pleocytosis (defined by >20% eosinophils) showed 16 cases of idiopathic eme, 4 cases of infectious disease (c. neoformans, n. caninum, baylisascaris procyonis), and 3 cases of ivdh. 83 the magnitude of pleocytosis or the percentage of eosinophils could not be used to distinguish infectious versus eme cases, although ivdh cases tended to have milder pleocytoses (<84 cells/μl). 83 in about half the eme cases, mri showed abnormal findings. 83 peripheral eosinophilia may or may not be present. highly suggestive of protozoal (toxoplasmosis, neosporosis), fungal (cryptococcosis), parasitic (including cuterebra spp., dirofilariasis), and algal (protothecosis) infections and also rarely in cases of canine distemper and rabies viruses. 10, 84 eosinophils have also been found in cases of granulomatous meningoencephalomyelitis (gme). 85 eosinophilic pleocytosis has been documented in bacterial encephalitis as well. 21 gondii infection tend to have neurological or neuromuscular signs. case reports are sporadic; documentation of mild acd (58 mg/dl) and also a report of mild lymphocytic or eosinophilic pleocytosis (35 cells/μl) with an elevated protein concentration of 77 mg/dl exist in the literature. 86 it is important to rule out other potential causes of the neurological signs because immunocompetent dogs tend to clear subclinical infections, and therefore paired serum igm-igg titers or sequential serum igg titers are preferable to a single serum igg titer. to the author's knowledge, no data on the life span of canine igg antibodies exist. reports in the literature are conflicting with regard to the cross-reactivity of t. gondii antibodies to other agents, such as n. caninum. 20,86 pcr testing for toxoplasma in serum, tissue, or csf is diagnostic. 86, 87 rabies. pleocytoses may be lymphocytic and of varying severity. ancillary antemortem diagnostics include viral pcr on saliva or csf and the saliva antigen latex agglutination test. in a study of 15 dogs under quarantine for suspected natural infection (subsequently confirmed positive), 13 of 15 were saliva-pcr positive, and 4 of 15 (27%) were csf-pcr positive. 88 all animals with positive results on csf were also positive on saliva, and interestingly 100% correlation was seen between positive csf-pcr and the dull clinical presentation (all aggressive clinical presentations were csf-pcr negative). 88 negative testing should never exclude diagnosis because viral load is highest within salivary glands and brain parenchyma. 88 frequently made by exclusion when coupled with appropriate clinical signs. csf and mri findings are variable and may be normal in the acute stage of disease before inflammation has peaked. 89 in a study of 32 dogs with noninflammatory distemper, half (15 of 32) had normal csf. 85 a study of eight dogs with natural infection (confirmed by cns tissue-pcr and histopathology) showed lymphocytic pleocytosis in all samples and normal protein concentrations. 90 another case (confirmed by tissue-pcr and csf-pcr) in a 7-month-old dog displayed marked (554 cells/μl) lymphocytic pleocytosis and a normal protein concentration. 89 because this is a demyelinating disease, myelin-like material, which is amorphous, granular, pink, foamy, and stains positively with luxol fast blue, may be present. 40 pcr testing of csf, serum, urine, epithelial or tonsillar tissue is available, and immunohistochemistry on biopsy specimens of nasal mucosa, haired skin, or footpad is 88% to 96% sensitive for detection of viral antigen. 91 is present, it is likely to be lymphocytic. pleocytoses are typically mild to moderate, but severe lymphocytic pleocytoses have been reported. 89 main differential diagnoses include other viral diseases, gme, or chronic bacterial infection. extranigral signs related to the gastrointestinal or the respiratory system, if present, may be helpful in distinguishing this disease from gme. 89 in a study comparing four dogs with chronic cdv, six dogs with acute cdv, and controls, dogs with chronic cdv had markedly elevated csf igg concentration. 92 the igg region was polyclonal, including a population of neutralizing antibodies for cdv. 92 fungus acquired through inhalation, and most cases in the united states are observed in the southwestern region of the country. signs tend to involve respiratory or skeletal systems, and cns involvement is rare. one dog had a mild to moderate lymphocytic pleocytosis. 57 complement fixation (detecting igg) or tube precipitation (detecting igm), or agar-gel immunodiffusion serological testing is recommended for confirmation. necrotizing meningoencephalitis. meningoencephalitis has been subcategorized as necrotizing meningoencephalitis (nme) and necrotizing leukoencephalitis (nle) on the basis of histopathological appearance. both nle and nme are believed to have an immune-mediated basis, and recent data support that in pugs with nme, canine leukocyte antigen gene aberrations exist. 93 meningoencephalitis is rapidly progressive and affects a variety of generally young to middle-aged toy-breed dogs, including the pug, shih tzu, papillon, maltese, chihuahua, yorkshire terrier, french bulldog, pekingese, west highland white terrier, boston terrier, japanese spitz, and miniature pinscher breeds. 20, 94 a study of csf from 14 pugs with nme showed 12 of 14 with pleocytoses of varying severity (mean 120 cells/μl). 95 of these dogs, 66% had a lymphocytic pleocytosis, 17% had a mononuclear pleocytosis, and 17% had a mixed cell pleocytosis (fig. 14. 13) 95 ; 11 of 14 dogs had elevated protein concentrations (mean 88.4 mg/dl). 95 another study of three dogs showed one with acd and two with moderate to marked (40-220 cells/μl) neutrophilic to lymphocytic pleocytosis. 94 mri findings may help support a diagnosis, but only histopathological examination of lesions provides definitive proof. other. four cats with ischemic encephalopathy had mild (<10 cells/μl) lymphocytic pleocytosis. 28 another study of feline ischemic encephalopathy reported one cat with normal csf and another with mononuclear to mixed pleocytosis (26 cells/μl). 96 a report of two cats with cerebrovascular disease (infarction or stroke) showed one with mononuclear pleocytosis and the other with acd. 97 cerebrovascular disease was correlated in several other cases (without csf data) to hepatic lipidosis or fip. 97 other. a case report of a pug with a mild mononuclear pleocytosis (8 cells/μl), mild elevation in protein concentration (89 mg/dl), evidence of hemorrhage, and direct visualization of angiostrongylus vasorum helminth larvae is found in the literature. 102 eosinophilia was not observed within the csf or peripheral blood. 102 the organism is endemic in europe and canada among foxes and canids, mainly causing respiratory signs or coagulopathy; neurological signs are typically caused by hemorrhage. 102 two dogs with paraparesis and pyogranulomatous lumbar masses (one intradural, one extradural) had lumbar csf with mild mixed cell pleocytosis (lymphocytes and nondegnerate neutrophils) or lymphocytic pleocytosis. 103 these patients were serologically pcr positive for bartonella vinsonii subsp. berkhoffi and presented with a nodular dermatosis. 103 a dog with neurological signs and hepatozoon canis infection showed marked lymphocytic pleocytosis (243 cell/μl) with mildly elevated protein concentration (37 mg/dl). 104 organisms were not visualized in csf but were found on cytology of peripheral blood, lymph node, bone marrow, and bony lesions. serology and positive pcr from a bone marrow sample were diagnostic. 104 intrathecal contrast administration. contrast media or pharmacological agents, such as epidural anesthetics, may introduce preanalytical error into csf samples, artificially raising tncc and protein concentrations. 105 in a study of 17 healthy dogs given either iopamidol or metrizamide for emg, same-day csf sampling showed that 8 of 17 developed a mild to moderate mononuclear to mixed mononuclear or neutrophilic pleocytosis (6 of 8 were from iopamidol). 106 in the same study, 3 of 17 (all metrizamide) developed mild protein elevation, but mean protein concentration for both groups stayed within the reference interval. 106 in dogs given metrizamide, 7 of 8 had an increased pandy score after emg, which was considered a false-positive result because of the contrast agent. 106 these data, plus histopathology from the same population, showed that the contrast agents caused low-grade leptomeningeal inflammation with no statistical difference between the two agents studied. 106 another similar study over 30 days showed that post-emg csf changes reversed after approximately 5 days. 107 granulomatous meningoencephalitis. gme is a progressive immune-mediated disease that is overrepresented in females, toybreed dogs, and terriers. 20 it is a diagnosis of exclusion and has clinical presentations and mri findings that may be similar to various infectious and neoplastic diseases. csf may be unaffected or may display a mononuclear to mixed pleocytosis and protein concentration elevations, both of varying severity (figs. 14.14 and 14.15). in a study of 188 csf samples from dogs with inflammatory neurological diseases, marked pleocytosis (>1000 cells/μl) was found in cases of srma, bacterial encephalitis, or gme. 85 pleocytosis may also be lymphocytic or neutrophilic. 10 csf protein electrophoresis may be helpful, as several cases have been shown with increased β-globulin and gammaglobulin fractions. 108 most of the diseases described previously in this chapter may manifest as mixed cell pleocytoses, depending on the time interval between disease onset and csf sampling, disease severity, and previous treatment administered. a mixed cell pleocytosis would be expected to occur during transition between different phases of the inflammatory response, where certain cells may predominate at specific times after injury. is typically rare and may involve chorioretinitis or focal cerebral granuloma in the cat. 109 a study of two dogs with systemic blastomycosis and neurologic signs showed mild mixed cell pleocytosis (8 cells/μl, mononuclear predominant; and 15 cells/μl, lymphocytic predominant). 110 using csf cytology or culture to diagnose the organism may be unrewarding. agar-gel immunodiffusion serologic testing has high sensitivity and specificity for canine antibodies and is recommended if appropriate clinical signs (respiratory signs or lymphadenopathy) are present. 57 agar-gel immunodiffusion testing is less sensitive (25%-33%) in the cat, as indicated by a limited number of reports. 57 urine antigen enzyme immunoassay (eia) has good sensitivity for dogs and has been used successfully on at least one cat. 109 eia may also be performed on csf. cytology of nasal, pulmonary, or dermal lesions is more likely to yield direct visualization of organisms. lymphoma. lymphocytic pleocytosis of inflammatory origin may be difficult to distinguish from lymphoma exfoliating into the csf (fig. 14.16 ). the size of lymphocytes and morphological atypia may be helpful, although these may be challenging to differentiate from artifactual morphological changes secondary to cytospin preparation. cats with neoplasia may have lymphocytic pleocytoses (suggestive of lymphoma), mild to moderate mononuclear to mixed cell pleocytoses (suggestive of nonlymphoma tumors), or normal csf. one study examined six cases of feline cns or multifocal lymphoma, which displayed pleocytoses of variable magnitude, absent to mildly elevated protein concentrations, and neoplastic cells visualized in 5 of 6 of the csf samples. 4 in this study, eight cats with cns signs that were ultimately diagnosed with nonlymphoma tumors (e.g., meningioma, carcinoma, nerve sheath tumor) had mild csf protein elevations and either normal tncc (1 of 8) or mild to moderate mononuclear or mixed cell pleocytosis (7 of 8). 4 another study of 11 cats with spinal lymphoma showed neoplastic cells visualized in one case and hemodilution, acd, or neutrophilic pleocytosis in the remainder of cases. 81 a case report of feline multiple myeloma involving lumbar vertebrae and associated soft tissues exhibited cisternal csf with an elevated protein concentration of 290 mg/dl and mild pleocytosis (8 cells/μl) consisting of a majority of neoplastic plasma cells. 111 diagnosis was further confirmed by abnormal urine protein electrophoresis and bone marrow aspiration. 111 histiocytic malignancies. malignant histiocytosis or histiocytic sarcoma tumor cells in canine csf have been documented in two recent case reports; csf cytology displayed marked mononuclear pleocytoses (>500 cells/μl) and mild to moderately elevated protein concentrations (<135 mg/dl). 112, 113 tumor cells phenotypically resembled macrophages, displayed multiple criteria of malignancy, and reacted positively to cd1c on immunocytochemistry, compatible with interstitial dendritic cell origin. 112, 113 necropsy was confirmatory and found no evidence of neoplasia outside of the cns. 112, 113 a case report of a gliomatosis cerebri (gc) neoplasm in a middle-aged poodle showed csf with a mild lymphocytic pleocytosis (20 cells/μl) and protein concentration elevation. 114 on histopathology, lymphocytelike perivascular cuffing and meningitis were noted. other case studies of canine gc have reported normal csf or mild acd. 115 in a study of 56 dogs with intracranial meningioma, in which csf analysis was performed, 29% had normal csf, 45% had acd, and 27% had pleocytosis (2 of 3 of these neutrophilic pleocytosis; 1 of 3 unspecified), with the overall incidence of neutrophilic pleocytosis at 18%. 116 in this study, a positive correlation existed between elevated tncc and anatomical localization of the lesion to the caudal (versus middle or rostral) portion of the cranial fossa, and no association between pleocytosis and necrosis within the lesion was found. 116 these findings contradict prior reports of a high percentage of abnormal csf findings in meningioma, and the authors reported that concurrent glucocorticoid therapy in some of the patients may have negatively biased the data. 11, 116 a study of 26 dogs with spinal meningioma showed no cases with exfoliating tumor cells, 62% with mild pleocytosis up to 47 cells/μl (mean 11 cells/μl), and normal or variably elevated protein concentrations up to 836 mg/dl (mean 212 mg/dl). 117 both cisternal and lumbar csf samples were evaluated in this study and not found to be significantly different. 117 interestingly, tumors of the lumbar region displayed higher mean tncc and protein concentrations compared with tumors of the cervical area (24 versus 4 cells/μl and 158 versus 98 mg/dl, respectively), which the authors postulated may be reflective of a higher number of lumbar csf samples with proximity to the lesion. 117 other neoplasms. a case report of canine csf with 240 cells/μl was characterized by atypical neoplastic round cells that were confirmed on immunocytochemistry and immunohistochemistry to be from a metastatic mammary carcinoma. 118 inflammatory cells were of low numbers and were of a mixed population. 118 a study of csf from 25 dogs with choroid plexus tumors showed direct observation of tumor cells in 47% of the cases of carcinoma. 119 mild to moderate mixed-cell pleocytosis was present in all cases of papilloma and in half of the carcinomas; when pleocytosis was present, no difference in magnitude existed between benign and malignant tumors. 119 all cases had elevated protein concentrations, with median concentration for carcinoma being significantly higher (108 mg/dl) than median concentration for papilloma (34 mg/dl). 119 a cutoff protein concentration of 80 mg/dl yielded a sensitivity of 67% and a specificity of 100% for detection of choroid plexus carcinomas. 119 another case report of canine choroid plexus carcinoma had a mononuclear pleocytosis of 165 cells/μl, mildly elevated protein concentration of 30 mg/dl, and numerous tumor cells visualized. 120 a rise in the availability of stereotactic brain biopsy has facilitated increased cytological assessments of cns lesions. this technique offers several advantages, although significant equipment investment and time to perfect techniques is required. stereotactic biopsy often offers application accuracy for targeting lesions that approximate 3 mm or less in all directions. in one study, diagnostic accuracy of stereotactic biopsy specimens submitted for histopathology (i.e., agreement with specimens obtained via open approaches) exceeded 90%. 121 in experienced hands, stereotactic biopsy is believed to be a relatively low-morbidity procedure. a b 20um 20um cytological interpretation of brain biopsy specimens acquired via stereotaxy or open approaches may be challenging and does require a tumor that exfoliates well, a surgeon willing to provide multiple samples, and a cytologist with expertise in this area. 122 a study of 42 canine and feline cases of biopsy-or necropsy-confirmed cns lesions showed squash-prep smear cytology to have 76% sensitivity in accurately determining diagnosis, with an additional 14% of cases having partial correlation between cytology and histopathology. for the remaining 10% of cases, cytological interpretation did not correlate with final diagnosis. 123 cytological interpretation of cns lesions may be very difficult, and biopsy with histopathological examination is recommended to confirm all diagnoses. it is important for cytological samples to be prepared in the same manner each time to avoid introducing additional cytological variations that the pathologist has to read through. some authors recommend wet-fixation of tissues followed by staining with hematoxylin and eosin (h&e). 122 at the authors' institution, cns cytological samples are air-dried and stained with diff-quik or a modified wright stain. the reader is referred elsewhere for a complete discussion of normal cns cytology. 124 clinical imaging findings, and signalment, should be considered carefully and may help the pathologist to formulate a list of potential differential diagnoses. it must be kept in mind that primary tumors may metastasize to the cns, and these should be included in the differential diagnoses, where appropriate. meningiomas are composed of neoplastic cells arising from the meningothelial cells of the leptomeninges of the cns. 125 these tumors are the most common primary cns tumors of dogs and cats. 126 histologically, these neoplasms are classified into at least nine subtypes based on appearance, and some tumors may be characterized by more than one pattern (fig. 14.17 ). 125 cytologically, smears are often characterized by spindle-shaped cells draped around vessels and arranged in large whorling structures (fig. 14.18 ). some cells may contain nuclei that display intranuclear cytoplasmic pseudoinclusions, but this is not a feature reliably seen on a majority of tumors (fig. 14.19 ). 127 as a whole, this group represents the second most common primary cns neoplasm seen in dogs and cats. 126 glial tumors are more a b common than meningiomas in brachycephalic breeds. 126 glial tumors arise from the supporting cells of the cns. astrocytomas are found most frequently in the cerebral hemispheres, although they have been reported to occur in various locations throughout the cns. 125 astrocytomas arise from transformed astrocytes and are characterized cytologically by high cellularity, a high degree of nuclear pleomorphism, and fibrillar cytoplasmic processes. 125 tumor cells will stain positively for glial fibrillary acid protein (gfap). 125 oligodendrogliomas are derived from transformed oligodendrocytes and are found within the gray or white matter of the cns, with the highest incidence in the cerebral hemispheres. 125 cytological preparations are characterized by large numbers of blood vessels surrounded by neoplastic cells (fig. 14.20) . 125 neoplastic oligodendrocytes have small amounts of eosinophilic cytoplasm surrounding uniformly round nuclei. 125 ependymomas are derived from the ependymal lining cells found on the surface of the ventricular 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immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection production of immunoglobulin g and increased antiviral antibody in cerebrospinal fluid of dogs with delayed-onset canine distemper viral encephalitis necrotizing meningoencephalitis of pug dogs associates with dog leukocyte antigen class ii and resembles acute variant forms of multiple sclerosis necrotizing meningoencephalitis in five chihuahua dogs epidemiology of necrotizing meningoencephalitis in pug dogs cerebrospinal cuterebriasis in cats and its association with feline ischemic encephalopathy feline cerebrovascular disease: clinical and histopathologic findings in 16 cats clinical characterization of a familial degenerative myelopathy in pembroke welsh corgi dogs detection of neospora caninum tachyzoites in canine cerebrospinal fluid detection of neospora caninum tachyzoites in cerebrospinal fluid of a dog following prednisone and cyclosporine therapy necrotizing cerebellitis and cerebellar atrophy caused by neospora caninum infection: magnetic resonance imaging and clinicopathologic findings in seven dogs angiostrongylus vasorum causing meningitis and detection of parasite larvae in the cerebrospinal fluid of a pug dog bartonella-associated meningoradiculoneuritis and dermatitis or panniculitis in 3 dogs hepatozoonosis in a dog with skeletal involvement and meningoencephalomyelitis cerebrospinal fluid response following metrizamide myelography in normal dogs: effects of routine myelography and postmyelographic removal of contrast medium cerebrospinal fluid changes after iopamidol and metrizamide myelography in clinically normal dogs transient leakage across the blood-cerebrospinal fluid barrier after intrathecal metrizamide administration to dogs cerebrospinal fluid protein electrophoresis: a clinical evaluation of a previously reported diagnostic technique cerebral blastomyces dermatitidis infection in a cat clinical and magnetic resonance imaging features of central nervous system blastomycosis in 4 dogs multiple myeloma with central nervous system involvement in a cat antemortem diagnosis of localized central nervous system histiocytic sarcoma in 2 dogs cerebrospinal fluid from a 10-yearold dog with a single seizure episode oligodendroglial gliomatosis cerebri in a poodle gliomatosis cerebri in six dogs characteristics of cisternal cerebrospinal fluid associated with intracranial meningiomas in dogs: 56 cases (1985-2004) canine intraspinal meningiomas: imaging features, histopathologic classification, and long-term outcome in 34 dogs neoplastic pleocytosis in a dog with metastatic mammary carcinoma and meningeal carcinomatosis choroid plexus tumors in 56 dogs (1985-2007) choroid plexus carcinoma cells in the cerebrospinal fluid of a staffordshire bull terrier ct-guided brain biopsy using a modified pelorus mark iii stereotactic system: experience with 50 dogs primary canine and feline nervous system tumors: intraoperative diagnosis using the smear technique squash-prep cytology in the diagnosis of canine and feline nervous system lesions: a study of 42 cases canine and feline cytology-e-book: a color atlas and interpretation guide tumors in domestic animals what is your diagnosis? intracranial mass in a dog a true "triphasic" pattern: thoracolumbar spinal tumor in a young dog key: cord-284262-lddmo1sv authors: li, linlin; mcgraw, sabrina; zhu, kevin; leutenegger, christian m.; marks, stanley l.; kubiski, steven; gaffney, patricia; dela cruz jr, florante n.; wang, chunlin; delwart, eric; pesavento, patricia a. title: circovirus in tissues of dogs with vasculitis and hemorrhage date: 2013-04-17 journal: emerg infect dis doi: 10.3201/eid1904.121390 sha: doc_id: 284262 cord_uid: lddmo1sv we characterized the complete genome of a novel dog circovirus (dogcv) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. dogcv was detected by pcr in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. co-infection with other canine pathogens was detected for 13/19 (68%) dogcv-positive dogs with diarrhea. dogcv capsid proteins from different dogs varied by up to 8%. in situ hybridization and transmission electron microscopy detected dogcv in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. the detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs. c ircoviruses are nonenveloped, spherical viruses with a single-stranded circular dna genome of ≈2 kb; they group as a genus within the family circoviridae, together with the proposed genus cyclovirus and the phylogenetically more distinct genus gyrovirus (1) . most of the known species in the genus circovirus infect birds and cause signs including malformations and necrosis of the integument, lymphoid depletion, and immunosuppression (2) . before 2012, the only circoviruses reported to infect mammals were the 2 closely related porcine circoviruses (pcvs) (3) . pcv2 is the primary pathogen associated with a spectrum of swine diseases called porcine circovirusassociated diseases that have been described in pigs worldwide. pcv2 infection causes severe economic losses because of increased mortality and reduced production, making it one of the most economically important viruses in the global swine industry. among lesions that have been attributed to pcv2 infection are pneumonia, enteritis, lymphadenitis, vasculitis, nephritis, and reproductive disease (4) . in cases for which pcv2 is considered causative, immunohistochemical and in situ hybridization (ish) analyses demonstrate large amounts of pcv2 antigen or nucleic acids in the cytoplasm of macrophages and dendritic cells in the depleted follicles in lymphoid tissues (4, 5) . naturally occurring porcine circovirus-associated diseases is often accelerated or exacerbated by concurrent viral or bacterial infections, and secondary infections often occur as a result of immunosuppression (6) . random nucleic acid amplification with or without prior enrichment for viral particle-associated nucleic acids (7, 8) , followed by deep sequencing and in silico similarity searches for sequences related to those of known viruses, have been highly productive in the field of animal virus discovery (9) (10) (11) . we used this technique to identify virus sequences in affected tissues from companion animals with diseases of unknown cause. we identified a canine circovirus in the liver of a dog that had necrotizing vasculitis and granulomatous lymphadenitis, both of which are described in pcv2-infected pigs (4) . we named this virus dog circovirus (dogcv) rather than canine circovirus to avoid confusion with the cacv notation used for canary circovirus (12, 13) , canine calicivirus (14, 15) , and capsicum chlorosis virus (16) . a closely related variant of dogcv was sequenced independently in canine serum samples and was published recently (17) ; however, no disease association was described with the virus. to determine whether dogcv could be associated with canine vascular disease, we identified additional dogs with vascular and granulomatous lesions and examined the distribution of dogcv by ish analysis. a 1-year-old castrated male dog that had been kenneled for 3 weeks was brought to the university of california, davis (uc davis), veterinary medical teaching hospital for evaluation of progressive vomiting and diarrhea with hematochezia. despite initial supportive therapy at the referring veterinarian, clinical signs worsened; at uc davis, the dog was treated for hypovolemic shock. because of suspected disseminated intravascular coagulation and a poor overall prognosis, the owner elected to have the dog euthanized and granted permission for routine necropsy, which was performed at the anatomic pathology service of the uc davis school of veterinary medicine. the clinical and postmortem workups for infectious causes in this case included negative test results for infectious causes of enteric disease, such as canine parvovirus, canine enteric coronavirus, salmonella spp., canine distemper virus, campylobacter spp., clostridium perfringens enterotoxin a gene, cryptosporidium spp., and giardia spp. histologic results showed extensive fibrinoid vascular necrosis, thrombosis, and hemorrhage throughout the gastrointestinal tract and kidneys, as well as granulomatous lymphadentitis of the mesenteric lymph nodes. special stains of histologic specimens revealed no detectable bacteria or other infectious agents. liver tissue samples were collected, stored in whirlpack bags, and frozen at −80°c until further processing. a liver tissue sample (≈25 mg) were immersed in 1 ml cold hank's balanced saline solution and disrupted with a tissue homogenizer for 30 sec on ice. the resulting homogenates were placed on dry ice for 5 min and then thawed at room temperature (18) . freezing and thawing were repeated twice. samples were clarified by centrifugation at 10,000 × g for 3 min; the supernatants were then filtered and underwent nuclease treatment as described (19) . viral nucleic acids were extracted by using the qiaamp viral rna mini kit (qiagen, valencia, ca, usa) and stored at −80°c. viral nucleic acid libraries were prepared as described (19) . the library of single-stranded dna fragments was sequenced by using the genome sequencer flx instrument (roche, indianapolis, in, usa). the pyrosequencing reads were sorted and trimmed as described (19) . trimmed reads from each sample were assembled de novo by using the mira assembly program (20) , with a criterion of >95% identity over 35 bp. the assembled sequences and singlets >100 bp were compared with the genbank nonredundant nucleotide and protein databases (www.ncbi.nlm.nih.gov/genbank) by using blastn and blastx, respectively (http://blast.ncbi. nlm.nih.gov/blast.cgi). potential viral sequences with significant hits (e-value <0.001) to known virus sequences were identified. pcr and sanger sequencing were used to confirm the presence of virus genome sequences assembled from deep sequencing reads. inverse pcr was then used to amplify the genome of target circoviruses with primers based on the sequences obtained by deep sequencing. virus genome sequences obtained were deposited in genbank (accession nos. kc241982-kc241984). putative open reading frames (orfs) with coding capacity >100 aa were predicted by vector nti advance 11 (invitrogen, carlsbad, ca, usa). the stem-loop structure was predicted by using mfold (21) . phylogenetic analyses based on aligned amino acid sequences from full-length replicate (rep) proteins were generated by using the neighbor-joining method in mega4 (22) , using amino acid p-distances with 1,000 bootstrap replicates. other tree-building methods, including maximum parsimony and maximum likelihood, were used to confirm the topology of the neighbor-joining tree. real-time pcr using 2 primers and a conventional hydrolysis probe with a 5-primer 6-fam and 3-primer tam-ra label and taqman universal pcr master mix (applied biosystems, foster city, ca, usa) was used to detect dogcv in dna extracts from 3 dog sample cohorts: 1) fecal samples from 204 healthy dogs; 2) fecal samples from 168 dogs with diarrhea; and 3) blood samples from 480 dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. primer pairs and probes for canine circovirus are given in the table. total nucleic acid was extracted by using the corbett x-tractor gene platform (qia-gen). real-time pcr was conducted by using the real-time pcr instrument lightcycler 480 (roche, indianapolis, in, usa) under these conditions: 50°c for 2 min, then 95°c for 10 min, followed by 40 cycles of 95°c for 15 sec and 60°c for 1 min. synthetic dna fragments (≈150 bp) of the corresponding regions were used to produce a standard curve and an analytical sensitivity of 10 molecules. a fourth sample cohort consisted of tissue samples from 21 necropsy cases of dogs whose clinical signs or microscopic lesions matched the sentinel animal (i.e., hemorrhagic diarrhea, vasculitis, and/or granulomatous disease); these samples were selected from the tissue archives of anatomic pathology at the uc davis veterinary medical teaching hospital. control tissues were obtained from 5 dogs whose cause of death was unrelated to vascular disease. tissue sections chosen for analysis were, in part, case dependent because the presence of vasculitis or inflammation among these cases was not limited to a single tissue type. spleen, lymph node, jejunum, and ileum were examined in all cases; other examined tissues were kidney, brain, adrenal gland, pancreas, duodenum, heart, and lung. tissues from the sentinel dog were included in this analysis. tissue sections were mounted on 3-aminopropyltriethoxylane-coated slides (fisher scientific, pittsburgh, pa, usa). a 25-nt oligomer (ctcagacagagacac-cgttgctatg) complementary to a segment of orf2 (capsid) was 3′-end labeled by the addition of a single digoxigenin-ii-dideoxy undine (eurofins mwg operon, huntsville, al, usa). a manual capillary-action work station (fisher scientific) was used to perform colorimetric dna in situ hybridization. tissue sections were deparaffinized and digested by incubation at 37°c for 10 min in 0.25% pepsin in 1× tris-buffered saline (ph 2.0); pepsin activity was stopped by a 5-min incubation at 105°c. nucleic acid was denatured by 5-min incubation at 105°c in 100% formamide. tissue sections were then incubated at 37°c in 10 µmol of the digoxigenin-labeled probe in hybridization buffer (22.5% deionized formamide, 7.5% chondroitin sulfate, 5× saline sodium citrate, 0.25% blocking reagent, and 50 mmol phosphate buffer). sections were incubated in an antidigoxigenin antibody solution (500:1 dilution) containing 2.5 ml buffer 1 with 5 μl of antidigoxigenin fab fragments conjugated with alkaline phosphatase (750 u/ml) (roche). sections were then washed and developed according to manufacturer instructions before counterstain with 1% fast-green fcf for 5 min. slides were mounted with immunohistomount (immunobioscience, mukilteo, wa, usa) and coverslipped with shur/mount (triangle biomedical sciences, durham, nc, usa). no background hybridization was seen when replicate tissue sections were incubated with an unrelated digoxigenin-labeled probe with similar guanine-cytosine content or when matched tissue from unaffected dogs were incubated with the dogcv-specific probe (23) (24) (25) . selected pieces of formalin-fixed lymph node tissue from the sentinel dog were post fixed in 2.0% glutaraldehyde and then routinely processed and embedded in epoxy resin (eponate12 kit; ted pella, inc., redding, ca, usa). selected thick sections were stained with toluidine blue, as described (26) . ultrathin sections from select areas of the lymph node were examined by using a zeiss (göttingen, germany) 906e transmission electron microscope. of ≈10,000 squesnce reads, 5 contigs and singlets composed of 52 sequence reads from the liver tissue had significant similarity to the rep protein of circoviruses (evalue <1 -10 ). two bocavirus sequences were also detected. because circoviruses have a circular genome, the full viral genome was then amplified by inverse nested pcr and the amplicon sequenced by primer walking. the assembled genome was named dogcv strain ucd1 (dogcv-ucd1). the complete circular genome of dogcv-ucd1 was 2,063 nt (genbank accession no. kc241982). analysis of the genome sequence showed characteristics typical of circoviruses, including an ambisense organization with 2 major inversely arranged orfs encoding the putative replication-associated (rep, 303 aa), and capsid (cap, 270 aa) proteins. a characteristic stem-loop structure with a conserved nonanucleotide motif (5′-tagtattac-3′, similar to the consensus of bird and pig circoviruses) was also found in the 5′-intergenic region (135 nt, between the start codons of the 2 major orfs). the 3′-intergenic region of dogcv-ucd1 between the stop codons of the 2 major orfs was 203 nt (figure 1, panel a) (27) . the complete genome of another dogcv strain (dogcv-ucd2, genbank accession no. kc241984) was amplified and sequenced from a fecal sample from a shelter-housed dog that had vomiting and diarrhea. this strain shared 95% overall genome nucleotide identity with dogcv-ucd1, and both strains showed 96%-97% nucleotide identity to the recently reported canine circovirus isolate 214 from blood (17). the putative rep proteins of dogcv-ucd1 showed 42%-54% amino acid identity to the rep proteins of porcine and avian circoviruses, with the closest identity to pcv1. the dogcv-ucd1 capsid showed <30% amino acid identity to known circovirus capsids. sequence alignment of the putative rep protein of dogcv-ucd1 with those of known species in the genus circovirus identified several highly conserved amino acid motifs, including wwdgy, ddfygw, and dryp. motifs associated with rolling circle replication (ftlnn, tphlqg, and csk) and the dntp binding (gxgks) were also identified. the n terminal region of the cap protein was highly basic and arginine-rich, as is typical for circoviruses. a phylogenetic analysis of the complete rep protein of dogcv strains and all known circoviruses was performed, with chicken anemia virus (genus gyrovirus) as the outgroup (figure 1 , panel b). the phylogenetic tree showed that dogcv strains grouped with pcv1 and pcv2, forming a distinct clade of mammalian circovirus, whereas circoviruses affecting birds clustered separately. dogcv was detected by real-time pcr in fecal samples from 14/204 healthy dogs and 19/168 dogs with diarrhea; the difference in prevalence was not significant (11.3% vs. 6.9%; p>0.05 by χ 2 test). of the 19 dogs with diarrhea who had dogcv detected in fecal samples, 13 (68%) were coinfected with >1 other pathogens, including canine enteric coronavirus, cryptosporidium spp., c. perfringens α toxin, giardia spp., salmonella spp., campylobacter jejuni, and campylobacter coli (tested by pcr). the prevalence of dogcv in blood samples from the cohort of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite was 3.3% (16/480), similar to that reported for canine serum samples (2.9%, 6/205) (17) . the partial rep and/or cap protein regions (≈350 bp) were amplified from 11/16 samples. all showed >96% nucleotide identity, except 1 amplicon, which had <90% nucleotide distance to dogcv-ucd1 and -ucd2. we sequenced the complete genome of this virus (dogcv-ucd3; genbank accession no. kc241983); it showed 91%-92% amino acid identity of the complete rep and cap proteins to dogcv-ucd1 and -ucd2 and to the published canine circovirus (cacv-1 strain ny214; genbank accession no. jq821392) (17) . to establish tissue distribution and investigate whether dogcv contributes to canine disease, we developed and validated an ish oligomeric probe and examined the sentinel dog and dogs from 21 suspected, retrospective cases that included >2 of these 3 signs: vasculitis, hemorrhage, or granulomatous disease. a wide spectrum of affected tissues was represented in this group; matching tissues were also examined from 5 control dogs in which these signs were not present. samples from the sentinel dog (dog 1) and 3 other dogs (dogs 2-4) were positive for dogcv by ish analysis. all other tissue samples from control dogs were negative by ish analysis. clinical signs, gross and histologic findings, and distribution of virus dna as determined by ish were used to examine a possible causal role for dogcv. dog 1 was a male beagle who had acute onset of vomiting and hemorrhagic diarrhea. dog 2 was a 5-yearold, female, spayed boston terrier who had vomiting and diarrhea. dog 3 was a 1-year-old, female, spayed boxer with a 5-day history of lameness and progressive tetraparesis. dog 4 was a 2-year-old greyhound found dead with bicavitary hemorrhage; blood smear and pcr showed this dog was co-infected with babesia conradae. gross examination revealed consistent lesions among these 4 dogs, including lymphadenopathy and hemorrhage. in dogs 1 and 2, the hemorrhage was associated with the gastrointestinal tract (figure 2 , panels a, c); dog 2 had additional multifocal to coalescing regions of hemorrhage in the kidneys (figure 2, panel b) . gross evidence of hemorrhage in dog 3 was restricted to the ventral surface of the brain along the basilar artery overlying the medulla; dog 4 had bicavitary hemorrhage. the common histologic lesion in all dogs was fibrinonecrotizing vasculitis, although the distribution of affected vessels and the amount of associated hemorrhage varied. in dog 1, segments of inflamed or necrotic vessels were seen in the intestine (multiple segments), urinary bladder, liver, spleen, and lungs. in dog 2, vasculitis was limited to the intestine and spleen, and in dog 3, vasculitis was in kidneys, intestine (figure 2, panel g) , heart, liver, spleen, and meninges. in dog 4, only a few vessels were affected, at the corticomedullary junction in the kidneys. for all dogs, histiocytic drainage or granulomatous lymphadenitis were seen in peyer's patches ( figure 2 , panels c, e) and in >1 lymph node. in addition, in dogs 2 and 3, multiple lymph nodes were severely necrotic. all dogs had microscopic lesions in the kidneys, but intensity and character varied widely. dogs 1 and 2 had tubular necrosis with little inflammation, dog 3 had a severe granulomatous interstitial nephritis, and dog 4 had multifocal hemorrhage and minimal lymphocytic, plasmacytic nephritis. dogs 1 and 3 had multifocal pancreatitis and adrenalitis. no multinucleate giant cells, common in pigs infected with pcv2, were seen. by ish analysis, abundant cytoplasmic viral nucleic acid was detected in macrophages within germinal centers and subcapsular and medullary sinuses of the mesenteric ultrastructural analysis of the mesenteric lymph node from dog 1 revealed macrophages laden with large numbers of intracytoplasmic inclusions (figure 3 , panel a). inclusions were round, oblong, or irregular; were <0.5 µm; and often clustered (up to 25 per cell) within the cytoplasm, (figure 3, panel b) . most inclusions were granular and electron-dense and had a distinct periphery but were nondelineated by membrane. some inclusions contained paracrystalline arrays of icosahedral virions that were 9-11 µm in diameter ( figure 3 , panel c). we identified a novel circovirus, dogcv, in the liver of a dog that had necrotizing vasculitis and granulomatous lymphadenitis. we characterized the genome of multiple dogcv strains, determined dogcv prevalence in dog fecal and plasma samples and tissue distribution in infected animals, and detected paracrystalline arrays in inclusion bodies in macrophages. real-time pcr analysis showed a prevalence of 11.3% and 6.9% in fecal samples from dogs with diarrhea and healthy dogs, respectively. dogcv dna was also found in 3.3% of blood samples from dogs with thrombocytopenia and neutropenia, fever of unknown origin, and past tick bite, which is approximately the same percentage as previously reported (17) . ish analysis of the sentinel dog and 21 additional dogs selected retrospectively from past necropsies detected viral nucleic acid in 4 dogs, including the sentinel dog, and the histopathologic features and distribution of virus in tissue samples from these dogs were evaluated. the organs affected varied even in this small set of animals, but all dogs had necrotizing vasculitis and hemorrhage, and all but 1 had lymphadenitis and granulomatous disease. because the tested retrospective animals were chosen to match the sentinel case, our sampling is biased, and the spectrum of diseases associated with this virus might be broader than we detected. among dogs positive by ish, disease signs varied, and clinical, gross, and microscopic features in some of the disease syndromes were similar to those associated with pcv2 infection (4, 5) . in particular, porcine dermatitis and nephropathy syndrome shares many of the histologic features seen in dogcv-positive dogs (28, 29) , and pcv2 has been reported to cause necrotizing lymphadenitis, vasculitis, or neurologic disease (4, 30, 31) . virus distribution, as assessed by ish analysis, is also similar between dogcv and pcv2. viral dna was consistently detected in the cytoplasm of macrophages and monocytes in lymphoid tissues of infected dogs. virus distribution in pigs infected with pcv2 is most consistently within lymphoid tissue, with sporadic reports of virus in other tissues (4, 23, 32, 33) . for example, in pigs with dermatitis and nephropathy syndrome, granulomatous inflammation of the kidneys is commonly reported; however, virus is detected in renal tissue in only a few cases by any method. in our study, dogcv dna was detected in lymphoid tissues, including peyer's patches, even for dogs 3 and 4, where no clinical or histologic enteric lesions were detectable. dogcv dna was also found in small, end-capillary endothelial channels of the intestinal lamina propria and the adrenal cortex; however, confirmation of these cells as endothelial will require further investigation. two distinct histologic features of pcv2 infection are viral inclusions and multinucleate giant cell formation (34) , neither of which was detected by routine histology in the dogs infected with dogcv. by electron microscopy, however, macrophages within the lymph node contained abundant cytoplasmic viral inclusions composed of dense granular or paracrystalline arranged virus. affected cells were found in both the sinus and medullary cords of the mesenteric lymph node examined. ultrastructural inclusions were similar to a subset of cytoplasmic inclusions that have been described in pcv2 infected tissues (35, 36) . numerous experimental and natural disease studies have indicated that pcv2 infection most often leads to clinical diseases in the presence of co-infection with other swine pathogens. pcv2 enhances viral (e.g., porcine parvovirus, porcine reproductive and respiratory syndrome virus), bacterial, protozoal, metazoal, and fungal infections in pigs (6) . among the dogs with diarrhea in our study, most (68%) of those positive for dogcv had co-infection with >1 enteric pathogens. also, for the small set of cases in which we identified virus in situ, 1 dog was co-infected with a canine bocavirus and 1 with babesia conradae. the role of co-infection in the pathogenesis of disease in these cases is unclear. in summary, dogcv should be considered in cases of unexplained vasculitis in dogs, although further studies will be required to ascertain whether and when dogcv causes disease. dogcv also could be a complicating factor in other canine infectious diseases, as is the case with pcv2, which is most dangerous in pigs co-infected with other pathogens. future research into the contribution of dogcv to disease should carefully consider these potential viral and host factors. multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces avian circovirus diseases: lessons for the study of pmws discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2 porcine circovirus type 2 (pcv2) infections: clinical signs, pathology and laboratory diagnosis porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention 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we thank scott fish and the california animal health food safety laboratory in davis for electron microscopy processing and expertise. key: cord-261955-6drue8i9 authors: schulz, b. s.; strauch, c.; mueller, r. s.; eichhorn, w.; hartmann, k. title: comparison of the prevalence of enteric viruses in healthy dogs and those with acute haemorrhagic diarrhoea by electron microscopy date: 2007-12-18 journal: j small anim pract doi: 10.1111/j.1748-5827.2007.00470.x sha: doc_id: 261955 cord_uid: 6drue8i9 objectives: to evaluate prevalence of enteric viruses in healthy dogs and to compare it with prevalences in dogs with acute haemorrhagic diarrhoea. methods: faecal samples were collected from 200 healthy dogs and examined by electron microscopy for presence of viral particles. data were compared with viral prevalences that had been determined retrospectively by electron microscopy for 936 dogs with acute haemorrhagic diarrhoea. results: there were significantly more negative faecal samples among the healthy dogs (82·0 per cent) compared with 55·8 per cent in dogs with acute haemorrhagic diarrhoea (p<0·001). with a prevalence of 17·5 per cent, significantly more healthy dogs were shedding coronavirus compared with 11·6 per cent in dogs with acute haemorrhagic diarrhoea (p=0·034). parvovirus was only detected in one healthy dog (0·5 per cent), thus with a prevalence that was significantly lower than 16·0 per cent detected in the dogs with acute haemorrhagic diarrhoea (p<0·001). paramyxovirus was not found in any of the healthy dogs but was found in 9·3 per cent of dogs with acute haemorrhagic diarrhoea (p<0·001). clinical significance: results suggest that shedding of parvovirus and paramyxovirus is strongly associated with acute haemorrhagic diarrhoea. however, coronavirus seems to be even more prevalent among healthy dogs, raising the need for further studies to investigate the strain‐associated pathogenicity of this virus. infections with enteric viruses are thought to play a significant role as an aetiological agent of acute enteritis in dogs. among the viral species commonly detected in dogs with diarrhoea are canine parvoviruses (cpvs), canine coronaviruses (ccovs) and paramyxoviruses (pvs). rotaviruses, caliciviruses and not further classified virus particles have also been described (hammond and timoney 1983 , evermann and others 1985 , biermann and others 1991 , finlaison 1995 , vieler and herbst 1995 , mochizuki and others 2001 , sokolow and others 2005 . prevalence of enteric viruses in dogs with diarrhoea seems to depend on age of dogs investigated, as well as on vaccination status of affected dogs; however, environmental factors are also thought to play an important role. high prevalences of cpv and ccov were detected in faecal samples of dogs with diarrhoea from animal shelters, indicating the influence of hygiene and infection pressure (stann and others 1984 , tennant and others 1993 , sokolow and others 2005 . furthermore, geographic differences may play a role in prevalence of enteric viruses in canine gastrointestinal disease. however, pathogenicity of some enteric viral species is a matter of discussion because viral agents were also identified in faecal samples of healthy dogs. while shedding of cpv seems to be associated with clinical signs of gastroenteritis, ccov can be detected in healthy dogs without signs of gastrointestinal disease (tennant and others 1993 , möstl and others 1994 , sokolow and others 2005 . antibody prevalence against ccov is high in dogs from animal shelters and breeding colonies in different geographic regions (tennant and others 1993 , naylor and others 2001b , yesilbag and others 2004 . however, there are studies suggesting a significant role for ccov in acute diarrhoea in dogs (tennant and others 1993, yesilbag and others 2004) . the present study was conducted to evaluate the prevalence of enteric viruses in healthy dogs and to compare the data with prevalences in samples obtained from dogs with acute haemorrhagic diarrhoea (ahd) to gain more information about incidence and importance of enteric viruses in the dog population in southern germany. faecal samples were collected prospectively from 200 healthy privately owned dogs. dogs belonged to students and employees or their families and friends (most of them living in southern germany) of the veterinary teaching hospital in munich, germany, or presented to the hospital's health service for routine vaccination. dogs were included in the study if they were clinically healthy and free of gastrointestinal problems (no history of vomiting, diarrhoea or anorexia) in the four weeks before sampling. currently, all dogs were on vaccination according to the owners, but no detailed information was obtained about time point and components of the last vaccination. information about deworming history and feeding management was not available. all dogs were housed indoors and were kept as pet dogs. detailed information about patient data and number of dogs sampled per household is displayed in table 1 . for the group of dogs with ahd, medical records of patients examined at the university of munich veterinary teaching hospital between 1991 and 2001 were searched, and records of dogs that had presented with an acute onset of bloody diarrhoea of less than 48 hours duration were reviewed. of these, dogs in which a faecal examination for virus detection had been performed by electron microscopy (em) were eligible for inclusion in the study. details regarding patient data are displayed in table 2 . samples were transferred to the institute for medical microbiology, infectious and epidemic diseases and examined by em (em 10; carl-zeiss). for this method, faecal material was diluted 1:5 in phosphate buffered saline and centrifuged at low speed. the supernatant was applied on copper grids and negative stained with tungstic acid. to obtain data on dogs with ahd, medical records of dogs examined at the veterinary teaching hospital within a period of 10 years were searched, and records of dogs presented with ahd were reviewed. in the years 1991 to 2001, 936 dogs in which a faecal examination for virus detection had been performed by (em) were eligible for inclusion in the study. viral prevalences obtained for the 200 healthy dogs were compared statistically with the prevalences determined for the 936 dogs with ahd. the parameter age was compared between the dogs shedding ccov and the virus-negative dogs in the healthy dog population; and age was also compared between the groups of dogs shedding different viruses in the group with ahd. furthermore, age comparison was performed between healthy dogs and dogs with ahd with a virus-negative status and between healthy dogs and dogs with ahd shedding ccov, respectively. gender distribution was compared between healthy dogs and dogs with ahd. statistical comparison was performed by use of commercially available statistical software (graphpad software, instat 3, viruses were detected in 36 of 200 (18 per cent) faecal samples from healthy dogs and in 414 of 936 (44á2 per cent) samples from dogs with ahd (fig 1) . there were significantly more negative samples among the healthy dogs compared with dogs with ahd (p,0á001; relative risk: 2á99). significantly, more healthy dogs were shedding ccov, with a prevalence of 17á5 per cent, compared with 11á6 per cent in dogs with ahd (p=0á034; relative risk: 1á46). cpv was only detected in one healthy dog, representing a prevalence of 0á5 per cent, which proved to be significantly lower than the prevalence of 16á0 per cent detected in the group with ahd (p,0á001; relative risk: 0á031). pv was found in 9á3 per cent of dogs with ahd and in none of the healthy dogs (p,0á001; relative risk: indeterminate). none of the healthy dogs was shedding more than one viral species in a single faecal sample. in the group of dogs with ahd, more than one enteric virus was present in 6á7 per cent of the samples; 4á2 per cent of the faecal samples showed ccov and pv, 1á2 per cent cpv and ccov, 0á9 per cent pv and cpv and 0á4 per cent pv, cpv and ccov. comparison of gender distribution between the group of healthy dogs and dogs with ahd did not show a significant difference between groups (p=0á1601). mean age of the healthy dogs shedding ccov was 2á9 years; mean age of the virus-negative healthy dogs was 5á2 years (details displayed in table 3 ). ccovpositive healthy dogs were significantly younger than virus-negative healthy dogs (p=0á002). mean age of the patients with ahd shedding ccov was 4á9 years; mean age of the virus-negative dogs with ahd was 5á5 years. there was no significant difference between ccov-positive and virus-negative dogs with ahd for the parameter age (p=0á222). when age distribution was compared between groups of dogs with ahd, dogs with cpv infection were significantly younger than dogs infected with ccov (p, 0á001), pv (p,0á001) or virus-negative (p,0á001). comparison of the parameter age for virus-negative healthy dogs and virusnegative dogs with ahd showed no statistical significance (p=0á6754). furthermore, no statistical significant difference was detected when age was compared between healthy dogs shedding ccov and dogs with ahd shedding ccov (p=0á0579). while several studies have investigated the role of enteric viruses in dogs with gastrointestinal disease, little information is available about their prevalence in healthy dogs. and because enteric viruses can be detected in a large number of dogs with diarrhoea, this raises the question as to if these viral species actually are the aetiological pathogens causing the gastrointestinal disease? or if they are an incidental finding of non-pathogenic organisms simply being shed by animals with diarrhoea as a result of another aetiology. therefore, the aim of the present study was to further investigate this question by evaluating privately owned pet dogs in southern germany and to compare viral prevalences in healthy dogs and in dogs with ahd in the same geographic region. in this study, cpv was detected in 16á6 per cent of dogs with ahd and in only one healthy dog. these data clearly suggest that shedding of cpv is associated with clinical disease. this assumption is underlined by the results of other studies in which cpv could not be detected in the faeces of healthy dogs (stann and others 1984 , hackett and lappin 2003 , sokolow and others 2005 . the only dog shedding cpv in this study was a one-year-old male hunting dog that had been adopted from a shelter six weeks earlier and since then had not displayed any gastrointestinal signs. a possible explanation could be a recent natural cpv infection before adoption because some naturally infected dogs have been shown to shed virus for up to 46 days after occurrence of first clinical signs (decaro and others 2005) . also, the dog could have ingested cpv contaminated faecal material leading to enteric passage of the virus without systemic infection. this could, for example, happen if the dog is protected by parvovirus vaccination against development of clinical signs but not against infection, thus leading to virus shedding in an asymptomatic animal, as described for puppies shedding ahd acute haemorrhagic diarrhoea, ccov canine coronavirus, cpv canine parvoviruses, pv paramyxovirus *dogs shedding more than one virus species were excluded from statistical comparison yage in years cpv despite presence of maternal antibody titres considered sufficiently high for protection (elia and others 2005) . the dog was fully vaccinated according to the owner. while this study confirms the role of cpv as a primary enteric pathogen, assessment of the role of ccov in canine diarrhoea seems to be more difficult. ccov was detected in 17á5 per cent of healthy dogs and only in 11á6 per cent of dogs with ahd, thus questioning the role of ccov as a primary intestinal pathogen in dogs. the prevalence of ccov in dogs with ahd is comparable with older german studies, which examined the faecal samples from dogs with diarrhoea by em, but so far no information is available about the prevalence of this virus in healthy dogs in southern germany. studies performed in europe and northern america showed high antibody prevalences of ccov in up to 59 per cent of healthy dogs from animal shelters (tennant and others 1993, sokolow and others 2005) . a study performed by tennant and others (1993) failed to detect ccov in the faeces of healthy pet dogs in england. there are several possible explanations for the high number of healthy dogs shedding ccov in this study. ccov belongs to one of three groups of the genus coronavirus and is genetically closely related to feline coronavirus (fcov) and transmissible gastroenteritis virus of swine. because of the high error frequencies of the rna polymerase, coronaviruses are prone to rapidly develop point mutations in coding and non-coding sequences, thus resulting in proliferation of different strains, serotypes and subtypes of the virus (dolja and carrington 1992) . at least two distinct genetic clusters of ccov can be identified in naturally infected dogs. type i ccov is classified as being genetically closely related to fcov and type ii as representing the ''typical'' ccov (pratelli and others 2003) , with both genotypes frequently being harboured by one dog (pratelli and others 2004) . because of the broad genetic variability and fast rate of recombination, even specific strains seem to develop within several weeks in the dog population of animal shelters (benetka and others 2006) . thus, dogs can be continuously re-infected with new virus variants exhibiting a broad range of antigenic and genetic variability, potentially evading the host's immune response. this might be an explanation why, as is known for fcov in cats, dogs are also suspected to become persistently infected with ccov. dogs were found to shed ccov for up to 156 days after natural infection, and the virus strains involved underwent several mutations during that period (pratelli and others 2002) . a persistent infection could therefore also explain the shedding of ccov in the healthy dogs in this study. follow-up faecal examinations and genetic sequencing would be interesting options in these patients to assess duration of shedding and strains involved. because of the wide genetic variation of ccov, different strains are also thought to have different characteristics concerning pathogenicity and virulence factors, possibly being responsible for variation of clinical signs associated with ccov infection. some strains seem to cause severe clinical signs of ahd predominantly in very young dogs (pratelli and others 2003 , evermann and others 2005 , buonavoglia and others 2006 , especially in cases of dual infection with other viral species others 1999, 2001) . although healthy dogs shedding ccov were shown to be significantly younger than virus-negative dogs in this study, this difference in age could not be demonstrated for the patients with ahd. therefore, it could be assumed that healthy young dogs are more likely to serve as hosts and carriers for ccov, but other factors are probably necessary to cause clinical disease related to ccov infection. no molecular analysis and differentiation of ccov strains were performed in this study, but it would be an interesting option for future studies to sequence and compare viral strains found in dogs with and without clinical signs. another factor that could not be investigated in this study was the immune system of the dogs. in a recent study investigating host immune response after oral or parenteral inoculation of ccov field or vaccine strains, decaro and others (2004b) found significantly higher ccov-specific faecal immunoglobulin a (iga) antibodies in dogs orally infected with ccov live-vaccine and in naturally infected dogs. further studies are needed to investigate if ccov-specific faecal iga as part of the immune response correlates with grade of protection. although the prevalence of pv in the dogs with ahd in this study was higher than that reported in previous studies (biermann and others 1991, mochizuki and others 2001) , no pv could be detected in the group of healthy dogs. little information is available about the role of pv in healthy dogs, but data from this study suggest a strong association of pv infection with clinical disease. canine distemper virus (cdv) is an important pv, and while earlier studies described respiratory and neurological signs in combination with enteritis in dogs infected with cdv, results from this study suggest that cdv might be involved in the aetiology of ahd without affecting other organ systems (decaro and others 2004a) . further studies are warranted to investigate the role of this viral species in canine enteritis as the em did not distinguish between different members of the paramyxoviridae family. in this study, em was used as the diagnostic technique for viral detection. one of the advantages of this diagnostic tool is the possibility to detect different viral species in one faecal sample in one diagnostic procedure and to evaluate the samples for concomitant infection with more than one virus. the disadvantages of this method are an implied relatively high cost, lack of quantitative results and a lower sensitivity and specificity compared with pcr assays (schunck and others 1995, pratelli and others 2000) . new pcr methods have been proven to be up to 4â10 4 times more sensitive than em and virus isolation for detection of ccov, thus being able to detect virus also in the faeces of low-grade shedding animals below 10 6 particles per gram of unprocessed faeces, which is considered the detection limit for em (naylor and others 2001a) . because of the lower sensitivity of em, the prevalence of enteric viruses might have been underestimated in this study, and at the same time, the prevalence of virus-negative animals might have been overestimated. in future studies investigating enteric viruses in the southern german dog population, strain differentiation, especially concerning ccov, would be desirable to obtain more information about its strain-associated pathogenicity and clinical characteristics. m gene analysis of atypical strains of feline and canine coronavirus circulating in an austrian animal shelter electron microscopic virus diagnosis in dogs, cats, calves, swine, and foals in the year 1989 canine coronavirus highly pathogenic for dogs canine distemper and related diseases: report of a severe outbreak in a kennel fecal immunoglobulin a antibodies on dogs infected or vaccinated with canine coronavirus clinical and virological findings in pups naturally infected by canine parvovirus type 2 glu-426 mutant evolution of positive-strand rna viruses antibody levels and protection to canine parvovirus type 2 isolation and identification of caliciviruses from dogs with enteric infections canine coronavirus-associated puppy mortality without evidence of concurrent canine parvovirus infection faecal viruses of dogs -an electron microscope study prevalence of enteric pathogens in dogs of north-central colorado an electron microscopic study of viruses associated with canine gastroenteritis recent epidemiological status of canine viral enteric infections and giardia infection in japan verbreitung und bedeutung von coronavirusinfektionen in heimischen hundepopulationen identification of canine coronavirus strains from feces by s gene nested pcr and molecular characterization of a new australian isolate canine coronavirus in australian dogs fatal coronavirus infection in puppies following canine parvovirus 2b infection diagnosis of canine coronavirus infection using nested-pcr severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type 1 and canine coronavirus m gene evolution of canine coronavirus in naturally infected dogs genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea a simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in faeces epidemiologic evaluation of diarrhea in dogs in an animal shelter clinical and pathologic features of parvoviral diarrhea in pound-source dogs studies on the epizootiology of canine coronavirus electron microscopic demonstration of viruses in faeces of dogs with diarrhea canine coronavirus infection in the turkish dog population the authors would like to thank all dog owners for participation and sample collection for the study. special thanks go to dr david morgan and dr alex german for their helpful comments on the manuscript. key: cord-347256-0ghflk81 authors: morgan, r. k.; cortes, y.; murphy, l. title: pathophysiology and aetiology of hypoglycaemic crises date: 2018-08-13 journal: j small anim pract doi: 10.1111/jsap.12911 sha: doc_id: 347256 cord_uid: 0ghflk81 hypoglycaemia is a common, life‐threatening complication that occurs as a component of a wide variety of disease processes. despite its frequent occurrence, information concerning the aetiology, characteristics and outcomes of hypoglycaemic crises in veterinary medicine is limited. this review summarises the current understanding of the pathophysiology of hypoglycaemia, the body's counter‐regulatory response, underlying aetiologies, diagnosis and treatment. disease mechanisms are discussed and published evidence in veterinary literature regarding prognostic indicators, prevalence, diagnosis and treatment is examined for hypoglycaemia‐related disease processes including insulinoma, glucose‐lowering toxins and medications. during homoeostasis, euglycaemia is maintained by a balance between glucose production, storage and release of glucose from its stored forms. glucose is the most abundant carbohydrate in the body and is the principal fuel for the brain and peripheral tissues. insulin is secreted from β-cells in the pancreas and is responsible for transporting glucose into the cells to be utilised immediately or stored as glycogen (klein 2012) . maintaining euglycaemia depends on the body's ability to secrete the appropriate hormone in response to changing concentrations of blood glucose (bg). in a hypoglycaemic state, glucagon and adrenaline levels rise within minutes to facilitate a transient increase in glucose production (cryer 2004) . hypoglycaemia causes the hypothalamus to stimulate the sympathetic nervous system to release adrenaline from the adrenal glands. adrenaline acts quickly to limit further secretion of insulin and increase secretion of glucagon. glucagon, which is secreted from the α cells of the pancreas, stimulates glycogenolysis and gluconeogenesis, resulting in increased glucose production from hepatic cells (koenig 2015) . glucagon inhibits glucose uptake by peripheral tissues and stimulates the release of gluconeogenic precursors (guyton & hall 2006) . however, the effects of glucagon are temporary and are quickly suppressed by increasing levels of insulin. cortisol and growth hormone are released more slowly, typically a few hours following the onset of hypoglycaemia. cortisol increases the rate of lipolysis and release of amino acids from muscle that also facilitate gluconeogenesis. growth hormone works synergistically by decreasing utilisation of glucose by peripheral tissues as well as promoting lipolysis (guyton & hall 2006) . a study examining these mechanisms in adrenalectomised dogs concluded that even when counter-regulatory hormones are absent, the body can utilise glucose auto-regulation and increased neural efferent signalling in order to stimulate hepatic production of glucose (gregory et al. 2017) . there is also evidence that central excitatory amino acids may also contribute to the maintenance of euglycaemia via activation of n-methyl-d-aspartate receptors and subsequent stimulation of the sympathoadrenal and hypothalamic-pituitary adrenal axis (molina & abumrad 2001) . in addition to increases in concentrations in plasma concentrations of counter-regulatory hormones, there is evidence of increases in concentrations of immunoreactive β-endorphins, adrenocorticotropin and cortisol within the cerebrospinal fluid (csf) in response to hypoglycaemia. however, the significance of these alterations in csf concentrations remains unknown (radosevich et al. 1988 ). hypoglycaemia, clinical signs may be minimal or absent until glucose levels are profoundly decreased, as a result of hypoglycaemic unawareness. the pathophysiology of hypoglycaemic unawareness is not fully understood but is thought to occur in patients whose brains have been conditioned to automatically upregulate cerebral glucose uptake as a result of prolonged, chronic or recurrent hypoglycaemia. this automatic mechanism of cerebral glucose uptake can actually lead to a decreased perception of peripheral hypoglycaemia and blunting of the body's counter-regulatory responses (weinstock et al. 2016) . while there is little evidence that this occurs in companion animals, it is important to keep in mind when evaluating patients with conditions that may place them at risk for chronic hypoglycaemia, including chronic insulin overdose of diabetes mellitus (dm) patients and insulinomas. the brain is particularly vulnerable to the effects of hypoglycaemia because it cannot manufacture its own glucose and is dependent on the constant availability of adequate amounts of glucose for energy (boyle 1997) . while it may briefly utilise ketones, the brain has a limited ability to utilise other forms of energy (boyle 1997) . in order to keep up with the brain's needs, glucose is continuously transported across the blood brain barrier from the peripheral bloodstream via facilitated transport. glucosensors within the ventromedial nuclei of the hypothalamus, intestinal tract, carotid body and hepatoportal area act to control the amount of glucose that is transported through the blood brain barrier (loose et al. 2008) . if arterial glucose levels fall, the diffusion gradient cannot be adequately maintained and neuroglycopenia may transpire. prolonged neuroglycopenia can lead to permanent brain injury and neurologic signs that may persist beyond resolution of the hypoglycaemia (kraje 2003, loose et al. 2008) . while transient hypoglycaemic episodes do not typically cause damage or persistent neurologic deficits, severe or repeated hypoglycaemic states can result in neurologic deficits that may resemble other acute neurological disease processes. this syndrome is referred to as hypoglycaemic encephalopathy (witsch et al. 2012 ). the prognosis for human patients is variable and ranges from reversible neurologic deficits (aoki et al. 2004 , bottcher et al. 2005 , kim et al. 2007 , maruya et al. 2007 to persistent neurologic deficits, persistent vegetative state and death (mori et al. 2006 , ma et al. 2009 , kang et al. 2010 , yaffe et al. 2013 , mehta et al. 2016 . in veterinary literature, a case report of a dog with hypoglycaemia secondary to insulinoma and refractory epilpesy has been described in which brain lesions within the superficial layers of the cerebral cortex and the dentate gyrus of the hippocampus were identified post mortem (shimada et al. 2000) . hypoglycaemia and the cardiovascular system numerous human case reports document an association between supraventricular, ventricular and bradyarrhythmias in association with hypoglycaemia (bolognesi et al. 2011 , celebi et al. 2011 , sanon et al. 2014 , with atrial fibrillation identified as the most common supraventricular arrhythmia. bradycardia and circula-tory collapse have also been documented in veterinary patients (little 2005) . the occurrence of these arrhythmias has led to concern that hypoglycaemic states may be pro-arrhythmogenic (sanon et al. 2014) . while hypoglycaemia commonly will lead to the release of catecholamines and secondary tachyarrhythmias, the cause of bradyarrhythmias is unknown. in order to demonstrate that clinical signs are secondary to hypoglycaemia whipple's triad must be observed: (1) clinical signs of hypoglycaemia; (2) documentation of a low blood glucose level at the time of clinical signs and (3) resolution of clinical signs with correction of hypoglycaemia (cryer 2013) . although the gold standard for the measurement of blood glucose is analysis by isotope dilution mass spectrometry, this is not available in a clinical setting (tonyushkina & nichols 2009 ). therefore, most glucose levels are measured with handheld point-of-care glucometers (pocg). pocgs that are typically designed for humans can demonstrate inconsistencies regarding accuracy in veterinary species (cohen et al. 2009 ). other pocgs marketed to veterinarians utilise internal algorithms that attempt to quantify species differences between free and haemoglobin-bound glucose (zini et al. 2009 ). the accuracy of handheld glucometers can be vulnerable to delays in separation of the serum from the red blood cells longer that 30 minutes after collection, leading to pseudohypoglycaemia as a result of continued consumption of glucose by the red blood cells in vitro (koenig & verlander 2015) . this effect can be prevented by collecting blood samples in sodium fluoride tubes if they cannot be processed via centrifugation within that time period. haemodilution and haemoconcentration can also affect the accuracy of glucose readings (solnica et al. 2012) . the mechanism behind this is believed to be a result of a reduction of the volume of plasma due to the presence of increased erythrocytes in a haemoconcentrated sample. this causes the volume of plasma that is able to penetrate the reagent layer to be reduced, resulting in a falsely reduced glucose measurement (tang et al. 2000) . conversely, a haemodiluted sample allows an abnormally large amount of plasma to contact the test strip reagent layer, which falsely elevates the glucose measurement (tang et al. 2000) . lane et al. (2015) proposed a formula to correct the glucose level given a known packed cell volume, but additional algorithms would need to be developed and validated for other glucometer models. less invasive alternatives to traditional monitoring of blood glucose for veterinary patients include the continuous glucose monitoring system (cgms; guardian real-time continuous glucose monitoring), in which measurements of interstitial glucose concentrations are monitored via a sensor implanted within the subcutaneous (sc) space (reineke et al. 2010 ). the sensor is comprised of a glucose diffusion limiting membrane which contains glucose oxidase. when the membrane is exposed to glucose in the presence of oxygen, it undergoes an oxidation reaction to produce gluconic acid and hydrogen peroxide. the hydrogen peroxide creates a current that reflects the glucose level within the interstitium via a reaction with the platinum electrode (reineke et al. 2010) . over the last 10 years, studies in experimental nondiabetic dogs, stable diabetic dogs and cats and those with diabetic ketoacidosis have, for the most part, demonstrated accuracy and agreement between glucose values obtained via cgms compared with those obtained via traditional glucometers (davison et al. 2003 , ristic et al. 2005 , wiedmeyer et al. 2005 , reineke et al. 2010 ). the causes of hypoglycaemia are multi-factorial and can be divided into groups: (1) excess insulin or insulin analogues; (2) inadequate glucose production; and (3) increased glucose consumption. fig. 1 shows a diagnostic algorithm that can be helpful in guiding the evaluation of a hypoglycaemic patient. both veterinary and human patients with dm are at risk for exogenous insulin overdoses, either secondary to insulin mishandling and administration errors or because of concurrent illness and transient remission. information regarding insulin overdoses in diabetic animals is relatively scarce, but a previous retrospective article identified that cats were more likely to experience overdose compared to dogs. furthermore, obese animals and cats that regularly received more than 6 units of insulin per injection were at increased risk (whitley et al. 1997) . diabetic animals that are experiencing gastrointestinal upset secondary to concurrent underlying illness were also reported to be at increased risk for experiencing hypoglycaemia secondary to concurrent administration of exogenous insulin (whitley et al. 1997) . in 2017, the authors of a second retrospective article reported that in a review of 28 cats with dm that had experienced hypoglycaemia, vomiting or reduced intake was reported in association with hypoglycaemia (viebrock and dennis 2017) . this study revealed that patients experiencing clinical improvement within 12 hours of presentation were more likely to survive, and that mental status and severity of hypoglycaemia on presentation were not negative prognostic indicators (viebrock and dennis 2017) . insulinomas are malignant insulin-secreting tumours of the pancreas which are most common in middle-aged to older dogs. these patients commonly present with weakness, collapse or seizures secondary to severe hypoglycaemia (polton et al. 2007) . diagnostic modalities include an amended or standard insulin glucose ratio, fructosamine levels, glucagon tolerance testing and oral glucose tolerance tests (greene & bright 2008 , melano & peterson 2013 . abdominal ultrasound has low sensitivity to detect these tumours and, in some cases, ct or abdominal exploratory is recommended for definitive identification (robben et al. 2005) . medical treatment options include stabilisation of refractory and life-threatening hypoglycaemia via administration of glucagon continuous infusions if indicated, frequent feeding and oral administration of glucocorticoids as well as other adjunctive medications such as diazoxide and somatostatin therapy to help alleviate clinical signs and maintain normoglycaemia. both diazoxide and somatostatin result in membrane hyperpolarisation and inhibition of normal insulin release (dunne & peterson 1991) . diazoxide may increase hepatic glucose production and reduce peripheral glucose uptake (altszuler et al. 1977) . somatostatin has inhibitory effects on several other hormones including glucagon, secretin and vasoactive inhibitory peptide. somatostatin analogues like octreotide are used more commonly due to their longer half-life (lamberts et al. 1996) . octreotide has been evaluated in healthy dogs and dogs with insulinomas (robben et al. 2006) : following administration, plasma insulin and glucagon concentrations decreased in healthy dogs but only insulin decreased in dogs with insulinomas. none of the dogs experienced significant adverse effects from the medication. evaluation of somatostatin receptors in canine insulinomas revealed that dogs only express one type of somatostatin receptor, in contrast to humans who can express several types (robben et al. 1997) . this can make it difficult to extrapolate information from human studies of somatostatin use in insulinomas to canine patients. there have been rare reports of insulinomas diagnosed in cats (mcmillan et al. 1985 , hawks et al. 1992 , elie & zerbe 1995 , kraje 2003 , greene & bright 2008 , but there are no reports of using somatostatins or diazoxide in cats. other medications include streptozocin, which selectively destroys pancreatic β cells. surgical removal and a partial pancreatectomy are required for more definitive control (moore et al. 2002) . median survival times for dogs following surgical resection alone has been reported as 785 days versus 196 days for medical management alone (polton et al. 2007) . ferrets can commonly also present with hypoglycaemia secondary to insulinomas. most ferrets develop clinical signs secondary to this condition around 4 to 5 years of age (caplan et al. 1996 , ehrhart et al. 1996 . normal fasting blood glucose in a ferret is 4•9 to 6•8 mmol/l, and in one case series evaluating 57 ferrets with a confirmed insulinoma, the mean blood glucose was less than 3•3 mmol/l (caplan et al. 1996) . ferrets can be treated in a similar way to dogs and glucocorticoids may aid in maintaining normoglycaemia. the recommended oral starting dose of 0•25 mg/kg prednisone is every 24 hours, which can then be titrated as needed (melano & peterson 2013) . diazoxide can also be prescribed, but this medication can be cost-prohibitive. surgery can also be pursued with published mean survival times of 668 days with a partial pancreatectomy versus 186 days with medical management alone (weiss et al. 1998) . one case report details the successful dose of 15 ng/kg/min glucagon constant rate infusion at in a 3-year-old female domestic ferret with an insulinoma (bennett et al. 2015) . in addition to numerous case reports in ferrets, there is one report of a pre mortem diagnosis and treatment of an insulinoma in a 5-year-old male guinea pig that was treated medically with a starting dose of 5 mg/kg diazoxide orally every 12 hours, eventually increasing to 25 mg/kg orally every 12 hours. the clinical signs were managed medically for 3 weeks before the patient presented with signs of constipation and abdominal distension and died the following day (hess et al. 2013) . any neoplasia can potentially cause a secondary hypoglycaemia. the most common tumours associated with this syndrome include hepatocellular carcinoma, leiomyomas and leiomyosarcomas (beaudry et al. 1995 , battaglia et al. 2005 , bergman 2007 ). although the exact cause is unknown, it is suspected that the cancerous cells have accelerated glucose consumption or can secrete insulin or insulin-like peptides (finotello et al. 2009 , singhal et al. 2017 ). in the case of hepatic neoplasia in particular, the tumour may affect the liver's endogenous ability to perform glycogenolysis or gluceoneogenesis. treatment of the hypoglycaemia is symptomatic with definitive treatment of the specific cancer required for adequate control. hyperinsulinaemic hypoglycaemic syndrome (hhs) is the most common cause of hypoglycaemia in human infants but has rarely been recognised in veterinary medicine. this syndrome is defined as excessive production of insulin due to hyperplasia within the β cells of the pancreas (senniappan et al. 2013 ). there is a case report proposing that hhs may have been the cause of refractory hypoglycaemia in two dogs, both of which were under 1 year of age: there was persistent hyperinsulinaemia in the face of hypoglycaemia and elevated insulin glucose ratios without evidence of any insulin-secreting tumours (breitschwerdt et al. 2014) . a recent report also documents for the first time hhs in a 6-year-old female spayed british shorthair cat secondary to suspect nesidioblastosis, a term which describes non-neoplastic β-cell hyperplasia within the pancreas (hambrook et al. 2016) . xylitol one of the most common toxins associated with hypoglycaemia in dogs is xylitol, an artificial sweetener that is commonly found in sugar-free gum, sweets, baked goods, nutritional supplements and dental care products, many of which are designed as sugar-free alternatives for humans with dm (dunayer 2004) . once ingested, peak plasma levels of xylitol in dogs are reached within 30 minutes. the majority of xylitol is metabolised in the liver and transformed to d-xylulose, which then becomes d-xylulose-5-phosphate (froesch & jakob 1974 , piscitelli et al. 2010 . xylitol is then converted to glucose, glycogen or lactate via the pentose phosphate pathway (piscitelli et al. 2010) . unlike humans, dogs experience a marked dose-dependent insulin surge following xylitol ingestion, leading to hypoglycaemia (xia et al. 2009 ). hepatic failure in dogs secondary to xylitol has also been described (dunayer & gwaltney-brant 2006 , todd & powell 2007 . a recent retrospective article found the most common clinical signs associated with xylitol ingestion in dogs were vomiting and lethargy, and 15% of dogs became hypoglycaemic during hospitalisation (duhadway et al. 2015) . none of the dogs developed hepatic failure and all survived to discharge. historically, 0•1 g/kg is the minimal dose believed to result in hypoglycaemia, while dogs ingesting 0•5 g/kg or greater are believed to be at increased risk of developing acute liver failure (dunayer & gwaltney-brant 2006) . there is also evidence that the development of hypoglycaemia and, especially, acute liver failure may be idiosyncratic rather than strictly dose-dependent, because previous cases series have documented dogs recovering after ingesting up to 16 g/kg of xylitol, while other dogs have died after ingesting far lower doses (dunayer & gwaltney-brant 2006) . there are no reports documenting xylitol toxicity in cats to our knowledge. ingestion of various types of alcohol compounds can also result in hypoglycaemia when it is. metabolised in the liver via alcohol dehydrogenase and aldehyde dehydrogenase to acetate. the oxidation of ethanol raises levels of nicotinamide adenine dinucleotide hydride (nadh), which then inhibits enzymes that regulate gluconeogenesis, leading to a decreased ability to release glucose from the liver (kandi et al. 2014) . the most common types of alcohols encountered include methanol, ethanol and isopropanol (also known as rubbing alcohol). previous case reports of poisoning from exposure to ethanol in veterinary medicine have included alcoholic beverages (ratclife & zuber 1977 , houston & head 1993 , keno & langston 2011 , various types of uncooked dough (thrall et al. 1984 , suter 1992 , means 2003 and uncooked apples (kammerer et al. 2001) . though ethanol administration has been documented to induce hypoglycaemia in dogs in the laboratory (lochner et al. 1967) , none of the cited case reports documented hypoglycaemia in the affected patients. however, hypoglycaemia is also intermittently described in human and veterinary patients that have ingested ethylene glycol, and is thought to occur secondary to depletion of glycogen stores and reduction of gluconeogenesis (brent et al. 1999 , garcia-ortuno et al. 2006 , kraut & kurtz 2008 . alpha lipoic acid (ala) is an acid that is found as a natural compound and acts as a biological cofactor (loftin & herold 2009 ). it has been investigated as a possible therapy in the treatment of a number of conditions in humans, including cancer (wenzel et al. 2005) , heavy metal intoxication (grunert 1960) , human immunodeficiency virus (baur et al. 1991 ) and multiple sclerosis (packer et al. 1995 , khamaisi et al. 1999 , yadav et al. 2005 , ziegler et al. 2006 . it has been utilised in veterinary medicine by alternative medicine practitioners as a potential therapy for patients with dm and cognitive dysfunction in dogs (means 2008) . the reported therapeutic dose in cats is 1 to 5 mg/kg with a maximum dose of 25 mg/day, and up to 80 to 200 mg/day in dogs. however, due to limited information on safety, its use in veterinary medicine remains controversial. however, ala has been reported to be a concerning potential toxicity in companion animals. there are three existing case reports of toxicity in dogs (means 2008 , loftin & herold 2009 , and ala has been noted to be 10 times more toxic in cats as compared to dogs, humans and rats (hill et al. 2004) . cats with ala toxicity have been noted to exhibit anorexia, ataxia, hypersalivation, vomiting and elevated hepatic enzymes with single doses of 60 mg/kg (loftin & herold 2009) . dogs can exhibit similar signs as well as hypoglycaemia, hepatic and renal failure (loftin & herold 2009 ). proposed mechanisms for hypoglycaemia include increased uptake of glucose, increased number of glucose transport proteins and inhibition of gluconeogenesis, among others (jacob et al. 1999 , loftin & herold 2009 ). there is one case report of oleander toxicity resulting in the development of hypoglycaemia in a 7-year-old female spayed maltese that presented after known ingestion of oleander leaves. other causes of hypoglycaemia were excluded and the patient recovered with supportive care (page & murtaugh 2015) . nerium oleander is a flowering shrub in the family apocyanaceae, and all parts of the plant contain cardenolides, which are cardiac glycosides (langford & boor 1996) . proposed mechanisms of action regarding oleander's ability to cause hypoglycaemia include disruption of the sodium gradient necessary for the transport of glucose via disruption of the na/k atpase pump, or inhibition of α-glucosidases within the small intestine responsible for hydrolysing saccharides to glucose (mwafy & yassin 2011 , page & murtaugh 2015 . neonatal and paediatric animals are susceptible to hypoglycaemia due to a combination of poor glycogen stores with which to initiate glycolysis and gluconeogenesis, and an underdeveloped liver (mcmichael 2005) . other factors that may predispose neonatal patients to the development of hypoglycaemia include decreased birth weight, premature birth and being born to a bitch with systemic illness or diabetes. there is some evidence to support neonatal hypoglycaemia as a predictor of higher mortality between 1 and 21 days of age (mila et al. 2017) . once weaned, paediatric patients can experience hypoglycaemia secondary to concurrent infections, immune system stimulation, exercise, decreased quality of nutrition, hypothermia, ongoing gastrointestinal losses and fasting (mcmichael 2015) . parvovirus is one of the most common causes of hypoglycaemia in young animals. a study by castro et al. (2013) evaluating puppies with either coronavirus or parvovirus showed that both groups commonly experienced hypoglycaemia, although they did not evaluate whether hypoglycaemia was a prognostic factor. in cats with parvovirus (feline panleukopenia virus), hypoglycaemia was much less common and not associated with prognosis (kruse et al. 2010) . a 2017 article comparing inpatient versus outpatient treatment protocols found that 50% of the outpatient group required dextrose supplementation, while 80% of the inpatient group required dextrose supplementation (venn et al. 2017) . other proposed causes of inadequate glucose production include what is often referred to as toy-breed hypoglycaemia, which is anecdotally reported either as a form of transient juvenile hypoglycaemia or to occur even in adults within this breed grouping. however, a thorough search of available literature reveals little evidence to describe and classify such a syndrome as distinct cause of hypoglycaemia. a recent case report by deavilla & leech (2016) described hypoglycaemia as a complication of refeeding syndrome in a 2-year-old male neutered domestic shorthair that was treated for starvation after being missing for 12 weeks. despite beginning with 24% of resting energy requirement on day 1 of hospitalisation, it developed hypoglycaemia 12 hours after refeeding was initiated and required supplementation via a dextrose constant-rate infusion. the cat went on to make a full recovery and theories regarding the cause of hypoglycaemia included reduced glycogen stores, depleted body fat secondary to starvation and exacerbation of inappropriate release of insulin secondary to dextrose supplementation (deavilla & leech 2016). hypoglycaemia that accompanies any septic focus, including pyothorax, pyometra, septic peritonitis or septic arthritis is caused by non-insulin-mediated increased consumption of glucose and decreased intake. increases in anaerobic glycolysis secondary to upregulation of inflammatory mediators such as tumour necrosis factor and other cytokines may be exacerbated by hypotension or hypoxia, further driving excess glucose consumption. although glucose can aid diagnosis of sepsis in patients with septic effusion, it may not be as helpful in those without effusion (koenig & verlander 2015) . other studies have found no significant difference in glucose between septic and non-septic dogs and no difference when comparing glucose in survivors versus non-survivors with sepsis (hauptman et al. 1997) . interestingly, in a recent retrospective evaluating septic cats, hypoglycaemia was not commonly seen or associated with outcome (brady et al. 2000) . in this cohort, hyperglycaemia was more common than hypoglycaemia. babesia is an example of a specific infection in which 20% of infected patients are hypoglycaemic. similar to sepsis, hypoglycaemia is associated with consumption of glucose by the parasite, depletion of hepatic stores or hepatic dysfunction, increased anaerobic glycolysis and hyperinsulinaemia (rees & schoeman 2010 , keller et al. 2004 . a prospective study examining the pituitary-adrenal and pituitary-thyroid axes in dogs with babesiosis found elevations in basal and post acth serum cortisol concentrations as well as decreases in body temperature, serum thyroxine and free thyroxine in those patients with hypoglycaemia, suggesting that adrenal insufficiency was not responsible for the alterations in glucose levels and that adrenal and thyroidal hormones respond differently to hypoglycaemia (schoeman & herrtage 2007) . some studies have reported that hypoglycaemia may be associated with more pathologic strains of babesia and that hypoglycaemia at presentation is a poor prognostic indicator (keller et al. 2004 , jacobson & lobetti 2005 , ayoob et al. 2010 ). according to one study, risk factors associated with the development of hypoglycaemia in dogs with babesia included collapsed state, youth, severe anaemia, vomiting and icterus (keller et al. 2004) . other parasitic infections also have the potential to cause hypoglycaemia in their hosts. a recent case report from france (deschamps et al. 2016 ) details refractory hypoglycaemia in a 2-year-old female intact mixed breed dog infected with trypanosoma congolense. the primary presenting complaint was seizure activity secondary to persistent hypoglycaemia. trypanosomes were visible in a routine blood smear and infection was confirmed by pcr. the infection responded to therapy with intramuscular injections of pentamidine at 48-hour intervals, the hypoglycaemia initially failed to improve despite aggressive therapy that included both intravenous supplementation of 30% glucose and administration of 70% sucrose via feeding tube. the authors speculated that glucose administration may have served as a food source for the trypanosomes as potential explanation for the persistent hypoglycaemia. a previous case report of a 6-yearold male intact dog with trypanosomiasis also documented a profound hypoglycaemia (gunaseelan et al. 2009 ). glucose homoeostasis is typically maintained by the liver's ability to facilitate gluconeogenesis and glycogenolysis. hepatic diseases such as hepatitis, hepatic lipidosis, hepatic neoplasia, cirrhosis, portosystemic shunt, glycogen storage disease and hepatotoxins can all induce hepatic dysfunction (walton 2002) . even if there is hepatic dysfunction, hypoglycaemia does not occur until more than 70% of hepatic function is lost (center et al. 1996) . once there is depletion of glycogen stores, the body utilises protein catabolism to supply amino acids from gluconeogenesis (weingarten & sande 2015) . a study by lester et al. (2016) evaluating dogs with acute liver failure from multiple aetiologies revealed that on presentation, 20% of these patients were hypoglycaemic. studies evaluating feline hepatic failure are sparse but in an evaluation of seven cats with benzodiazepine-associated liver failure, three were hypoglycaemic but the remaining four had blood glucose levels at the lower end of the reference interval (hughes et al. 1996) . hypoadrenocorticism may cause hypoglycaemia due to cortisol depletion since glucocorticoids stimulate hepatic gluconeogenesis. studies evaluating hypoadrenocorticism in dogs showed hypoglycaemia in 3 to 37% of cases (rakich & lorenz 1984 , melian & peterson 1996 . the percentage of dogs that go on to develop symptomatic neuroglycopenia, including seizures, appears to be quite low (levy 1994 , melian & peterson 1996 . apart from hypo-glycaemia, other clinicopathologic abnormalities that may be noted included azotaemia, hyponatraemia, hyperkalaemia, hypochloraemia and the absence of a stress leukogram (syme & scott-moncrieff 1998 , feldman & nelson 2004 . clinical signs may include gastrointestinal signs such as vomiting, diarrhoea and anorexia in addition to polyuria, polydipsia, bradycardia and dehydration (feldman & nelson 2004) . animals may be asymptomatic until confronted with the stress of concurrent illness or other physical sources of stress, including anaesthetic episodes (lane et al. 1999) . hypoadrenocorticism is less common in cats but appears to rarely cause hypoglycaemia (peterson et al. 1989 , stonehewer & tasker 2001 . emergency treatment of the hypoglycaemic crisis the mainstay of therapy for the patient experiencing hypoglycaemic crisis is intravenous administration of dextrose, typically using an initial bolus of 0•5 to 1 g/kg bolus of 50% dextrose diluted 1:1 with 0•9% nacl followed by a constant-rate infusion of 1•25 to 10% dextrose diluted with an isotonic crystalloid fluid (loose et al. 2008) . complications of intravenous dextrose include phlebitis secondary to its hypertonicity and so placement of a central line is recommended for administration of dextrose solutions with concentrations greater than 5%. additional complications of dextrose administration include rebound hypoglycaemia, as the influx of dextrose can cause pancreatic β cells to release insulin in response to the acute hyperglycaemia and may induce recurrence of hypoglycaemia (goutal et al. 2012 ). rebound hypoglycaemia is particularly a risk during treatment of insulinomas, prompting recommendations to avoid aggressive dextrose bolusing in favour of small frequent meals and medical management, as discussed previously (table 2) . glucagon is a 29-amino acid polypeptide hormone that is produced by the α cells of the pancreas in order to facilitate glycoge-nolysis and gluconeogenesis (zeugswetter et al. 2012 , datte et al. 2016 . in cases of hypoglycaemia that are refractory to dextrose administration, glucagon is also utilised as a therapy in human and veterinary medicine. in a recent retrospective study of nine dogs with hypoglycaemia due to a variety of causes including insulinoma and atypical hypoadrenocorticism, all patients demonstrated a statistically significant increase in blood glucose with administration of glucagon infusions compared to levels at intake (datte et al. 2016) . the only adverse effect reported in this study attributable to glucagon was mild hyperglycaemia. a recent prospective study by zeugswetter et al. (2012) investigated the potential for use of sc glucagon as part of an emergency kit intended for patients with dm that experience hypoglycaemic crises. the authors administered 1 mg synthetic glucagon intravenously in one group of healthy beagles and sc in another group. after treatment the levels of insulin-like immunoreactivity (insulin-imr), glucose, acth and cortisol were compared between the two groups. the authors concluded that sc glucagon did increase the glucose and insulin-imr levels, with peak glucose concentrations reached in 20 minutes. administration of sc glucagon did not increase cortisol or acth levels. although the peak concentration in the sc glucagon group was lower than those reached in the intravenous group, the authors concluded that it could potentially be beneficial in the initial at-home treatment of hypoglycaemic crises in dogs with dm, although it would not be useful in the treatment of animals with suspect hypocortisolism (zeugswetter et al. 2012) . apart from mild sedation, no other potential adverse effects were observed (graf et al. 1999 , fall et al. 2008 . hypoglycaemia is a common presenting problem in small animal practice and has a large variety of possible underlying causes. it is important that veterinarians are able to recognise the vast array of symptoms that a hypoglycaemic patient may exhibit. fortunately, hypoglycaemic patients commonly respond well to initial symptomatic treatment although the prognosis for the underlying diseases vary considerably and it is important that owners are adequately informed on the prognosis of their individual animal. advances in at-home monitoring for patients with dm in the form of cgms and potential future development of sc glucagon emergency kits may help owners to recognise hypoglycaemic events in the early stages and provide initial therapy before the episode becomes prolonged. additional novel methods of early detection that may one day be extrapolated from human medicine include development of a method to analyse breath samples to detect the presence of volatile organic compounds which may signal hypoglycaemia in patients with dm (siegel et al. 2017) . on the mechanism of diazoxideinduced hyperglycemia reversible hyperintensity lesion on diffusion-weighted mri in hypoglycemic coma clinical management of canine babesiosis hypoglycaemia as a paraneoplastic syndrome associated with renal adenocarcinoma in a dog alpha-lipoic acid is an effective inhibitor of human immuno-deficiency virus (hiv-1) replication hypoglycemia in four dogs with smooth muscle tumors constant rate infusion of glucagon as an emergency treatment for hypoglycemia in a domestic ferret (mustela putorius furo) paraneoplastic syndromes marked sinus bradycardia and qt prolongation in a diabetic patient with severe hypoglycaemia localized reversible reduction of apparent diffusion coefficient in transient hypoglycemia-induced hemiparesis alteration in brain glucose metabolism induced by 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monitoring system in diabetic dogs hypoglycemia associated with refeeding syndrome in a cat refractory hypoglycaemia in a dog infected with trypanosoma congolense retrospective evaluation of xylitol ingestion in dogs: 192 cases hypoglycemia following canine ingestion of xylitol-containing gum acute hepatic failure and coagulopathy associated with xylitol ingestion in eight dogs potassium selective ion channels in insulinsecreting cells: physiology, pharmacology and their role in stimulus secretion coupling pancreatic beta cell tumor in ferrets: 20 cases (1986-1994) insulinoma in dogs, cats, and ferrets. compendium of continuing education for the practicing veterinarian glucagon stimulation test for estimating endogenous insulin secretion in dogs hypoadrenocorticism (addison's disease). in: canine and feline endocrinology and reproduction pancreatic islet cell tumor secreting insulin-like growth factor type-ii in a dog canine pancreatic islet cell tumours secreting insulin-like growth factor type 2: a rare entity the metabolism of xylitol clinical-pathological diagnosis of ethylene glycol poisoning: a case report insulinoma in dogs: a review pharmacokinetic and glucodynamic comparisons of recombinant and animal-source glucagon after iv, im, and sc injection in healthy volunteers insulinoma in a cat glucose autoregulation is the dominant component of the hormone-independent counterregulatory response to hypoglycemia in the conscious dog the effect of dl-a-lipoic acid on heavy-metal intoxication in mice and dogs haemato biochemical changes in a case of canine trypanosomiasis in: textbook of medical physiology. 11th edn hyperinsulinaemic, hypoglycaemic syndrome due to acquired nesidioblastosis in a cat evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs insulin-secreting pancreatic (islet cell) carcinoma in a cat diagnosis and treatment of an insulinoma in a guinea pig (cavia porcellus) formulation and validation of a predictive model to correct blood glucose concentrations obtained with a veterinary point-of-care glucometer in hemodiluted and hemoconcentrated canine blood samples oleander toxicity: an examination of human and animal toxic exposures retrospective evaluation of acute liver failure in dogs hypoglycemic seizures attributable to hypoadrenocorticism in a dog hypoglycaemic bradycardia and circulatory collapse in a dog and a cat ethanol-induced hypoglycemia: i. the acute effects of ethanol on hepatic glucose output and peripheral glucose utilization in fasted dogs therapy and outcome of suspected alpha lipoic acid 818 toxicity in two dogs hypoglycemia and its effect on the brain mr imaging of hypoglycemic encephalopathy: lesion distribution and prognosis prediction by diffusion-weighted imaging rapid improvement of diffusion weighted imaging abnormalities after glucose infusion in hypoglycaemic coma pediatric emergencies. veterinary clinics of north america critically ill neonatal and pediatric patients functional pancreatic islet cell tumor in a cat bread dough toxicosis in dogs ataxia and vomiting in a german shepherd association of hypoglycemia with subsequent dementia in older patients with type 2 diabetes mellitus treatment of insulinoma in the dog, cat, and ferret diagnosis and treatment of naturally occurring hypoadrenocorticism in 42 dogs monitoring of the newborn dog and prediction of neonatal mortality contribution of excitatory amino acids to hypoglycemic counter-regulation streptozocin for treatment of pancreatic islet cell tumors in dogs: 17 cases(1989-1999) hypoglycemic encephalopathy with extensive lesions in the cerebral white matter antidiabetic activity evaluation of glimepiride and nerium oleander extract on insulin, glucose levels and some liver enzyme activities in experimental diabetic rat model alpha-lipoic acid as a biological antioxidant hypoglycemia associated with oleander toxicity in a dog primary hypoadrenocorticism in ten cats pretreatment clinical and laboratory findings in dogs with hypoadrenocorticism: 225 cases (1973-1993) xylitol toxicity in dogs. compendium on continuing education for the practising veterinarian improved survival in a retrospective cohort of 28 dogs with insulinoma effects of insulin-induced hypoglycemia on plasma and cerebrospinal fluid levels of ir-beta-endorphins, acth, cortisol, norepinephrine, insulin and glucose in the conscious dog clinical signs and laboratory abnormalities 902 in 23 dogs with spontaneous hypoadrenocorticism acute ethyl alcohol poisoning in dogs accuracy of a continuous glucose monitoring systemin dogs and cats with diabetic ketoacidosis plasma insulin concentrations in hypoglycaemic dogs with babesia canis rossi infection evaluation of a continuous glucose monitoring system in cats with diabetes mellitus in vitro and in vivo detection of functional somatostatin receptors in canine insulinomas comparison of ultrasonography, computed tomography, and single-photon emission computed tomography for the detection and localization of canine insulinoma effect of octreotide on plasma concentrations of glucose, insulin, glucagon, growth hormone and cortisol in healthy dogs and dogs with insulinoma hypoglycemia from a cardiologist's perspective the response of the pituitary-adrenal and pituitary-thyroidal axes to the plasma glucose perturbations in babesia canis rossi babesiosis the molecular mechanisms, diagnosis and management of congenital hyperinsulinism hypoglycaemic 946 brain lesions in a dog with insulinoma analyzing breath samples of hypoglycemic events in type 1 diabetes patients: towards developing an alternative to diabetes alert dogs paraneoplastic hypoglycaemia: a rare manifestation of pelvic gastrointestinal stromal tumour the effect of hematocrit on the results of measurements using glucose meters based on different techniques hypoadrenocorticism in a cat presumed ethanol intoxication in sheep dogs fed uncooked pizza dough chronic hypoglycaemia in a hunting dog clue to secondary hypoadrenocorticism effects of different hematocrit levels on glucose measurements with handheld meters for point-of-care testing ethanol toxicosis secondary to sourdough ingestion in a dog xylitol intoxication associated with fulminant hepatic failure in a dog glucose meters: a review of technical challenges to obtaining accurate results evaluation of an outpatient protocol in the treatment of canine parvoviral enteritis hypoglycemic episodes in cats with diabetes mellitus: 30 cases (2013-2015) severe liver disease acute liver failure in dogs and cats risk factors associated with severe hypoglycemia in older adults with type 1 diabetes insulinoma in the ferret: clinical findings and treatment comparison of 66 cases a-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant o2 -generation insulin overdose in dogs and cats: 28 cases (1986-1993) evaluation of a continuous glucose monitoring system for use in veterinary medicine hypoglycemic encephalopathy: a case series and literature review on outcome determination experimental acute toxicity of xylitol in dogs lipoic acid in multiple sclerosis: a pilot study association between hypoglycemia and dementia in a biracial cohort of older adults with diabetes mellitus metabolic and hormonal responses to subcutaneous glucagon in healthy beagles oral treatment with α-lipoic acid improves symptomatic diabetic polyneuropathy: the sydney 2 trial evaluation of a new portable glucose meter designed for the use in cats none of the authors has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. none of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. key: cord-023503-zco0zpax authors: miller, paul e. title: the glaucomas date: 2009-06-05 journal: slatter's fundamentals of veterinary ophthalmology doi: 10.1016/b978-072160561-6.50015-0 sha: doc_id: 23503 cord_uid: zco0zpax nan the glaucomas are a diverse group of diseases united only by the fact that intraocular pressure (iop) is too high to permit the optic nerve and, in some species, the retina to function normally. characteristic changes of glaucoma include disrupted axoplasmic flow in the optic nerve head, death of retinal ganglion cells and their axons, cupping of the optic disc, and visual impairment or blindness. the production and drainage of aqueous humor are influenced not only by the anatomy of the anterior segment but also by a large number of endogenous compounds, including neurotransmitters, hormones, prostaglandins, proteins, lipids, and proteoglycans. indeed, so many factors influence the production and drainage of aqueous humor that it is difficult to identify a single pathway or drug that is capable of dramatically lowering iop in every patient. aqueous humor is produced in the ciliary body by both active (selective transport of larger or charged molecules against a concentration gradient) and passive processes (diffusion and ultrafiltration). in diffusion, lipid-soluble substances enter the aqueous humor by passing through the ciliary epithelial cell membrane in proportion to their concentration gradient across the membrane. ultrafiltration is the passage of water and water-soluble substances (which are generally limited by their size or charge) through theoretical micropores in the cell membrane in response to an osmotic gradient or hydrostatic pressure. many substances in the blood pass by ultrafiltration from the ciliary capillaries into the stroma of the ciliary processes before accumulating behind the tight junctions of the nonpigmented ciliary epithelium (the site of the blood-aqueous barrier). some substances, such as sodium and chloride ions, are then actively pumped across the membrane into the posterior chamber, thereby drawing water passively along this concentration gradient. this process may account for the majority of actively formed aqueous. aqueous humor is also produced via the enzyme carbonic anhydrase, which catalyzes the formation of carbonic acid from carbon dioxide and water as follows: co 2 + h 2 o g h 2 co 3 g hco 3 -+ h + carbonic acid then dissociates, allowing negatively charged bicarbonate ions to pass to the aqueous. although exactly how this leads to aqueous humor production is unclear, it appears that positively charged sodium ions, and eventually water, follow negatively charged bicarbonate ions into the posterior chamber. drugs that inhibit carbonic anhydrase therefore decrease aqueous production and reduce iop. aqueous exits the eye via several routes. in the conventional or traditional outflow route aqueous humor passes from the posterior chamber, through the pupil, and into the anterior chamber. because of temperature differences between the iris and cornea, thermal convection currents occur in the anterior chamber, with aqueous near the iris rising and aqueous near the cornea falling. this is one reason cells and particulate matter in the anterior chamber may settle on the inferior corneal endothelial surface. aqueous humor then leaves the anterior chamber by passing between the pectinate ligaments to enter the ciliary cleft, which contains the trabecular meshwork . after filtering between the beams of the spongelike meshwork, aqueous crosses through the endothelial cell membranes of the meshwork to enter a series of radially oriented, blood-free collecting vessels collectively called the angular aqueous plexus. from there it enters an interconnected set of blood/aqueous-filled vessels (the scleral venous plexus) before draining either anteriorly via the episcleral and conjunctival veins or posteriorly into the vortex venous system and into the systemic venous circulation (figure 12 -2). contraction of smooth muscle fibers of the ciliary muscle that insert into the trabecular meshwork are probably capable of increasing drainage of aqueous from the eye by enlarging the spaces in the trabecular meshwork. in most species the majority of aqueous humor (about 50% in horses, 85% in dogs, and 97% in cats) leaves the eye via the traditional outflow route. the remainder of the aqueous humor leaves the eye via the uveoscleral pathway (see figure 12 -1). in this route aqueous humor passes through the root of the iris and interstitial spaces of the ciliary muscle to reach the supraciliary space (between the ciliary body and the sclera) or the suprachoroidal space (between the choroid and the sclera). from these locations aqueous humor may pass through the sclera into the orbit either via pores in the sclera where blood vessels and nerves enter the eye or between the scleral collagen fibers themselves. outflow via this route may substantially increase in certain disease states and in response to certain antiglaucoma drugs, such as the prostaglandin derivatives. iop is the result of a delicate balance between production and outflow of aqueous humor (figure 12 -3). in glaucoma both production and outflow are altered. usually a large percentage of the outflow pathway (perhaps as much as 80% to 90%) needs to be impaired before iop starts to rise. if the outflow system is impaired to the point that iop begins to increase, the eye usually attempts to compensate by reducing the passive production of aqueous humor. active secretion, however, typically continues at a relatively normal rate, perhaps because if it did not, the avascular tissues of the eye that rely on aqueous humor for their nutrition would starve. because the glaucomatous eye is functioning on a greatly diminished percentage of its normal levels of aqueous humor outflow and production, and because it has exhausted its usual compensatory pathways, pathologic processes or drugs that alter production or outflow only a small amount can have dramatic effects on iop. this characteristic is one reason that glaucomatous eyes are typically more responsive to antiglaucoma drugs than normotensive eyes, but it also explains why iop can rapidly rise to very high levels in a matter of 1 to 2 hours in some patients. often it is difficult to empirically predict the effect a given drug or its antagonist will have on iop because many compounds affect both aqueous humor production and outflow-sometimes in complex and contradictory ways. for example, stimulation of b-adrenergic receptors in the ciliary processes increases intracellular cyclic adenosine monophosphate (camp), resulting in greater aqueous humor production. b-adrenergic blocking drugs (e.g., timolol, betaxolol) decrease camp, thereby lowering aqueous humor production and ultimately reducing iop. b-blockers reduce iop, however, only if the patient is awake and adrenergic tone is present. this means that although a drug such as timolol can reduce iop in a cat when it is awake, the agent may not control iop for the more than 20 hours a day the cat is sleeping. as expected, b-adrenergic drugs such as epinephrine and its derivative dipivefrin may transiently increase iop, presumably by increasing aqueous humor production via stimulation of camp. a few minutes after application of these drugs, however, iop begins to decrease, and it stays reduced for several hours. this is because epinephrine also increases aqueous outflow via b 2 receptors in the trabecular meshwork, and does so to a greater degree than it increases aqueous humor production. epinephrine may also lower iop by (1) reducing blood flow to the ciliary body (thereby lowering aqueous production) and (2) increasing uveoscleral outflow by relaxing the ciliary muscle and recruiting prostaglandins. the latter means, which can be blocked by topical nonsteroidal antiinflammatory drugs, may result in further increases in uveoscleral outflow and additional decreases in aqueous humor production. complex interactions such as this are but one reason why both b-adrenergic agonists and b-blockers lower iop in many species. when one considers species and individual differences in the density, distribution, and type of receptors as well as differences in the cause of the glaucoma, it is easy to see why it can be difficult to precisely predict what effect a given drug will have on iop in a particular patient. figure 12-2. a, the scleral venous plexus is often visible in normal animals as a series of interwoven blood vessels several millimeters posterior to the limbus. b, prominent episcleral and, to a lesser extent, conjunctival venous injection in a dog with glaucoma. increased intraocular pressure compresses the intrascleral blood vessels, which drain posteriorly. this forces more blood through the episcleral and conjunctival veins-one reason the eye appears injected in glaucoma. iop varies slightly with time of day in many species, being the greatest in the morning and gradually declining over the course of the day in dogs and humans. the opposite phenomenon has been suggested to occur in cats, rabbits, and nonhuman primates. both production and outflow of aqueous humor tend to decline with age, but production declines at a little faster rate than outflow in most individuals. in humans, aqueous production and iop tend to decline after 60 years of age, although this tendency varies considerably with ethnic background and the presence of other diseases, such as systemic hypertension and obesity. similarly, iop in cats has been shown to decline approximately 1 mm hg per year after 7 years of age. in a small percentage of humans, and perhaps animals, however, aqueous humor outflow is reduced to a greater degree than aqueous humor production, resulting in increased iop with age. disorders associated with substantially lower blood flow to the eye (e.g., dehydration, hypovolemic shock, cardiogenic shock) tend to result in lower iop. a dog collar can significantly increase iop if the dog is pulling against a leash or if the collar is too tight. dogs with glaucoma probably should be exercised with a harness rather than a collar. in addition to the numerous antiglaucoma drugs that alter iop, other drugs also may affect iop. most general anesthetics and tranquilizers cause iop to fall. ketamine may temporarily increase iop, presumably owing to extraocular muscle spasm. both spontaneous and surgically induced inflammation lower aqueous production and iop. a profound reduction in iop is an important diagnostic clue to the presence of intraocular inflammation, especially uveitis. measurement of and normal values for iop are discussed in chapter 5. it is suggested that the reader refer to that discussion before proceeding with this chapter. despite its disadvantages, the most economical instrument in general veterinary practice is the schiøtz tonometer with the human conversion tables. surprisingly, dog-specific conversion tables for the schiøtz tonometer do not agree as well with the more accurate applanation and rebound tonometers, and dog specific tables should not be used to convert schiøtz scale readings to iop estimates in dogs or cats. two handheld tonometers that are more accurate and easier to use than the schiøtz instrument are the tono-pen applanation tonometer and the tonovet rebound tonometer. the ability to perform tonometry is essential to every veterinarian engaged in small animal practice. tonometry minimizes the chances of making an important or even catastrophic error in diagnosis. iop should be determined in every red eye with an intact cornea and sclera. direct and indirect ophthalmoscopy may be used to examine the optic nerve head for cupping of the optic disc, which is the hallmark of glaucoma. the red-free filter (green light) on many of these instruments facilitates examination of the optic nerve and retinal nerve fiber layer. iridocorneal angle, and to evaluate the response to therapy (figure 12-4 the effects of increased iop on ocular tissues are similar regardless of the cause of the elevation. it is essential to con-sider whether the lesions and clinical signs observed are associated with or result from the cause of the increased pressure. glaucoma is one of the most commonly misdiagnosed eye conditions. failure of owners to recognize the disease early in its course may prevent effective treatment of the first eye. failure of clinicians to recognize onset in the second eye may prevent retention of sight. the clinical signs of glaucoma in the dog are summarized in figure 12 -12. the signs present in a particular animal depend on the duration, intensity, and cause of the pressure elevation. in general the most obvious signs are associated with end-stage clinical signs of canine glaucoma: 1, descemet's streaks (advanced cases); 2, aphakic crescent; 3, luxated lens (some cases); 4, corneal edema; 5, iris atrophy; 6, enlarged episcleral vessels; 7, fixed, dilated pupil; 9, shallow anterior chamber; 11, cupping of the optic disc; 12, retinal atrophy and vascular attenuation; 13, buphthalmos. not shown: 8, increased intraocular pressure; 10, partial or complete loss of vision; 14, ocular pain; 15, loss of corneal sensitivity. disease in which there is no hope of preserving vision. in the very early stages of glaucoma, in which there is a chance of preserving vision, the eye may appear normal and iop may or may not be elevated. in some patients there is only a history of intermittent episcleral injection (especially in the evening) that spontaneously resolves, and iop is normal on examination in the office. glaucoma may be detected in these animals only by performing tonometry when the eye is red or, occasionally, by repeatedly measuring iop over 24 hours. in other patients the eye may appear to be essentially normal and the only finding is increased iop on tonometry. in these patients it is essential to differentiate glaucoma from increased iop measurements associated with an uncooperative patient, technical problems with measuring iop (excessive tension on the eyelids, a collar that is too tight, compression of the jugular veins during restraint, etc.), and malfunction of the instrument. specialist assistance may be required to make the diagnosis of glaucoma in its early stages. an acute increase in iop to 50 to 60 mm hg or more is typically described by a human as "the worst headache of my life." it is likely that animals experience a comparable degree of pain with pressures in this range. if the iop rise is acute, the dog may be blepharospastic, depressed, less active, timid, or, in rare cases, more aggressive. some sleep more, eat less, vomit, and are less interested in play. on occasion they rub at the eye, but this behavior is an unreliable sign of glaucoma. application of pressure to the affected eye through the upper lid or to the surrounding area may cause severe pain. if the condition is not treated, severe pain and blepharospasm are replaced by signs of chronic pain that many owners may not properly recognize as being attributable to glaucoma. frequently the owner believes that the pet is simply "getting old" and this is why it is less active, sleeps more, and is less playful. a surgical procedure that alleviates the increased pressure (and accompanying pain) almost invariably results in a comment from the owner that the pet "acts like a new dog." elevated iop should be considered to be painful even if the disease is chronic and the animal outwardly appears normal. engorgement of episcleral veins (see figure 12 -2, b) is one of the more common signs of increased iop. episcleral engorgement arises because the increased iop reduces flow through the ciliary body to the vortex veins, and increased flow passes forward via anastomosing episcleral veins at the limbus (see figure 12 -1). conjunctival capillaries may also be engorged, but usually to a lesser degree. episcleral vascular engorgement is a sign of intraocular disease (anterior uveitis or glaucoma) and may be differentiated from superficial conjunctival vessel engorgement (which indicates ocular surface disease) by the following features: • episcleral vessels are larger, darker red, and more visible, and pass over a conjunctiva that is usually white or slightly pink. superficial conjunctival vessels are brighter pink to red and cover a larger portion of the sclera. • episcleral vessels do not typically branch the closer they get to the limbus, whereas superficial conjunctival vessels do. • episcleral vessels blanch slowly or not at all after the application of topical 1% epinephrine, whereas superficial conjunctival vessels typically blanch within 1 to 2 minutes. increased iop impairs the function of the corneal endothelium, resulting in corneal edema. typically the entire cornea is diffusely edematous in glaucoma, and the edema can be quite dramatic in acute glaucoma when iop is very high (figure 12 -13). in advanced cases subepithelial bullae may form, which can lead to corneal ulceration if they rupture. in chronic glaucoma both superficial and deep vascularization, scarring, and pigmentation are common. chronic increases in iop results in stretching of the cornea and sclera and enlargement of the globe (buphthalmos; figure 12 -14). buphthalmos may be especially pronounced in young animals and in shar-peis, who have a more easily distended cornea and sclera than most adult dogs. buphthalmic eyes are almost invariably blind, although limited vision may be retained for a while in some puppies and shar-peis. buphthalmos is irreversible even if the pressure is later reduced, although a variety of surgical procedures are available to restore a cosmetically acceptable appearance. by the time severe stretching has occurred, atrophy of the ciliary body may have reduced the iop to normal and pain may be lessened. as the cornea stretches, linear ruptures in descemet's depth of the anterior chamber (distance between cornea and iris) is evaluated with an oblique focal source of light or, better yet, by biomicroscopy. decreased depth of the anterior chamber is often associated with impediments to outflow through the pupil (because the lens and iris are in greater contact) and the iridocorneal angle (because the anterior chamber is more crowded). a shallow anterior chamber is an especially prominent sign in cats in which aqueous humor is misdirected into the vitreal cavity (resulting in a forward displacement of the lens and iris) and in any animal in which the lens is anteriorly luxated or subluxated. therefore a shallow anterior chamber should alert the clinician to the possibility of glaucoma. glaucoma may also be associated with an abnormally deep anterior chamber in animals with posterior lens luxation or in buphthalmic eyes. as iop rises, the pupillary constrictor muscle becomes ischemic and the pupil dilates to midrange or larger (figure 12-16) . a dilated pupil, along with episcleral injection and pain, may be among the first signs noticed by the owner. mydriasis is not an invariable sign of glaucoma-the pupil may be normal in mild iop elevations, and miosis may be present in uveitis-induced glaucoma. in these latter cases, a careful examination is necessary to distinguish glaucoma from uveitis, and it is possible for both to be present in the same eye. in chronic glaucoma, or when iop is acutely markedly elevated, the direct and consensual pupillary light reflexes are usually greatly impaired or absent. the longer glaucoma remains unresolved, the greater the chance that peripheral anterior synechiae will form and permanently block the drainage angle by fixing the peripheral iris in position. although a dilated, unresponsive pupil is consistent with glaucoma, it may be due to other diseases (e.g., progressive retinal degeneration, sudden acquired retinal degeneration syndrome, optic neuritis) and is not by itself diagnostic for glaucoma. lens luxation in glaucoma may be either primary or secondary. a glaucomatous eye with a luxated, cataractous lens may have reached this state by one of several ways: • cataract (variety of etiologies) ae lens-induced uveitis ae glaucoma ae buphthalmos ae tearing of zonules ae lens luxation • zonular malformation ae lens luxation (or subluxation) ae glaucoma ae cataract • glaucoma (variety of etiologies) ae buphthalmos ae tearing of zonules ae lens luxation ae cataract lens luxation or subluxation may be recognized from the following signs: • presence of the lens in front of the iris (anterior luxation) • presence of an aphakic crescent in the pupil (most frequent in subluxation) • movement of the iris (iridodonesis) or lens (phacodonesis) • abnormally shallow or deep anterior chamber • vitreous strands in the pupil if a luxated lens enters the anterior chamber and touches the corneal endothelium, a focal area of corneal edema may result. this opacity is frequently permanent, even if the lens is later removed. the continuous presence of a luxated lens in the anterior chamber damages the endothelium over a wider area and lowers the probability of successful surgical removal of the lens. the recognition of how the final state was reached is important in determining which combination of therapeutic methods is required. history and signalment are critical factors in differentiating between these various possibilities. in all three pathways the lens may be displaced anteriorly or posteriorly or may be in the plane of the iris (either superiorly or inferiorly). an aphakic crescent is formed when the lens zonules have broken for a portion of the circumference of the lens, and it is possible to visualize the tapetal reflex through a crescentshaped space between the lens equator and the pupillary border ( figure 12 -17). after luxation the lens frequently, but not invariably, becomes cataractous. primary lens luxation, as occurs in terriers and certain other breeds (box 12-1), may result in pupillary block with acute elevations in iop. the presence of vitreous strands in the anterior chamber in the absence of buphthalmos suggests primary lens luxation. in these animals the lens may be completely luxated or only partially luxated (subluxation), and usually the lens is not cataractous until it becomes luxated . primary glaucoma tends to occur in middle-aged to somewhat older dogs of certain breeds (box 12-2), and the lens notice that the anterior chamber is deeper superiorly than inferiorly, indicating that the lens has shifted position. the iris and lens also "trembled" when the eye moved (iridodonesis and phacodonesis). subluxation or luxation does not occur until the globe has become buphthalmic and the lens zonules are stretched beyond the breaking point (secondary luxation). similarly, primary cataract formation in a wide variety of breeds is frequently followed by lens luxation and glaucoma. lens-induced uveitis from a secondarily luxated lens that has become cataractous from elevated iop, and decreased iop from the uveitis further complicate diagnosis and treatment. thus the combination of glaucoma, cataract, and lens luxation in any particular eye may occur through several mechanisms and may be associated with a variety of iop values at any given moment. lens luxation in a glaucomatous eye does not necessarily mean that luxation was the inciting cause of the glaucoma. the luxation may have resulted from the glaucoma. loss of some or all vision is a common sequela of glaucoma. in the early stages peripheral vision may be lost ( figure 12-20) , and it is difficult, if not impossible, to detect these changes in most animals. complete vision loss can occur in a very short period (hours to a day) if the increase in iop is very high, or over a period of weeks to months if the pressure increase is more insidious. preservation of vision depends on control of iop. cupping, or posterior bowing of the optic disc through the lamina cribrosa, is the hallmark of glaucoma. retinal nerve fibers run parallel to the surface of the retina and then turn 90 degrees to enter the multilayered, fenestrated meshwork of the lamina cribrosa before exiting the eye. glial cells, blood vessels, and collagen beams form variably sized pores through which the optic nerve fibers pass. when iop rises the scleral lamina cribrosa bows posteriorly, distorting the alignment of the pores and compressing the optic nerve fibers. although this change may initially be so subtle as to not be detected ophthalmoscopically, it is sufficient to mechanically interfere with axonal axoplasmic flow and also probably with blood supply to the optic nerve head. very large increases in iop may also interfere with blood flow to the choroid and produce vision loss through ischemic damage to the photoreceptors and outer retinal layers. in acute glaucoma the optic disc may appear swollen in response to ischemia. within a day or two the increased pressure may cause the disc to appear pale and compressed. as ganglion cell axons die, optic nerve head tissue is lost and pressure forces the lamina cribrosa outward (figures 12-21 to 12-23). this change indicates irreversible damage to the optic nerve. wallerian degeneration of the optic nerve follows ( figure 12 -24). in advanced glaucoma, profound retinal atrophy with increased tapetal reflectivity occurs together with attenuation or complete loss of retinal vessels, atrophy of the pigment epithelium in the nontapetal fundus, and optic atrophy (grayish-white appearance; figure 12 -25). these findings are also present in advanced progressive retinal degeneration (progressive retinal atrophy). in progressive retinal degeneration the other signs of glaucoma are lacking, the disease is usually bilateral, the optic disc is not cupped, and differential diagnosis may be determined by the tionally by depressed electroretinograms, and in some patients it is possible to visualize wedge-shaped defects in the retina that correspond to pressure-induced infarction of the choroidal blood supply (figure 12-26) . early in glaucoma, if the pressure elevation is acute and very large, the photoreceptors in the retina undergo necrosis. in the next few days they begin to die by apoptosis as well. ophthalmoscopically the cell death is seen as increased tapetal reflectivity. as in any other severe retinal atrophy, the condition is irreversible. it has now been recognized that increased iop may initiate a chain of events that can continue to impair vision despite return of iop to within normal limits (figure 12-27) . in human primary open-angle glaucoma (poag), in which the rise in iop is more insidious and of usually smaller magnitude than in acute canine primary angle-closure glaucoma (pacg), vision loss is usually attributed mainly to retinal ganglion cell degeneration. pressure-associated alterations in microcirculation and/or axoplasmic flow at the level of the lamina cribrosa may play a role in the death of ganglion cells in this form of glaucoma. dying ganglion cells may then release glutamate and other excitatory compounds that initiate a self-perpetuating circle of apoptotic cell death in previously unaffected neighboring ganglion cells. in dogs with acute pacg and more rapid/marked increases in iop, one study found retinal damage to extend well beyond the ganglion cell layer. within the first few days of an attack of acute pacg ganglion cell necrosis and segmental full-thickness areas of retinal attenuation consistent with infarction were apparent. as ganglion cell and retinal necrosis decreased over the ensuing days retinal cell death by apoptosis markedly increased. this finding suggests that in acute pacg, the marked increase in iop not only interferes with axoplasmic flow through the lamina cribrosa but also causes ischemic necrosis of the retina. again, as these cells die they initiate a vicious circle of progressive cell death due to apoptosis that continues despite normalization of iop. this hypothesis would explain the clinical observation that even though iop is controlled in some dogs with pacg, progressive vision loss still occurs. although this sequence of events is discouraging, it does offer the possibility for the development of additional therapeutic avenues for the treatment of glaucoma, including neuroprotective agents that prevent cell suicide via apoptosis, drugs that help maintain retinal/optic nerve blood flow and minimize ischemia, and modalities that interrupt reperfusion injuries when iop is reduced from very high levels to normal. these differences in the histologic appearance among the various forms of glaucoma also reinforce the concept that glaucoma is not a single entity and that there are likely to be important differences in the cellular events leading to vision loss in patients with glaucoma. glaucoma almost invariably is the result of impaired aqueous humor outflow. in fact, in most patients with glaucoma aqueous humor production is less than normal (but still excessively high in view of the outflow capacity of the eye). the mechanism of this impairment may be etiologically classified as primary or secondary. primary glaucomas have no consistent, obvious association with another ocular or systemic disorder, are typically bilateral, have a strong breed predisposition, and hence are believed to have a genetic basis (see box 12-2). primary glaucoma is subdivided into two main forms, primary openangle glaucoma, in which the drainage angle appears gonioscopically normal (presumably because the impediment to aqueous outflow is deep to the pectinate ligaments) and primary angle-closure glaucoma, in which the drainage angle appears gonioscopically narrowed or closed (figures 12-28 through 12-33). in the dog, pacg is at least eight times more common than poag. acute pacg also is two times more common in female dogs than in male dogs. a similar sex predisposition has been found in humans with pacg and has been attributed to a generally shallower anterior chamber in women than in men. secondary glaucomas are at least twice as common as primary glaucomas in dogs (and even more common in cats) and are associated with other ocular or systemic disorders that alter aqueous humor dynamics. secondary glaucoma may be unilateral or bilateral and may or may not be inherited; the physical width of the gonioscopically visible drainage angle may also be classified as open or closed. often the exact mechanism by which outflow is impaired in secondary glaucoma is unclear. because glaucoma is almost always due to the impaired flow of aqueous humor, it can be very useful to classify glaucoma according to the location(s) of those impediments (box 12-3). impediments to the normal flow of aqueous humor commonly occur at the level of the ciliary body, pupil, trabecular meshwork, angular aqueous plexus, scleral venous system, or episcleral veins. frequently the obstruction to outflow starts at one place (for example, the lens-pupil interface), but as the disease progresses, impediments to outflow also develop in more anterior structures (for example, at the iridocorneal angle; figure 12 -34), further worsening the problem. therefore the longer the increased iop persists, the more difficult it will be to successfully treat the patient. the keys to successful therapy for glaucoma are early recognition of the problem, correct identification of the location of the impediment to outflow, and circumvention of that obstruction before additional impediments to outflow develop. poag is a bilateral disorder in which iop tends to increase in a slow, insidious fashion simultaneously in both eyes in young to middle-aged dogs of certain breeds, most notably the beagle and the norwegian elkhound (see figures 12-28 and 12-29) . initially the gonioscopically visible angle is open. over time the angle closes, the globe becomes buphthalmic, and the lens may subluxate. the precise mechanism of poag in dogs is unclear, but it most likely results from subtle biochemical alterations in the trabecular meshwork that ultimately lead to greater resistance to aqueous outflow and increased iop. in beagles, the defect appears to be inherited in an autosomal recessive fashion and may involve the glycosaminoglycan accumulation in the trabecular meshwork. the optic nerve is depressed from the surface of the fundus (cupped), has little myelin, and is darker than normal. the area surrounding the optic disc also has altered reflectivity. pacg is also a bilateral disorder but it tends to manifest as an initially unilateral, rapid, marked increase in iop in middleaged to older dogs of certain breeds (see figures 12-30 and 12-31, box 12-2, and table 12-1 ). an overt attack of glaucoma usually occurs in the initially normotensive fellow eye a median of 8 months after disease in the first eye becomes apparent. again, the precise mechanism by which pacg occurs is uncertain, but there is a clear association with congenital pectinate ligament dysplasia (pld or goniodysgenesis; see later). it is also associated with a female sex predisposition (approximately 2:1 female-to-male ratio), periods of stress or excitement, and dim light. women also have a similarly higher risk of pacg than men, which has been attributed to a smaller, somewhat more "crowded" anterior chamber in women. whether a similar phenomenon explains the female sex predisposition to pacg in dogs is unclear. some dogs also experience transient, selflimiting episodes in which iop spikes upwards but spontaneously returns to normal. pld is a condition in which the normally fine pectinate ligaments are replaced by tissues that range from a few broadbased, thick pectinate ligaments to large sheets of dysplastic tissue that cover varying amounts of the trabecular meshwork and deeper structures of the iridocorneal angle (see . large sheets of tissue may be punctuated by variably sized perforations ("flow holes") that permit aqueous humor to enter the trabecular meshwork. the deeper tissues of the iridocorneal angle may or may not be normal. because the spaces within the trabecular meshwork tend to segmentally interconnect beneath the sheets of dysplastic pectinate ligaments, iop tends to be normal even if only a few flow holes are present. although virtually any breed of dog can be affected by pld, the disorder is especially common in the basset hound, bouvier des flandres, american and english cocker spaniels, norwegian elkhound, siberian husky, dachshund, miniature poodle, welsh terrier, wirehaired fox terrier, and chihuahua. pld, however, is only one risk factor for pacg and in and of itself it is insufficient to cause glaucoma in all but the most extreme and rare case in which the dog is born with glaucoma (congenital glaucoma). evidence for this view comes from the observation that even though pld is present at birth, glaucoma in the aggregate, these observations suggest that pld is only the first step in a multistep process leading to pacg. recent imaging of the anterior segment in dogs experiencing an acute episode of pacg has led to a mechanistic theory, which holds that the event that initiates an attack may be impaired outflow at the level of the pupil (see [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . according to this theory, stress or excitement may raise heart rate and increase the difference between the systolic and diastolic blood pressures in the choroidal blood vessels. the increases in heart rate and pulse pressure result in a faster and larger forward "push" by the choroidal blood vessels on the posterior vitreous during systole. this force ultimately is transferred through the vitreous to the aqueous humor in the posterior chamber, causing an additional small bolus of aqueous humor to be forced through the pupil into the anterior chamber during systole. in the normal eye (or if the pulse pressure is normal) this fluid would simply flow back into the posterior chamber during diastole or, if trapped in the anterior chamber, it would force the iris more posteriorly, thereby opening the iridocorneal angle and allowing the additional fluid to exit via an expanded trabecular meshwork. in the eye at risk for pacg, however, exit of this small bolus of additional aqueous humor is impeded by abnormal pectinate ligaments, which may also prevent the angle from "popping open" in response to increased pressure in the anterior chamber. alternatively, or perhaps in combination with pld, age-associated declines in trabecular meshwork facility may also prevent the additional aqueous humor from escaping the anterior chamber. this results in a transient pressure differential in which pressure is slightly greater in the anterior chamber than in the posterior chamber. if the pupil is midrange in size the iris can be pressed more firmly against the lens, resulting in a "ball-valve" effect and a so-called reverse pupillary block (see figure 12 -34). a midrange to somewhat dilated pupil (as occurs in dimmer light or during excitement) is more floppy and readily pressed against the lens than a very large or very small pupil. very large pupils tend to cause the iris to "slide" off the more highly curved equatorial region of the lens, and very small pupils tend to have an iris that is taut and more resistant to compression against the lens. the next systole results in the forcing of a little more aqueous humor from the posterior chamber into the anterior chamber, further increasing iop. this process continues until iop reaches a physiologic maximum (typically 60 to 80 mm hg) that is related to systemic blood pressure and the resistance of the intraocular tissues. intermittent, spontaneously resolving attacks may occur if reverse pupil block develops but the pupil dilates to the point at which it can "slide" off the more highly curved equatorial region of the lens and break the block at the pupil; this block would then allow the excess aqueous humor to flow back through the pupil into the posterior chamber. if the block is not broken at the pupil, the iridocorneal angle and ciliary cleft may further collapse, thereby worsening the attack and making effective therapy much more difficult even though the whole process is of relatively short duration. pacg may also be classified as having the following potentially overlapping phases: • latent: the fellow, normotensive eye has all the risk factors that the overtly affected eye exhibits, except that iop has not increased. this eye should receive prophylactic therapy, because in 50% cases the fellow eye will experience overt pacg in 8 months if untreated. • intermittent: characterized by transient (minutes to hours) increases in iop that spontaneously resolve. • acute congestive: characterized by very rapid, marked (50 to 80 mm hg) increases in iop with overt clinical signs. • postcongestive: refers to an eye that has been successfully treated for acute congestive glaucoma and now has a normal or subnormal iop. • chronic: iop is chronically elevated. this state may follow an acute congestive episode that does not respond to therapy. less commonly, multiple episodes of intermittent angle closure may slowly close the angle and create a clinical course that is characterized by multiple transient spikes in iop and a gradually rising iop between the spikes. • absolute: end-stage disease. vision is lost, the eye is usually buphthalmic, and many secondary changes are typically present (lens luxation, corneal ulceration, etc.). the iridocorneal angle may be of normal width and simply filled with cells or substances that impair outflow (so-called secondary open-angle glaucoma) or the angle may very gradually narrow until closed by peripheral anterior synechia, fibrovascular membranes, and so on (so-called secondary closed-angle glaucoma). in general, secondary open-angle glaucomas carry a somewhat better prognosis than secondary closed-angle glaucomas because the anatomy is less severely deranged. examples of materials that may obstruct the trabecular meshwork are uveal cysts, neoplastic cells (especially melanocytes), inflammatory cells and debris, scar tissue (following chronic uveitis or intraocular surgery), red blood cells, macrophages filled with lens debris after capsule rupture (phacolytic glaucoma), vitreous, new blood vessels (preiridal fibrovascular membranes), air, viscoelastic materials used during intraocular surgeries, and epithelial cells originating from the cornea or conjunctiva. in many patients blocks may also exist at other locations in the eye (figure 12-37) . in traditional pupillary block the flow of aqueous humor from the posterior chamber to the anterior chamber is impaired. this may result from direct physical adhesions between the iris and lens (iris bombé; figure 12 -38) due to chronic anterior uveitis or may simply reflect a condition in which the iris and lens are in tight apposition to each other but not physically fused (physiologic iris bombé). pupillary block in the absence of physical adhesions commonly occurs in eyes in which the lens is very large (intumescent) or luxated into the pupillary aperture or when a portion of the lens zonules is disrupted and vitreous is able to move forward and occlude the pupil. in all forms of pupillary block glaucoma, however, aqueous accumulates in the posterior chamber, thereby increasing iop. very often, secondary angle-closure glaucoma complicates the latter stages of the process as the root of the iris is pushed forward into the angle. these apposed but not fused tissues (appositional closure) quickly lead to permanent adhesions and peripheral anterior synechia (synechial closure). chronic low-grade uveitis due to lens movement also often leads to secondary angle closure. sometimes called "aqueous humor misdirection" or "malignant glaucoma," ciliary body-vitreous-lens block glaucoma develops when aqueous humor flows posteriorly into the vitreal cavity or between the vitreous and the retina (figures 12-39 and 12-40) . the remaining vitreous is forced anteriorly, compressing its proteins and forcing them between the ciliary body and the lens. this tends to impair the forward flow of aqueous humor at the level of the ciliary body and to displace the entire lens-iris diaphragm anteriorly, shallowing the anterior chamber. pupillary block is common, and in the later stages of the process secondary angle closure develops as well. a syndrome of aqueous humor misdirection, shallow anterior chamber, pupil dilation, and glaucoma is common in older cats. it can be differentiated from a luxated/subluxated lens by the absence of iridodonesis and phacodonesis. glaucoma also can occur via a combination of mechanisms, and in some patients it is not yet possible to definitively ascertain the mechanism by which iop increases. the higher the iop and the longer it remains increased, the less the chance that vision can be restored; 24 to 72 hours of very high iop usually results in irreversible vision loss. the specific actions of the antiglaucoma drugs are discussed in chapter 3. ideally the primary cause of the glaucoma should be treated directly. in some outflow obstructions, however, direct treatment may not be possible, and the surgeon is forced to treat the problem indirectly by reducing the production of aqueous humor. regardless of cause, urgent therapy is required if vision is to be preserved. often a combination of medical and surgical therapy is required, and the specific drugs and procedures chosen depend on the cause and stage of glaucoma and, to a significant extent, on the clinician's personal experiences. effective client education is essential in the therapy of glaucoma. many owners' sole experience with glaucoma has been with poag in older humans. in this disorder there is no pain, the rise in iop is very slow and generally mild, and vision can often be maintained for the remainder of the person's life with medical therapy alone. the clinician should be careful to explain to the owner of an animal with glaucoma that there are many types of glaucoma and that treatment strategies used for poag in older people are not appropriate for the vast majority of cases of glaucoma in animals (or other forms of glaucoma in humans, for that matter). because many forms of glaucoma initially manifest with only one eye affected but are bilateral disorders, it is also imperative to inform the client about the clinical signs that should prompt him or her to seek medical attention in the event of an attack in the animal's fellow eye. primary glaucoma is a bilateral disease in dogs. once a diagnosis of glaucoma has been made in one eye, the remaining eye should receive prophylactic medication and regular pressure checks. the first step in the treatment of the animal with newly diagnosed glaucoma is to determine whether (1) the disorder is acute and the eye still has the potential for vision or (2) the problem is chronic and the eye is irreversibly blind (figure 12-41) . regardless of the cause of the glaucoma, aggressive, potentially toxic, and expensive medical therapy is generally of limited to no value in patients with end-stage disease and an irrevocably blind eye. the clinician can more effectively treat such patients by identifying the cause of the glaucoma (neoplasia, lens luxation, primary angle closure, hyphema, etc.) and then performing the appropriate surgical procedure (cyclodestruction, enucleation, evisceration with intrascleral prosthesis, etc.) in conjunction with evaluating the risk of glaucoma in the remaining eye. if the affected eye has the potential for sight, however, aggressive attempts to lower iop should be instituted. in these cases the next step is to determine the inciting cause of the glaucoma and directly address that cause, if possible. glaucoma is usually treated by a veterinary ophthalmologist after the family veterinarian has made the initial diagnosis and provided emergency therapy. early identification of the cause of the glaucoma and rapid reduction of iop are essential to prevent permanent damage; box 12-4 summarizes emergency treatment for pacg in an eye that still has the potential for vision. although the initial response to medical therapy may be dramatic, definitive treatment, usually surgical, must follow medical therapy in order to control iop in the long term in most patients. except in very specific circum-stances, medical therapy alone is generally not effective in the long-term control of most forms of glaucoma in animals and humans. reduction in pressure with this regimen is usually rapid (1 to 2 hours) but temporary (12 to 36 hours). if the eye responds to latanoprost, this medication should be continued every 12 hours until the patient can be evaluated by a specialist. mannitol is very potent but it also can be quite toxic, so its use is limited to eyes with the potential for vision. if iop remains elevated after a single injection of mannitol the 1.0 g/kg dose may be repeated in 4 hours if necessary, but long-term use should be avoided. because mannitol solution is at or near the saturation point it may need to be heated or put through a 5-mm filter to avoid intravenous injection of crystals and potentially fatal consequences. mannitol lowers iop by dehydrating the vitreous along with the rest of the animal. side effects include headache, osmotic diuresis, and worsening of dehydration, renal failure, or cardiovascular disease. deaths due to pulmonary edema also have been reported if mannitol is given to animals anesthetized with methoxyflurane. a hyperosmotic agent should be used with caution if the blood-ocular barrier is not intact (uveitis, hyphema), because a leaky barrier may allow mannitol to enter the vitreous, thereby pulling water into the vitreal cavity and increasing iop. oral glycerin at 1 to 2 ml/kg orally is an alternative to mannitol, although it is a less reliable ocular hypotensive drug and frequently induces vomiting. the probability of vomiting may be reduced by dividing the dose into thirds and giving it chilled or mixed with food. glycerin is contraindicated in diabetic patients. on rare occasions glycerin may be used every 8 hours for up to 5 days if toxicity is not significant. glycerin is occasionally dispensed for the owner to administer to treat a sudden 1. latanoprost 0.005%: 1 to 2 drops topically and recheck intraocular pressure in 1 to 2 hours if latanoprost is unavailable or ineffective: 1. mannitol (1.0 to 1.5 g/kg iv): 5.0 to 7.5 ml/kg of 20% solution over 15 to 20 minutes 2. methazolamide or dichlorphenamide: 2.2 to 4.4 mg/kg orally every 8 to 12 hours for dogs 3. pilocarpine (2.0% drops): 1 drop every 10 minutes for 30 minutes, then every 6 hours water should be withheld for several hours after administration of mannitol. systemic dexamethasone (0.1 mg/kg iv) or topical 0.1% dexamethasone (every 6 to 8 hours) may be useful as well if pressure-induced ischemia has resulted in significant intraocular inflammation. if the other eye is still normotensive, prophylactic therapy consisting of demecarium bromide (0.25% every 24 hours at bedtime with a topical corticosteroid) or betaxolol 0.5% every 12 hours should be instituted. box 12-4 ͉ emergency therapy for primary angleclosure glaucoma in an eye with the potential for vision attack of glaucoma immediately before seeking professional assistance. boxes 12-5 through 12-7 summarize emergency treatment for glaucoma associated with, or due to, specific circumstances or diseases. in most cases, definitive therapy for glaucoma is surgery (cyclocryotherapy, laser cyclophotocoagulation, gonioimplantation, evisceration with intraocular prosthesis insertion, or enucleation). if required, antiglaucoma drugs may supplement surgery and fine-tune iop control. certain types of glaucoma (e.g., uveitis-induced or hyphema-associated) may be treated medically first; if medical therapies fail, surgical methods may then be used. glaucoma following primary lens luxation may be controlled medically if the lens remains posterior to the iris. particular attention should also be paid to the patient's general physical health before surgery, because alterations in hydration, electrolyte, and acid-base status are common in animals that have malaise, inappetence, and so on from the pain associated with high iop or that have received antiglaucoma drugs. blood gas and acid-base status along with serum potassium levels may also need to be assessed before induction of anesthesia, especially if a systemic carbonic anhydrase inhibitor (cai) has been administered recently. preoperative rehydration may be necessary in animals that have received mannitol. glaucoma procedures used to treat eyes with the potential for vision are classified according to whether they increase aqueous humor outflow (e.g., gonioimplantation, filtering procedures) or decrease aqueous humor production (cyclophotocoagulation, cyclocryosurgery). a combination of outflowenhancing and inflow-reducing procedures may be more effective than either one alone at controlling iop and preserving vision. in current clinical practice gonioimplantation, cyclophotocoagulation, and cyclocryosurgery are by far the dominant surgical procedures used to treat an eye with the potential for retaining vision. if the eye is irreversibly blind, enucleation, evisceration with intrascleral prosthesis, and perhaps a cyclodestructive procedure are more appropriate. historically a number of procedures to increase outflow (iridencleisis, corneoscleral trephination, cyclodialysis, and sclerectomy) have been used alone or in combination in an effort to address glaucoma due to impaired outflow of aqueous humor. these procedures would theoretically address the root cause of the glaucoma and allow for more normal nutritional support for the cornea and lens, because they would enable aqueous humor production to continue at more normal levels. full-or partial-thickness holes in the sclera, however, have been plagued by fibrosis over the filtering site and long-term failure to control iop in most patients. artificial aqueous humor shunts (gonioimplants) with or without pressure-sensitive valves (to prevent iop from getting too low) have also been used to try to create a pathway for aqueous to drain from the eye, but these also have the problem of development of a scar tissue-lined, cystlike space that again becomes relatively resistant to the flow of aqueous humor (figure 12-42) . in an effort to avoid fibrosis around the drainage device, some surgeons have placed the distal end of the tubing into the frontal sinus, parotid salivary duct, nasolacrimal duct, or the orbit. none of these approaches, however, has been demonstrated to be more effective than subconjunctival drainage, and endophthalmitis is always a risk if the tube is placed in structures that communicate with the outside environment. use of an antimetabolite such as mitomycin c or 5-fluorouracil may limit fibrosis over the body of the implant and improve its long-term filtering capacity. adjunctive medical antiglaucoma therapy, or a limited cyclodestructive procedure, may also be used to fine-tune iop control once control is achieved grossly with the implant. although these procedures do not address the underlying reason for the glaucoma (impaired outflow), they can be quite effective at lowering iop. techniques for destroying the portion of the ciliary body that make aqueous humor include cyclocryotherapy with either liquid nitrogen or nitrous oxide, cyclophotocoagulation (cyclophotoablation) with either a diode or neodymium:yttrium-aluminum-garnet (nd:yag) laser, cyclodiathermy, focused ultrasound, and chemical ablation. in practical terms, however, only cyclocryosurgery and cyclophotocoagulation are reliable and used with any regularity today. these are relatively crude procedures because they require the surgeon to estimate both the degree of outflow impairment and the amount of cyclodestruction necessary to match that impairment. often the outflow facility is so severely compromised that the eye is highly sensitive to even minor alterations in aqueous production, resulting in a relatively narrow margin for error in these estimates. too little destruction can result in persistence of the glaucoma, and too much can lead to phthisis bulbi. it is also not uncommon for outflow to be so severely impaired that aqueous humor production must be reduced to levels that cannot maintain normal ocular health, resulting in cataract formation or corneal endothelial decompensation and vision loss even though iop is controlled. failure to control iop in the long term with these procedures is the result of inadequate destruction of the ciliary body, regeneration of the ciliary epithelium, and progressive angle closure with loss of additional outflow capacity. despite these limitations, however, a cyclodestructive procedure is more appealing as a single procedure than a gonioimplant or filtering procedure because it is faster, technically easier, less expensive to perform, and repeatable. cyclodestruction is indicated in cases of medically uncontrollable primary glaucoma in an eye that still has the potential for vision and for the relief of chronic ocular pain in an irreversibly blind eye in an animal whose owner wishes to preserve the globe. the success rate is much lower in eyes with glaucoma secondary to chronic anterior uveitis, preiridal fibrovascular membrane formation, or retinal detachments. relative contraindications include intraocular neoplasia, hyphema, and anterior lens luxations. controlled application of intense cold to the sclera overlying the ciliary body causes necrosis of the ciliary body and reduced aqueous production. both liquid nitrogen and nitrous oxide are acceptable cryogens, but some surgeons believe liquid nitrogen to be a more reliable agent, perhaps because it achieves a colder temperature than nitrous oxide. preoperatively dexamethasone (0.1 mg/kg iv) and flunixin meglumine (0.1 mg/kg iv) are administered in anticipation of the severe uveitis than may follow cyclocryosurgery. precise application of the cryoprobe over the ciliary processes and avoiding the 3 and 9 o'clock positions is essential. if the globe is approximately normal size, a 3-mm (diameter) nitrous oxide glaucoma cryoprobe is centered 5 mm posterior to the limbus (figure 12-43 ). if the globe is enlarged, the cryoprobe is centered 5.5 to 6.0 mm posterior to the limbus. gentle pressure on the globe, slightly indenting it, enlarges the extent of the ciliary destruction by shortening the distance between the cryoprobe and target tissue and by reducing blood flow to the area. usually six to eight spots are frozen for 2 minutes when nitrous oxide instrumentation is used. timing begins when the probe achieves a temperature of -70°to -80°c, a range that correlates with a temperature in the ciliary body of at least -10°c, which is necessary to cause cyclodestruction. if liquid nitrogen is used, the probe is placed in the same location, but the cryogen is circulated through the probe until the ice ball extends 1 mm past the limbus into clear cornea, after which the freeze is terminated. the larger size of the tip (2.5 ¥ 6.5 mm) and the more profound freeze usually allows fewer sites to be frozen (perhaps as few as two to four). at the conclusion of the procedure a subconjunctival injection of 0.5 to 1.0 mg of dexamethasone or other suitable corticosteroid may be given. systemic analgesics may be necessary in some animals because freezing can induce significant ocular pain. the marked chemosis that follows freezing can result in exposure conjunctivitis and/or keratitis, so a partial temporary tarsorrhaphy may also be performed at the conclusion of the procedure. marked conjunctivitis, chemosis, and uveitis should be expected. topical 0.1% dexamethasone/triple antibiotic ophthalmic ointment is administered every 4 to 6 hours, depending on the degree of inflammation. antiglaucoma drugs are continued as before surgery, and if the eye has the potential for vision, the iop is carefully followed for several days, and then at 1 and 2 weeks. if the eye is irreversibly blind, antiglaucoma drugs are continued for 10 to 14 days, after which the patient is reevaluated. marked postoperative iop spikes can persist for days after surgery, and occasionally aqueocentesis may be necessary to control iop in the immediate postoperative period. tapping the anterior chamber, however, can be detrimental because doing so exacerbates the uveitis, risks introducing bacteria or damaging the lens, and probably increases the chance of reperfusion injury to the retina and optic nerve. if iop is well controlled 2 weeks postoperatively, the antiglaucoma medication dosage may be gradually tapered. the timing of further followup examinations varies according to response to therapy and whether the eye has the potential for vision. complications include the aforementioned iop spike, uveitis, exposure keratoconjunctivitis, neurotrophic keratitis if the long posterior ciliary nerves are damaged, hyphema, retinal detachment, recurrence of glaucoma, and phthisis bulbi with a cosmetically unacceptable globe. the relatively high frequency of these complications indicates that cyclocryosurgery should not be performed as a prophylactic measure in the normotensive fellow eye of an animal with glaucoma. success rates vary with the duration of follow-up, whether iop control or preservation of vision was the goal, and whether the owner permits more than one freezing episode. if iop control, not vision, is the goal and the owner will allow multiple procedures to be performed, cyclocryosurgery can have a success rate as high as 90%. if the eye has the potential for vision at the outset, the rates of vision preservation may be as high as 60% at 6 months postoperatively. unfortunately, as for all glaucoma procedures, the success rate declines with the length of followup. if iop begins to rise again additional medical and or surgical therapy is required. in general cats seem to have a lower success rate than dogs. certain breeds (cocker spaniel, siberian husky, norwegian elkhound, chow chow, and shar-pei) may require more aggressive ciliary body destruction to ensure long-term iop control. an alternative method of destroying the ciliary body processes is transscleral irradiation of the ciliary body with a diode or nd:yag laser. laser therapy has the advantages of being more controllable and potentially causing less reaction than cyclocryotherapy. it can also be repeated with less risk of hypotony. it suffers from the disadvantage of frequently requiring more than one treatment and of having a higher failure rate than cyclocryotherapy. laser cyclophotocoagulation is exclusively performed by ophthalmic surgeons trained and experienced in its use. the combination of a limited cyclodestructive procedure and a gonioimplant (with or without adjunctive medical therapy) offers some attractive theoretical advantages in treating glaucomatous eyes with the potential for vision. they include (1) blunting of the postoperative iop spike that often accompanies a cyclodestructive procedure and can destroy the last vestiges of vision the patient has, (2) allowing for a greater level of aqueous humor production postoperatively so as to improve intraocular nutrition and reduce the chance a blinding cataract will occur, and (3) allowing for a finer control of iop in the postoperative period. in one retrospective study a combination of the two procedures appeared to be more effective than a single procedure and allowed more than 50% of patients to retain vision for at least 1 year after an overt attack of angle-closure glaucoma. combining procedures also allowed for a greater percentage of patients to maintain iop within the normal range, even though vision was ultimately lost either because of progressive retinal and optic nerve degeneration secondary to an apoptotic cascade or because of cataract. the frequent follow-up visits, additional expense, and potentially greater complications of a combined procedure, however, do not allow for it to be advocated for the treatment of irrevocably blind eyes, for which the goal of therapy is simply pain relief. the clinician should be aware that primary lens luxation is bilateral and usually hereditary, although very commonly the patient initially presents with an overt luxation in only one eye. an acute episode of glaucoma associated with lens luxation is managed as previously described in this chapter. if the eye is irreversibly blind the clinician should consider enucleation, evisceration with intrascleral prosthesis, or perhaps a cyclodestructive procedure. lens extraction is seldom indicated in blind eyes because it is more costly than other procedures and because other impediments to outflow (e.g., at the angle) are usually present and cause glaucoma to persist postoperatively. longer-term therapy for an eye with the potential for vision and glaucoma attributable to a subluxated or luxated lens depends on the position of the lens and whether or not other impediments to outflow are present. if lens luxation is acute and the lens has luxated posteriorly, the eye may be treated with miotics to ensure that the lens does not enter the anterior chamber. many animals tolerate a lens in the vitreous for long periods without recurrences of glaucoma, provided that medications are continued. if the lens is opaque and interferes with vision in the vitreous or has very recently become luxated, or if the pupil will not effectively constrict, intracapsular lens extraction may be performed, although the prognosis is guarded even when the procedure is performed by experienced surgeons. if the lens has luxated into the plane of the pupil or anterior chamber, most surgeons prefer to remove it by either intracapsular lens extraction or phacoemulsification. alternatively, the pupil may be dilated and an attempt made to get the lens to fall back into the vitreous. if the lens does fall into the vitreous, miotics may then be used in an effort to ensure that it remains there. if it does not, it should be surgically removed. the longterm success of any of these treatment strategies hinges on whether the patient has either poag or peripheral anterior synechia and secondary angle-closure glaucoma in addition to the lens luxation. unfortunately, both of these conditions commonly occur in patients with primary lens luxations thereby greatly reducing the probability of maintaining a comfortable and sighted eye over the long term. gonioscopy is performed once emergency therapy has been implemented and the inflammation has been reduced. if the angle is open, medical therapy may be slowly reduced in accordance with control of the uveitis. the ability of topical dexamethasone 0.1% to increase iop in normal dogs and dogs with poag is of uncertain importance in the treatment of dogs with uveitis-induced glaucoma. in a clinical setting the relatively small rise in iop attributable to topical corticosteroids is masked by the much more dramatic changes in iop induced by inflammation of the ciliary body and the compromise of the drainage angle (both of which may be returned to more normal values by the use of topical corticosteroids). therefore it seems reasonable to use topical corticosteroids for the treatment of uveitis-induced glaucoma, although these agents should not be employed indiscriminately. if peripheral anterior and posterior synechiae are present, and pressure does not fall with emergency therapy, the prognosis for retaining vision is very poor. if the eye still has vision and an open iridocorneal angle, laser iridotomy may be attempted to create a new hole in the iris to allow aqueous to bypass the occluded pupil; however, the iris holes usually seal closed with time. laser iridotomy is much less effective in eyes that also have peripheral anterior synechia and angle closure because it does not resolve this additional impediment to outflow at the level of the angle. gonioimplantation may also be attempted, but frequently this procedure fails because the tube rapidly occludes with inflammatory debris and the subconjunctival filtering bleb rapidly scars. a cyclodestructive procedure may also be attempted although it frequently exacerbates the uveitis, possibly leading to even more synechia and outflow impairment. if the eye is irreversibly blind the clinician should consider enucleation (with histopathology to determine the cause of the uveitis), evisceration with an intrascleral prosthesis (again with histopathology; it should not be performed if neoplastic or infectious causes of the uveitis are suspected), or, in carefully selected cases, a cyclodestructive procedure. melanoma of the iris or ciliary body is a relatively common cause of secondary glaucoma in dogs and a less common one in other species. in most cases, enucleation, with or without an orbital prosthesis, is the treatment of choice. in very select cases iridocyclectomy (removal of a portion of the iris and ciliary body), cyclocryotherapy, or laser photocoagulation is successful in treating circumscribed tumors. by the time glaucoma is present the tumor is usually too advanced for this type of therapy. glaucoma secondary to lymphosarcoma may respond to medical antiglaucoma therapy and definitive systemic chemotherapy. in general evisceration with placement of an intrascleral prosthesis is to be avoided in patients with presumed intraocular neoplasia. absolute glaucoma is the end stage of chronic, increased iop with buphthalmos, severe degenerative changes in most ocular tissue, blindness, and, almost invariably, pain. although the patient with absolute glaucoma frequently shows no pain on palpation of the eye, and the owner may not believe the animal has pain, enucleation of the affected eye almost invariably results in increased playfulness and improvements in the patient's demeanor. this observation leaves little doubt that chronic glaucoma is a painful condition in the vast majority of animals. the goal of therapy for absolute glaucoma is to provide pain relief and address any cosmetic concerns the owner may have. eyes with end-stage glaucoma are best treated by enucleation (with or without an intraorbital prosthesis), evisceration with intrascleral prosthesis, or a cyclodestructive procedure. evisceration with intrascleral prosthesis is indicated if the owner desires to maintain a more cosmetically pleasing eye. after a careful assessment of the eye (box 12-8), the globe is eviscerated via removal of the internal contents through a limbal incision, leaving a scleral and corneal shell. after hemorrhage is controlled a silicone prosthesis is inserted (figure 12-44) . the enlarged globe shrinks to the size of the prosthesis over the next 3 to 4 weeks. during this time the cornea may vascularize and appear red. this appearance eventually resolves, and the cornea assumes its final gray or black color. the extent of pigmentation is impossible to predict, and owners are so advised before surgery. prostheses may also be used after severe injury, when phthisis bulbi is beginning, to preserve a chronic glaucoma ± buphthalmos prevention of phthisis bulbi blinding ocular trauma (may be used even after penetrating corneal wounds) chronic, noninfectious uveitis intraocular neoplasia panophthalmitis ulcerative keratitis senile degenerative keratopathy degenerative corneal disorders foci of bacterial infection (e.g., severe untreated dental disorders, discospondylitis, otitis externa) box 12-8 ͉ indications and contraindications for intraocular prosthesis insertion cosmetically acceptable eye. prostheses have been successfully inserted into equine eyes with glaucoma previously unresponsive to medications and cyclocryotherapy. although this procedure is generally quite successful, complications include ocular pain in the immediate postoperative period, ulcerative keratitis (potentially with exposure or extrusion of the prosthesis), keratoconjunctivitis sicca, infection, and recurrence of an unsuspected tumor. because of the last possibility, all excised tissue should be histologically examined. once an eye has been thoroughly evaluated and a diagnosis of absolute glaucoma with pain has been made, the owner may decide to have the eye removed. an intraorbital prosthesis may or may not be placed, depending on the owner's wishes. see chapter 17 for the technique. all enucleated eyes should be examined by an experienced veterinary ophthalmic pathologist. the general principles of glaucoma therapy also apply to feline glaucoma. in general, normal feline iop tends to be greater than that of the dog and to decline with age. one study found normal iop for young cats with the tono-pen to be 20.2 ± 5.5 mm hg with a range of 9 to 31 mm hg, whereas the tono-pen yielded readings of 12.3 ± 4.0 mm hg (range 4 to 21 mm hg) in cats 7 years or older. the exact incidence of glaucoma in cats is unclear, although data from the veterinary medical data base suggested that 1 in 367 cats presenting to a university teaching hospital had glaucoma. in contrast, a prospective evaluation in a feline exclusive private practice found that 0.9% of cats 7 years or older had abnormally high iop on tonometric screening. secondary glaucoma, most frequently due to chronic uveitis or intraocular neoplasia, is approximately 19 times more common than primary glaucoma in cats. inherited congenital poag has been described in siamese cats, but acute pacg as seen in dogs is rare to nonexistent in cats. the rise in iop in the vast majority of cats tends to be slow and insidious, and the condition is usually unilateral. many cats with glaucoma initially present for another ocular disorder (chronic uveitis, iris color change, intraocular mass). another common presentation, especially for those with aqueous humor misdirection syndrome (see earlier description in this chapter), is anisocoria with slowly progressing buphthalmos. the buphthalmos can be quite extreme in some animals. ocular pain also tends to not be as obvious as in dogs, perhaps because the rise in iop is typically not as abrupt or as high as in dogs, but there is no reason to believe that the condition is not painful in cats like it is in other species. often the inciting cause is difficult to identify by the time the patient is first seen. common causes of glaucoma in cats include feline aqueous humor misdirection syndrome, chronic low-level lymphocytic plasmacytic uveitis with the formation of preiridal fibrovascular membranes, and neoplasia such as diffuse iris melanoma and uveal lymphoma. in one study toxoplasma was implicated in 79%, feline corona virus in 27%, feline immunodeficiency virus in 23%, and feline leukemia virus in 6%. the most common clinical signs are dilated pupil, lens luxation, buphthalmos, exposure keratitis, and retinal degeneration. cats with uveitis and prominent lymphoid nodules in the iris and iris erythema are considered to be at high risk for eventual development of glaucoma. cats with positive toxoplasma titers are more effectively treated with a combination of clindamycin and topical corticosteroid than with either drug alone. medical therapy for glaucoma in cats is similar to that in the dog, although cats tolerate some glaucoma medications poorly and may respond differently to antiglaucoma drugs. for example, latanoprost and the other commercially available prostaglandins do not lower iop in cats, although they can induce profound miosis. the topical cai brinzolamide did not lower iop in normal cats when administered every 12 hours but may do so when given every 8 hours to cats with glaucoma. a related topical cai, dorzolamide given every 8 hours, is effective in lowering iop in glaucomatous cats. as in dogs, topical application of dexamethasone or 1% prednisolone acetate has increased iop in cats, but the clinical significance of this finding is unclear. additionally, unilateral topical administration of 0.5% tropicamide can raise iop an average of approximately 3.5 mm hg in both the treated and untreated eyes, and in some cats this increase may be as much as 17 to 18 mm hg in the treated and untreated eyes. these observations reinforce the concept that cats are anatomically and physiologically distinct from dogs and that some therapies appropriate for the dog may not be transferable to the cat. although surgical therapy for feline glaucoma is similar to that for dogs, cyclocryotherapy must be quite aggressive if used, and liquid nitrogen is recommended as the cryogen to limit treatment failures. cyclodestructive procedures are often unsuccessful in the long term in cats, perhaps because of the nature of their glaucoma. evisceration with insertion of an intrascleral prosthesis may be performed, although the cosmetic results with a black silicone ball are less satisfactory than that achieved with dogs because of the normally brightly colored feline iris and vertically oriented slit pupil. varying the color of identify underlying cause and directly address it if possible systemic dexamethasone (0.1mg/kg iv) or flunixin meglumine (0.1 mg/kg iv) carbonic anhydrase inhibitors either topically (dorzolamide 2% alone or in combination with timolol, or brinzolamide 1%, both every 8 hours) or systemically (methazolamide or dichlorphenamide: 2.2 to 4.4 mg/kg orally every 8 to 12 hours for dogs and 1 to 2 mg/kg every 8 to 12 hours for cats) usually, pilocarpine, latanoprost, and systemic hyperosmotics should be avoided medical and surgical management of the glaucoma patient use of high-resolution ultrasound as a diagnostic tool in veterinary ophthalmology combined cycloablation and gonioimplantation for treatment of glaucoma in dogs: 18 cases (1992-1998) implantation of filtering devices in dogs with glaucoma: preliminary results in 13 eyes development of glaucoma after cataract surgery in dogs: 220 cases (1987-1998) pectinate ligament dysplasia and narrowing of the iridocorneal angle associated with glaucoma in the english springer spaniel the feline glaucomas: 82 cases (1995-1999) neodymium:yag laser treatment of iris bombé and pupillary block glaucoma veterinary ophthalmology vitreous body glutamate concentration in dogs with glaucoma histomorphometry of optic nerves of normal dogs and dogs with hereditary glaucoma seroepidemiologic and clinical observations of 93 cases of uveitis in cats diode laser transscleral cyclophotocoagulation for the treatment of glaucoma in dogs: results of six and twelve months' followups cullen frontal sinus valved glaucoma shunt: preliminary findings in dogs with primary glaucoma equine glaucoma: a retrospective study of 13 cases presented at the western college of veterinary medicine from 1992-1999 putative aqueous humor misdirection syndrome as a cause of glaucoma in cats: 32 cases (1997-2003) effect of topical ophthalmic latanoprost on intraocular pressure in normal horses phacoemulsification and intraocular lens implantation: a study of surgical results in 182 dogs a histopathological study of iridociliary cysts and glaucoma in golden retrievers correlation of morphologic features of the iridocorneal angle to intraocular pressure in samoyeds age-related changes in ocular distances in normal eyes of samoyeds heritability of the depth of the opening of the ciliary cleft in samoyeds the canine glaucomas prevalence of the breed-related glaucomas in pure-bred dogs in north america secondary glaucomas in the dog in north america changes in intraocular pressure associated with topical dorzolamide and oral methazolamide in glaucomatous dogs effects of different dose schedules of latanoprost on intraocular pressure and pupil size in glaucomatous beagles the ocular hypertensive effects of topical 0.1% dexamethasone in beagles with inherited glaucoma comparative doppler imaging of the ophthalmic vasculature in normal beagles and beagles with inherited glaucoma the intracapsular extraction of displaced lenses in dogs: a retrospective study of 57 cases (1984-1990) comparison of the use of new handheld tonometers and established applanation tonometers in dogs effect of topically applied demecarium bromide and echothiophate iodide on intraocular pressure and pupil size in beagles with normotensive eyes and beagles with glaucoma effect of hyaluronidase on aqueous outflow resistance in normotensive and glaucomatous eyes of dogs effects of topical l-epinephrine and dipivalyl epinephrine on intraocular pressure and pupil size in the normotensive and glaucomatous beagle the effect of topical pilocarpine on intraocular pressure on pupil size in normotensive and glaucomatous beagles intraocular silicone prostheses in dogs: a review of the literature and 50 new cases primary glaucoma in burmese cats effect of topical 1% atropine sulfate on intraocular pressure in normal horses possible association of glaucoma with pectinate ligament dysplasia and narrowing of the iridocorneal angle in shiba inu dogs in japan an evaluation of a rebound tonometer for measuring intraocular pressure in dogs and horses intraocular pressure measurements obtained as part of a comprehensive geriatric health examination from cats seven years of age or older development of glaucoma after phacoemulsification for removal of cataracts in dogs: 22 cases (1987-1997) scanning electron microscopic examination of selected canine iridocorneal angle anomalies gonioscopy and anatomical correlations of the drainage angle of the dog primary glaucoma in the dog glaucoma in the basset hound evaluation of glutamate loss from damaged retinal cells in dogs with primary glaucoma intraocular silicone prosthesis implantation in eyes of dogs and a cat with intraocular neoplasia: 9 cases intraocular silicone prosthesis in a horse the efficacy of topical prophylactic antiglaucoma therapy in primary closed angle glaucoma in dogs: a multicenter clinical trial mechanisms of acute intraocular pressure increases phacoemulsification lens extraction in dogs evaluation of two applanation tonometers in horses comparison of the human and canine schiøtz tonometry conversion tables in clinically normal cats comparison of the human and canine schiøtz tonometry conversion tables in clinically normal dogs description of ciliary body anatomy and identification of sites for transscleral cyclophotocoagulation in the equine eye equine glaucoma: clinical findings and response to treatment in 14 horses light-microscopy evaluation of zonular fiber morphology in dogs with glaucoma secondary to lens displacement effect of topical atropine on intraocular pressure and pupil diameter in the normal horse eye treatment of glaucoma by use of transscleral neodymium:yttrium aluminum garnet laser cyclocoagulation in dogs surgery for lens instability the use of transscleral cyclophotocoagulation with a diode laser for the treatment of glaucoma occurring post intracapsular extraction of displaced lenses: a retrospective study of 15 dogs effects of the application of neck pressure by a collar or harness on intraocular pressure in dogs equine glaucoma: a retrospective study of 11 cases comparison of the effects of topical administration of a fixed combination of dorzolamide-timolol to monotherapy with timolol or dorzolamide on iop, pupil size, and heart rate in glaucomatous dogs pectinate ligament dysplasia (pld) and glaucoma in flat coated retrievers. i: objectives, technique and results of a pld survey canine goniodysgenesis-related glaucoma: a morphologic review of 100 cases looking at inflammation and pigment dispersion feline glaucoma: a retrospective study of 29 clinical cases two cases of intraocular silicone prostheses in eyes with traumatic corneal lacerations cyclocryotherapy. part i: evaluation of a liquid nitrogen system cyclocryotherapy. part ii: clinical comparison of liquid nitrogen and nitrous oxide cryotherapy on glaucomatous eyes cyclocryotherapy and noncontact nd:yag laser cyclophotocoagulation in cats morphologic features of the aqueous humor drainage pathways in horses orthograde rapid axoplasmic transport and ultrastructural changes of the optic nerve part ii: beagles with primary open angle glaucoma golden retriever uveitis: 75 cases combined transscleral diode laser cyclophotocoagulation and ahmed gonioimplantation in dogs with primary glaucoma: 51 cases effects of risk factors and prophylactic treatment on primary glaucoma in the dog ocular hypertension following cataract surgery in dogs: 139 cases (1992-1993) unconventional aqueous humor outflow of microspheres perfused into the equine eye effects of 0.005% latanoprost solution on intraocular pressure in healthy dogs and cats the effects of intracameral carbachol on postoperative intraocular pressure rises after cataract surgery in dogs the effects of a timolol maleate gel-forming solution on normotensive beagle dogs normal variation in, and effect of 2% pilocarpine on, intraocular pressure and pupil size in female horses treatment of equine glaucoma by transscleral neodymium:yttrium aluminum garnet laser cyclophotocoagulation: a retrospective study of 23 eyes of 16 horses implantation of intraocular prostheses in dogs. comp cont ed pract vet morphologic features of degeneration and cell death in the neurosensory retina in dogs with primary angle-closure glaucoma glaucoma in horses equine glaucoma advances in topical glaucoma therapy effects of topical administration of 0.005% latanoprost solution on eyes of clinically normal horses pectinate ligament dysplasia and glaucoma in flat coated retrievers. ii: assessment of prevalence and heritability box 12-5 ͉ emergency therapy for uveitis-induced glaucoma 1 . identify underlying cause and directly address it if possible. 2. topical dexamethasone (0.1% every 2 to 4 hours) or prednisolone acetate (1.0% every 2 to 4 hours) 3. carbonic anhydrase inhibitors either topically (dorzolamide 2% alone or in combination with timolol or brinzolamide 1%, both every 8 hours) or systemically (methazolamide or dichlorphenamide: 2.2 to 4.4 mg/kg orally every 8 to 12 hours for dogs and 1 to 2 mg/kg every 8 to 12 hours for cats) or together 4. if additional intraocular pressure lowering is required, consider adding in topical timolol 0.5% every 8 to 12 hours, epinephrine 1% every 6 to 8 hours, or dipivefrin 0.1% every 6 to 8 hours. 5. usually, systemic hyperosmotics should be avoided. 6. the use of topical pilocarpine or atropine is controversial.box 12-6 ͉ emergency therapy for hyphemaassociated glaucoma 1 . if lens in anterior chamber, dilate pupil with atropine 1% or tropicamide 1.0%. 2. topical dexamethasone (0.1% every 2 to 4 hours) or prednisolone acetate (1.0% every 6 to 8 hours) if ineffective: 1. mannitol (1.0 to 1.5 g/kg iv): 5.0 to 7.5 ml/kg of 20% solution over 15 to 20 minutes 2. carbonic anhydrase inhibitors either topically (dorzolamide 2% or brinzolamide 1%, both every 8 hours) or systemically (methazolamide or dichlorphenamide: 2.2 to 4.4 mg/kg orally every 8 to 12 hours for dogs and 1 to 2 mg/kg every 8 to 12 hours for cats) 3. if additional intraocular pressure lowering required, consider adding topical epinephrine 1% every 6 to 8 hours or dipivefrin 0.1% every 6 to 8 hours. 4. if the lens is in the anterior chamber, pilocarpine, timolol, and latanoprost should be avoided. referral to a specialist for further evaluation is advisable.box 12-7 ͉ emergency therapy for lens luxation-associated glaucoma the sphere and tattooing a slit pupil onto the cornea can improve the postoperative appearance of the globe. enucleation, with or without the placement of an intraorbital prosthesis, is a reasonable procedure in cats. there are some suggestions, however, that cats may reject an intraorbital sphere more frequently than dogs. normal equine iop is higher than a cat's or dog's, averaging approximately 23 mm hg and ranging up to the low to mid 30s. glaucoma is less commonly recognized in horses than in dogs or cats, perhaps because the uveoscleral pathway constitutes a greater percentage of the equine outflow pathway. although primary glaucoma appears to occur in horses, the most common form is glaucoma secondary to chronic anterior uveitis or intraocular neoplasia. appaloosas, horses with concurrent equine recurrent uveitis, and horses older than 15 years are at greater risk of glaucoma. clinical signs of equine glaucoma include corneal striae (caused by rupture of descemet's membrane), buphthalmos, decreased vision, lens luxation, loss of the pupillary light reflex, mild anterior uveitis, optic nerve atrophy, optic disc cupping, and elevated iop. because many horses with glaucoma also have anterior uveitis, the pupil is often miotic or normal in size and is not dilated as is common in other species. a feature that complicates both the diagnosis and therapy of equine glaucoma is that the iop fluctuates markedly, and frequent measurements may be necessary to demonstrate the presence of glaucoma and the effects of treatment. the reason for this fluctuation is unclear but it may involve compression of the globe by the orbicularis oculi or extraocular muscles. auriculopalpebral nerve block may be required to obtain accurate applanation tonometry in fractious horses, and sedatives may significantly decrease iop. the principles of medical and surgical therapy for glaucoma in other species apply to horses with glaucoma, although the response to antiglaucoma medications in horses may be different from that in dogs and cats. studies of antiglaucoma drugs in horses often yield conflicting results, suggesting that there may be considerable interindividual variations in the responsiveness of this species to many antiglaucoma drugs. for example, topical pilocarpine given alone can increase iop in many, but not all, horses. the mechanism for this finding is unclear but may involve exacerbation of preexisting uveitis, pupillary block, or a reduction in the uveoscleral outflow pathway. atropine, which stabilizes the blood aqueous barrier and may increase uveoscleral outflow, can reduce iop in many normal horses and in horses with glaucoma secondary to chronic uveitis. atropine can, however, also raise iop in some horses. the prostaglandin derivative latanoprost does not lower iop in normal horses (or does so only by 1 to 2 mm hg) and can be quite irritating. other studies have indicated that topical prostaglandins exacerbate elevated iop in horses with glaucoma. only timolol or the topical cais seem to consistently lower iop in horses. systemic cais may be prohibitively expensive in horses, and their efficacy and safety has not been determined. antiglaucoma therapy in the horse often involves a combination of antiglaucoma and antiinflammatory drugs. unfortunately, the therapy of primary equine glaucoma is largely empirical owing to our lack of understanding of the pathogenesis of the condition.a cyclodestructive procedure (cyclocryotherapy, laser cyclophotocoagulation) may be used in equine eyes that have the potential for vision and in an attempt to maintain a comfort-able, but blind eye. one study suggested that an effective key: cord-317153-2la3hkzv authors: kauhala, kaarina; kowalczyk, rafal title: invasion of the raccoon dog nyctereutes procyonoides in europe: history of colonization, features behind its success, and threats to native fauna date: 2011-10-01 journal: curr zool doi: 10.1093/czoolo/57.5.584 sha: doc_id: 317153 cord_uid: 2la3hkzv we aimed to review the history of the introduction and colonization of the raccoon dog nyctereutes procyonoides in europe, the features behind its successful expansion and its impact on native fauna. the raccoon dog quickly colonized new areas after being introduced to the european part of the former soviet union. today it is widespread in northern and eastern europe and is still spreading in central europe. features behind its success include its adaptability, high reproductive potential, omnivory, hibernation in northern areas, multiple introductions with > 9000 individuals from different localities, and tendency to wander enabling gene flow between populations. firm evidence of the raccoon dog’s negative impact on native fauna, such as a reduction in bird populations, is still scarce. raccoon dogs may destroy waterfowl nests, although a nest predation study in latvia did not confirm this. predator removal studies in finland suggested that the raccoon dog’s impact on game birds is smaller than expected. however, raccoon dogs may have caused local extinction of frog populations, especially on islands. raccoon dogs may compete with other carnivores for food, for example for carrion in winter, or for the best habitat patches. in northern europe potential competitors include the red fox vulpes vulpes and the badger meles meles, but studies of their diets or habitat preferences do not indicate severe competition. the raccoon dog is an important vector of diseases and parasites, such as rabies, echinococcus multilocularis and trichinella spp. and this is no doubt the most severe consequence arising from the spread of this alien species in europe. invasive species have many ecological effects and may threaten biological diversity (e.g., ebenhard, 1988; hulme, 2007; vilà et al., 2010) . alien species may alter habitat, and predate on or compete with native fauna or be important vectors of diseases and parasites. they may also hybridize with native species and thus affect their genetic variability. besides ecological effects, they may have considerable economic impacts on invaded areas (vilà et al., 2010) . there are 44 alien mammal species in europe, 33 of which are considered established, i.e. they form self-sustaining populations (genovesi et al., 2009) . these include several carnivore species, such as the american mink neovison vison, raccoon procyon lotor and raccoon dog nyctereutes procyonoides. they were brought to europe because of their valuable fur or as pets and either escaped or were intentionally introduced into the wild (genovesi et al., 2009) . these carnivores are widespread in europe, occurring in over 10 european counties. the raccoon dog is one of the most successful alien carnivores in europe. it has spread rapidly into many european countries after being introduced by russians during the first half of the 20 th century (e.g., lavrov, 1971; lever, 1985; helle and kauhala, 1991) . the raccoon dog has been suspected of causing damage to native fauna through predation, but firm evidence of this is scarce (lavrov, 1971; nasimovič and isakov, 1985; kauhala, 2004) . raccoon dogs may also compete with native medium-sized carnivores, such as the eurasian badger meles meles and the red fox vulpes vulpes (jędrzejewska and jędrzejewski, 1998; kowalczyk et al., 2008) . furthermore, the raccoon dog is an important vector of zoonoses and parasites, such as rabies echinococcus multilocularis and trichinella spp (e.g., oivanen et al., 2002; deplazes et al., 2004; holmala and kauhala, 2006; romig et al., 2006) . its role as a vector of diseases and parasites is likely to cause considerable ecological and economic impacts and may also cause health problems to humans. our aim was to review the published literature on the history of the introduction and colonization of the raccoon dog, the features behind its success and its ecological effects in europe. the raccoon dog originates from the far east. six subspecies are usually distinguished: n. p. procyonoides (gray, 1834) in most of china and northern vietnam, n. p. orestes (thomas, 1923) in the mountainous region of yunnan in china, n. p. koreensis (mori, 1922) in korea, n. p. ussuriensis (matschie, 1907) in the amur and ussuri regions of siberia and eastern china, n. p. viverrinus (temminck, 1838) in japan (except hokkaido) and n. p. albus (beard, 1904) in hokkaido (ellerman and morrison-scott, 1951; ward and wurster-hill, 1990) . the climate in the original distribution area varies from the subtropical regions of japan, northern vietnam and southern china to a harsh continental climate with cold winters in mongolia and southeast siberia. accordingly, raccoon dogs in different areas have adapted to different climates, habitats and diets, which can be seen in their body size, fat reserves, thickness of fur, and their behavioral and dental characteristics (kauhala and saeki, 2004a) . a total of about 9100 individuals of the ussuri raccoon dog n. p. ussuriensis were introduced, mainly to european parts of the former soviet union, between 1929 and 1955 (lavrov, 1971 ). this subspecies is now widespread in northern and eastern europe (mitchell-jones et al., 1999; kauhala and saeki, 2004b) . its original range in south-east siberia covers the valleys of the amur and ussuri rivers and the khankai lowland, the shores of the sea of japan and also areas as far inland as komsomol'sk (novikov, 1962) . in its native range the winters are cold with thick snow cover, and raccoon dogs are forced to hibernate. it has thick winter fur and accumulates large fat deposits in autumn to survive through the harsh winter (stroganov, 1969) . the ussuri raccoon dog was thus pre-adapted to survive in areas with long winters in northeast europe. its thick fur was the reason that russians introduced it into the western parts of their country. russians first bred the animals in fur farms and then released them deliberately so as to have a new valuable fur animal in the wild. the first introductions in 1928 or 1929 (415 pregnant females) were made to transcaucasia, abkhazia, southern ossetia and karatalinia (lever, 1985) . in many areas, especially on the asian side of the caucasus, the populations did not flourish but remained small or vanished completely. more raccoon dogs were released in the mid-1930s e.g., in leningrad, novgorod and kalinin provinces, in north caucasus, ryazan province south of moscow, kirgizia and ukraine ( fig. 1; lavrov, 1971) . raccoon dogs were introduced to astrakhan between 1936 and 1939 and to moldavia between 1949 and 1954 (lever, 1985 . they were also released in estonia in the 1950s, in pskov in 1947 and in the karelian isthmus near finland in 1953 (lavrov, 1971) . one hundred raccoon dogs were released in belarus in 1963 (lever, 1985) . some individuals were also introduced further north, to the kola peninsula in 1936 and to archangel in 1950 -1953 (lavrov, 1971 ). many introductions made to the european part of the soviet union were successful and the populations started to increase rapidly. populations spread at a rate of 40 km per year (and even up to 120 km per year) from the introduction sites (lavrov, 1971 ). the first wandering raccoon dogs were seen in finland in the 1930s and 1940s (siivonen, 1958; suomalainen, 1950; fig. 1) . the raccoon dog started to truly colonize finland in the mid-1950s, but there was a time-lag of about 10 years until rapid population increase started in the mid-1960s. the phase of rapid population growth lasted for another decade and by the mid-1970s most of southern and central finland was inhabited (helle and kauhala, 1991) . after this phase of increase the population growth seemingly ceased and numbers fluctuated for 10-15 years until they started to increase again. the raccoon dog population is still increasing, and today the raccoon dog is the most common medium-sized carnivore in finland (kauhala, 2007) . the hunting bag increased from 818 in 1970/71 to 172,000 in 2009 (finnish game and fisheries research institute, 2010 1 ). raccoon dogs were found all over estonia in the 1950s (lavrov, 1971) . their numbers remained low, however, because of numerous wolves canis lupus and lynx lynx lynx, the natural enemies of raccoon dogs in estonia. in contrast, wolves and lynx were very scarce in finland in the 1960s and 1970s (ermala, 2003) , which may have contributed to the rapid spread of raccoon dogs in the country. raccoon dogs were found all over lithuania in the late-1950s and thus colonized the country in about 10 years (lavrov, 1971; fig. 1) . in latvia 1000 raccoon dogs were observed or hunted as early as 1951. the first raccoon dogs were seen in sweden in 1945 (notini, 1948) but after that raccoon dogs were observed only occasionally in norrbotten, until they started to spread a few years ago (p.-a. åhlèn, pers. com.). the reason for this long time-lag between the first observations and the rapid population increase in sweden is not known. in norway (finnmark) the first records of raccoon dogs are from 1983 (wikan, 1983) . there were no other observations in norway until winter 2007/2008 when a few raccoon dogs were shot in central norway (r. andersen, pers. com.). they most probably invaded central norway via sweden. raccoon dogs were first noticed in poland in 1955 and in east germany in 1961 (dehnel, 1956 nowak and pielowski, 1964; nowak, 1984) . fifteen years later, i.e. by the end of the 1960s, almost all of poland, with the exception of higher parts of the mountains in the south, was occupied by the species (fig. 2) . nowadays the raccoon dog is among the most common carnivores in some areas of the country (jędrzejewska and jędrzejewski, 1998) . over 11,000 raccoon dogs are caught in poland annually, and the hunting bag increases from year to year. in germany, the raccoon dog population remained sparse until it started to increase in the 1990s, thirty years after the first observations, especially in eastern parts of the country (ansorge and stiebling, 2001) . the hunting bag has increased exponentially since the early 1990s in germany (drygala et al., 2008a, b) , and in brandenburg (eastern germany) alone the hunting bag increased from 398 in 1995/1996 to (baagøe and jensen, 2007) . the first observations of raccoon dogs were made in eastern and central europe between 1951 and 2002 (nowak and pielowski, 1964; artois and duchêne, 1982; nowak, 1984; lever, 1985; weber et al., 2004; fig. 1 ). for instance, in france the first observation of the species was made in 1975 or 1979 and the first case of reproduction was observed in 1988 (léger and ruette, 2005) . in northern italy raccoon dogs were seen and photographed in 2005 (p. genovesi, in litt.) , showing that the species has managed to cross the alps. today the raccoon dog is also found occasionally in the netherlands, moldova, slovenia, croatia, bosnia-herzegovina, and serbia. it has also been seen once in macedonia (ćirović and milenković, 1999; mitchell-jones et al., 1999; ćirović, 2006) . one raccoon dog was run over by car in se spain in 2008 (anon, 2008 4 ). the northern limit of the raccoon dog's distribution is determined by climate. it can live in areas where the mean annual temperature is above 0°c, the thickness of snow cover is < 80 cm, the snow cover lasts < 175 days and the length of the growing season for plants is at least 135 days (lavrov, 1971) . today the northern limit of its permanent distribution lies at the arctic circle (helle and kauhala, 1991) . raccoon dogs will possibly widen their distribution area northwards due to climate change. increased spring precipitation in the form of snow at higher latitudes may, however, compensate for the effect of global warming (melis et al., 2010) . one of the main factors responsible for the successful expansion of raccoon dogs in europe was mass introduction over a wide area coupled with a large amount of genetic variation. together with their natural tendency to disperse and high migratory ability this allowed raccoon dogs to invade neighboring areas in a relatively short time. more research on the colonization process should be done, because the exact details are still poorly known. genetic data on raccoon dogs is still scarce. so far, populations from several locations in finland and germany have been investigated by pitra et al. (2010) . reconstructed phylogenies reveal two major clades in european raccoon dogs, which diverged approximately 457,800 years ago. in total, nine haplotypes were found in raccoon dogs in europe with a sequence divergence of 0.2%-3.2% (mean 1.3%). as suggested by pitra et al. (2010) , a combination of factors including multiple translocations with use of individuals from different geographical areas, secondary contact and admixture of two co-occurring separate maternal lineages with divergent evolutionary histories are probably the main determinants of the genetic variability of raccoon dogs in europe. it was probably a sequential, two-step process that included reduction in genetic variation due to the founder effect and population bottlenecks during initial introductions in the european part of the former soviet union in 1929-1955, followed by an increase in genetic variation by hybridization of individuals from multiple native-range sources representing divergent haplogroups (pitra et al., 2010) . however, according to ansorge et al. (2009) there was no indication of the founder effect or inbreeding in the european populations, indicated by the same level of variability that can be seen in the native amursk population. additionally, phylogenetic analysis indicates different invasion corridors of the species in the western range of raccoon dog distribution in central europe, as earlier suggested by ansorge et al. (2009) , with the secondary contact zone between previously geographically and genetically different source populations in germany (pitra et al., 2010) . an important factor behind the raccoon dog's success is the very high plasticity of the species. they are true omnivores and eat anything they can catch (reviewed in sutor et al., 2010) . in białowieża forest, the index of food niche breadth for raccoon dogs was 6.25, nearly twice as high as in the next species with the widest niche -the red fox (3.77; jędrzejewska and jędrzejewski, 1998) . the diet of raccoon dogs varies between areas and seasons, according to the availability of different food sources (e.g., ivanova, 1962; nasimovič and isakov, 1985; sutor et al., 2010) . in many areas, small mammals form the bulk of their diet in all seasons (bannikov, 1964; nasimovič and isakov, 1985; kauhala et al., 1998a; bao et al., 2005; sutor et al., 2010) . carrion may reach up to 76% of biomass consumed during harsh winters (jędrzejewska and jędrzejewski, 1998; sidorovich et al., 2000) . frogs, lizards, invertebrates and birds are also frequently consumed (e.g., barbu, 1972; jędrzejewska and jędrzejewski, 1998; sutor et al., 2010) . raccoon dogs eat berries and fruit, especially in late summer and autumn because they serve as an important food source when raccoon dogs fatten themselves before entering winter dormancy (e.g., nasimovič and isakov, 1985; kauhala et al., 1993a; kauhala, 2009; sutor et al., 2010) . the preferred habitats of raccoon dogs are wet open habitats: damp meadows and forests with sparse canopy but abundant undergrowth, marshlands, river valleys and gardens. however, they may occupy various habitats from continuous forests to open agricultural landscapes and suburban areas (jędrzejewska and jędrzejewski, 1998; drygala et al., 2008c) . however, habitat preferences at the western and southern edge of the distribution area are poorly known. the raccoon dog has a high reproductive capacity; higher than expected for a medium-sized carnivore species (kauhala 1996a ). this has also contributed to its success. mean litter size is 8-10 in areas with favourable conditions, both in native and introduced ranges (judin, 1977; helle and kauhala, 1995; ). the maximum litter size at birth in a sample of 203 adult females from southern finland was 16. the maximum number of embryos was 18, and that of corpora lutea was 23 . raccoon dogs are monogamous and the male participates in pup-rearing by warming and guarding the pups when the female is foraging (ikeda, 1983; yamamoto, 1987; kauhala et al., 1998b; drygala et al., 2008a) . females can thus spend a lot of time foraging and produce enough milk for a large litter. the raccoon dog's opportunistic diet further contributes to the large litters and also survival rate of juveniles. the survival rate is highest when the crop of berries is good . the mean annual mortality rate of juveniles during their first year of life is as high as 88% -89% in southern finland (helle and kauhala, 1993) . raccoon dogs thus produce many pups, but most of them die early in their life, and the mortality rate of juveniles is the major mortality factor of the species. the mortality rate is lowest (ca. 43%) among middle-aged (2-4 year old) raccoon dogs and increases after 5 years of age. only one per cent of individuals reach the age of 5 years, with the maximum life span being about 8 years . raccoon dogs generally reach sexual maturity at the age of 10 months, but the reproductive value is highest among 2-3-year old females which thus produce most of the pups in the population . in europe, raccoon dogs are found from the warmer conditions of hungary and the balkans to the much harsher conditions of northern europe (kauhala and saeki, 2004b) . in cold climates raccoon dogs hibernate during winter. during hibernation their body temperature is 1.4 -2.1 °c lower than during summer. this habit is unique among canids, and may also have contributed to the successful spread of raccoon dogs in northern europe (mustonen et al., 2007) . in winter, raccoon dogs settle in shelters which protect them against cold and predation (kowalczyk and zalewski, 2011) . in poland, active badger setts are most often selected by raccoon dogs. badger setts can also be used as breeding dens (kowalczyk et al., 2008) . hibernation usually lasts from november until march (in finland), but when the winter is mild raccoon dogs may be active even in mid-winter. they usually sleep when the air temperature is < -10 °c, snow depth > 35 cm and day length < 7 h . most raccoon dogs are active when the temperature is above zero, there is no snow and day length is > 10 h. for instance, in germany raccoon dogs do not usually hibernate (drygala et al., 2008b) . raccoon dogs are very well adapted to a long period of food deprivation in winter . they fatten themselves during autumn and almost double their body weight between early summer and late autumn (korhonen, 1988a, b; kauhala, 1993; mustonen et al., 2007) . fattening in autumn and sleeping in winter are regulated by hormonal changes . thyroid hormone levels are low during winter and the animal thus adapts its general metabolism to the availability and requirements of energy (korhonen, 1987 1 ). in fur farms, raccoon dogs lose their appetite when air temperature decreases to -5 °c, which indicates an endogenous behavioural pattern: when it is too cold raccoon dogs stop eating and hibernate. another feature behind the success of raccoon dogs is their tendency to wander (nasimovič and isakov, 1985) . after introduction in europe, raccoon dogs wandered as far as 300 km in a year (nasimovič and isakov, 1985) or 500 km within three years (nowak, 1973) . adults may disperse in a colonizing population (sutor, 2008) whereas in stable populations only juveniles usually disperse (nasimovič and isakov, 1985; kauhala et al., 1993b; kauhala and helle, 1994) . the mean dispersal distances, estimated on the basis of home range sizes, were 14 km for females and 19 km for males in south-east finland, with the mean maximum distances being 48 km and 71 km respectively . the maximum straight line distance in a couple of months was 145 km in southern finland (kauhala and helle, 1994) . in north-east germany, the mean and maximum dispersal distances of both sexes were 13.5 km and 91 km respectively (drygala et al., 2010) . very little is still known about dispersal routes and more research on the subject is required. the successful expansion of raccoon dogs in europe was also possible due to the secretiveness of the species and low persecution at the beginning of invasion. raccoon dogs are nocturnal animals, utilising mainly wet habitats covered with dense vegetation and showing inactivity in winter. these facts decrease their vulnerability to persecution kowalczyk et al., 2008) . in many countries the species was only persecuted once it had successfully established its population. in some areas hunters are not very interested in hunting raccoon dogs because the fur of wild animals has low value. population density varies according to the structure of the landscape. in finland, described an inverse relationship between home range size and the proportions of meadows and gardens in the home range. habitat richness (number of habitat patches per ha) also affected home range size: home ranges were small in areas where the landscape was a small-scale mosaic consisting of meadows with abundant undergrowth, gardens where raccoon dogs could find fruit and berries and small patches of mixed forests . because home range size and population density tend to be negatively correlated in monogamous canids with exclusive home ranges (trewhella et al., 1988; contesse et al., 2004; woodroffe et al., 2004) , population density can be estimated from home range size. the maximum density is then two adults in each home range in pup-rearing season when overlap of home ranges is smallest (kauhala et al., 1993b) . for instance, in the 'best' areas of southern finland home ranges are only 100 ha and raccoon dog density can be up to two adults per km 2 , whereas in poorer areas with large spruce forests pup rearing home ranges are about 260 ha and the corresponding density is < 0.8 adults per km 2 . in northern germany pup rearing home ranges are about 200 ha (drygala et al., 2008b) and the density would thus be up to one adult per km 2 . the area is a mosaic of mixed forests, wetlands and maize fields. as the raccoon dog population in germany is still increasing and spreading, population density may also increase. in forests of suwałki landscape park, nw poland, raccoon dog density was estimated to be only 0.37 individuals per km 2 (goszczyński, 1999) . in białowieża primeval forest (poland) density was 0.5 -0.7 ind./km 2 , and mean home range size 5.0 km 2 (jędrzejewska and jędrzejewski, 1998; kowalczyk et al. unpublished) . hunters in particular have suspected that raccoon dogs destroy the nests of game birds (lavrov, 1971 ). according to naaber (1971 naaber ( , 1984 , raccoon dogs robbed 85% of waterfowl nests in some areas of estonia. ivanova (1962) found remains of birds (mainly water birds) in 45% of raccoon dog scats collected in a river valley in voronez. when the raccoon dog population increased rapidly in russia, it was thought to be very harmful but, according to lavrov (1971) , this was not based on fact. raccoon dogs were accused of causing the decline of grouse populations even in areas where raccoon dogs did not occur (lavrov, 1971 ). even today robust scientific studies clearly demonstrating that raccoon dogs cause damage to native birds are scarce. birds eaten by raccoon dogs are mainly passerines (kauhala, 2009; sutor et al., 2010) . they are more important for raccoon dogs when voles are scarce than during vole peak population times (ivanova, 1962; judin, 1977; kobylińska, 1996) . it is not known whether raccoon dogs have caused a decline in passerine populations. the occurrence of birds in the diet increases with latitude, i.e. birds are consumed especially in northern europe (sutor et al., 2010) . the increase of carnivory with increasing latitude has also been detected among other omnivorous mammals (vulla et al., 2009) . according to diet studies (84 data sets from different parts of native and introduced ranges) it is unlikely that raccoon dogs affect game bird populations in general. this is because, excluding some finnish data, there were remains of waterfowl or grouse in only 0-5% of the feces or stomachs of raccoon dogs (kauhala, 2009) . although raccoon dogs may prey on ground-nesting birds such as waterfowl (barbu, 1972; włodek and krzywiński, 1986; schwan, 2003; sutor et al., 2010) , they probably consumed many of the non-passerine birds as carcasses (novikov, 1962; barbu, 1972; woloch and rozenko, 2007; kauhala and auniola, 2001) . waterfowl, especially female eiders somateria mollissima, occurred commonly in the feces of raccoon dogs in the sw archipelago of finland (kauhala and auniola, 2001 ). however, a viral disease killed many brooding eiders during the years (1998) (1999) when the data were collected and raccoon dogs probably found most of the eiders as carcasses. raccoon dogs also catch sick or injured birds left behind by hunters (samusenko and goloduško, 1961; pavlov and kiris, 1963; barbu, 1972; naaber, 1974; viro and mikkola, 1981; kauhala et al., 1993a) . remains of egg shells, including those of domestic poultry, occurred in 0 -41% of the samples (kauhala, 2009) . in most studies, they are not mentioned at all. it is thus hard to know the extent of egg consumption by racoon dogs. opermanis et al. (2001) found, however, that raccoon dogs destroyed only 0.3% of waterfowl nests in a wetland area of latvia. amphibians (e.g., rana spp., bufo spp., bombina spp. and triturus cristatus) commonly occur in the diet of raccoon dogs in spring and summer (e.g., ivanova, 1962; lavrov, 1971; barbu, 1972; viro and mikkola, 1981; kauhala et al., 1993a kauhala et al., , 1998a jędrzejewska and jędrzejewski, 1998; sutor et al., 2010) . both adult frogs and tadpoles are easy prey for raccoon dogs and this may cause a decline in frog populations, especially on islands and in other fragmented or isolated areas (kauhala and auniola, 2001; sutor et al., 2010) . frogs were scarce in the diet of raccoon dogs in the outer archipelago in southern finland, although they occurred commonly in the diet on the mainland (kauhala and auniola, 2001) . it is possible that raccoon dogs had already caused a decline in the frog populations of the archipelago. predator removal studies do not provide firm evidence of the harmfulness of raccoon dogs to game bird populations. studies in finland indicated that the breeding success of ducks improved in only one study area in northern finland where raccoon dogs occurred only occasionally even at the beginning of the experiment (kauhala, 2004) . the most frequently removed predators in the area were red foxes and pine martens martes martes. in southern finland where raccoon dogs are common, the breeding success of dabbling ducks was positively correlated with a raccoon dog index. furthermore, the breeding success of ducks improved in the predator protection area of southern finland during the experiment (kauhala, 2004) . in the predator removal area chick production declined at the end of the study with a simultaneously increasing fox population. the most frequently removed predators in this area were raccoon dogs, which may have resulted in an increase in fox populations which, in turn, may have affected the breeding success of ducks. the raccoon dog index also correlated positively with the reproductive success of black grouse in southern finland (kauhala et al., 2000) . another predator removal study in finland indicated, however, that raccoon dog removal might have had some effect on the breeding success of ducks (väänänen et al., 2007) , but the change was not significant. furthermore, chick production of, for example, mallards anas platyrhynchos and coots fulica atra, increased at first but then declined after the second year of raccoon dog removal. this happened simultaneously with the decline in the raccoon dog index. these results resemble those of the predator removal study described above and probably relate to the interactions between different predators. on the other hand, when different areas were compared there was a negative relationship between the breeding success of mallards and raccoon dog abundance index, so more research is needed on this topic in order to understand these contradictory outcomes. raccoon dogs consume carrion during all seasons if available, but carcasses are especially important for them in winter when other food sources are scarce (jędrzejewska and jędrzejewski, 1998; sidorovich et al., 2000 sidorovich et al., , 2008 . raccoon dog scavenging was recorded on 47% of carcasses available in białowieża forest (poland; selva et al., 2005) . according to sidorovich et al. (2000) raccoon dogs compete with native carnivores for carcasses in belarus in late winter. this competition can be so severe that the increasing raccoon dog population appears to have caused a decline in native carnivore populations, including the red fox, brown bear ursus arctos and pine marten. the polecat mustela putorius has probably suffered most from competition with raccoon dogs (sidorovich et al., 2000) . this information is, however, based only on correlative data and firm evidence is lacking. however, in białowieża forest, the rate of food niche overlap was very high (59%) among raccoon dogs and polecats in spring and autumn (jędrzejewska and jędrzejewski, 1998) . the polecat population has also decreased in finland during recent decades. the probable reasons for this include habitat changes and competition with other carnivores (liukko et al., 2010) . in northern europe, the red fox and the badger might compete directly or indirectly with raccoon dogs for food, habitats or den sites. correlative data from finland showed that when raccoon dogs were heavily hunted and their population decreased, the fox population started to increase (kauhala, 2004) . this may be a coincidence, but it can also indicate that raccoon dogs and red foxes compete for some resources in finland. in southern finland, there was some overlap in the diet of raccoon dogs, badgers and foxes but differences also existed: the badger consumed more invertebrates and the fox more mammals and birds than the raccoon dog (kauhala et al., 1998a) . furthermore, female foxes in finland have become more carnivorous after the arrival of the raccoon dog, as revealed by a study on dental morphology (viranta and kauhala, 2011) . this case of character displacement points to the conclusion that foxes and raccoon dogs have competed for food resources in finland. however, in winter when food is scarcest both raccoon dogs and badgers hibernate and, hence, no competition for food between the raccoon dog and other carnivores exists in this season in northern areas. in north-eastern poland, the diet of raccoon dogs overlaps 41% with red foxes and 35% with badgers in spring and summer. in winter, diet overlap between raccoon dogs and red foxes increases to 62%, when both species utilize more carrion. other species that raccoon dogs may compete with are semi-aquatic species (american mink -38% and river otter lutra lutra -33%; jędrzejewska and jędrzejewski, 1998) . a habitat preference study from southern finland indicated that the habitat preferences of raccoon dogs and badgers differed to some extent: raccoon dogs favored meadows (including clear-felled areas) and open woodlands with abundant and tall undergrowth as well as gardens, whereas badgers favored forests with a thick canopy but sparse undergrowth . management of forests using clear felling (common in finland) may thus benefit raccoon dogs at the expense of native badgers. both species were, however, flexible in their habitat use and when the most favored habitats were not available they used the same habitats, such as fields . whether they compete in these circumstances for the best habitat patches is unknown and certainly requires further research. in north-eastern poland the habitat niche of raccoon dogs overlapped 91% with that of the fox and 77% with that of the polecat (jędrzejewska and jędrzejewski, 1998) . raccoon dogs commonly use badger setts kowalczyk et al., 2008) . raccoon dogs thus benefit from the occurrence of badgers in the area. the habit of using badger setts has probably facilitated the invasion of raccoon dogs in europe (kowalczyk et al., 2008) , because deep and complex badger setts might offer refuge against the cold and predation (kowalczyk and zalewski, 2011) . common use of burrows may lead also to intra-guild predation. in białowieża forest, the killing of raccoon dog pups by badgers was recorded. raccoon dogs may also influence badger breeding success, as no concurrent breeding of badgers was recorded in badger setts in which raccoon dogs bred (kowalczyk et al., 2008). 9 raccoon dogs as vectors of diseases and parasites although the red fox has been the main terrestrial wildlife rabies vector in europe since the 2 nd world war (e.g., anderson et al., 1981) , the significance of the raccoon dog as a vector of rabies has recently increased who, 2009 ). seventythree percent of the observed rabies cases were in raccoon dogs during an epizootic of sylvatic rabies in finland in -1989 (nyberg et al., 1990 westerling, 1991; westerling et al., 2004) . in the baltic states as well, the raccoon dog is an important secondary host of rabies: the number of observed rabies cases in raccoon dogs in 2009 was 24 in estonia and 28 in lithuania (who, 2009) . the corresponding figures for fox cases were 24 and 17. in lithuania, the prevalence of rabies in raccoon dogs increased almost 2.5 times from 11.8% in 1994 to 28.9% in 2004 28.9% in (mačiulskis et al., 2006 . in russia, belarus and ukraine rabies also occurred in raccoon dogs in 2009 (who, 2009) . in poland, the raccoon dog is the second (after red fox) most important vector of rabies among wild animals. north-eastern poland (24,000 km 2 ), 131 cases of rabies were recorded in raccoon dogs (siemionek et al., 2007) . when oral vaccination was used, the number of rabies cases in raccoon dogs in poland declined to 7−15 in the whole country (smreczak et al., 2006 (smreczak et al., , 2007 (smreczak et al., , 2008 smreczak and żmudziński, 2009) . the role of the raccoon dog as a vector of rabies may further increase in europe, because the raccoon dog population is still growing and spreading (ansorge and stiebling, 2001; drygala et al., 2008a, b) . the total number of reported wildlife rabies cases (excluding bats) in europe in 2009 was 4114, 302 of which were in raccoon dogs (who, 2009) . bait vaccinations against rabies have proved to be effective and have resulted in many rabies-free countries in europe (wandeler, 1988; artois et al., 2001; pastoret et al., 2004) . the increasing raccoon dog population may, however, alter the situation: the current strategies to control wildlife rabies may not be effective enough in a community of two important vector species; the red fox and the raccoon dog singer et al., 2009) .the hibernation of the raccoon dog may further complicate the situation (schneider et al., 1988) . indeed, a modelling study showed that epizootics in the community of two species were stronger than expected for single species (singer et al., 2009) . rabies could persist in the community, even if the disease was not spreading in an individual vector species due to low density. in the community of two vector species raccoon dogs were usually the major rabies host, and the number of cases in fox populations depended on raccoon dog density. when raccoon dog density was high, invasive raccoon dogs could even outcompete native foxes (singer et al., 2009) . additionally, badgers may act as a spill-over species and suffer from rabies epizootics. other viruses also dangerous to humans, including sars (severe acute respiratory syndrome) and avian h5n1 viruses have been found in raccoon dogs in china (guan et al., 2003; changchun et al., 2004; qi et al., 2009) . raccoon dogs have additionally fallen victim to canine distemper virus (cdv) in japan (machida et al., 1993; aoyaki et al., 2000) . cdv may have the most far-reaching consequences of all infectious agents for free-living carnivores (deem et al., 2000) . data from germany indicates the possible transmission of cdv between wild carnivores and the domestic dog canis familiaris (frölich et al., 2000) . the prevalence of the virus was much higher in urban and suburban foxes than in rural ones. to our knowledge there is no data showing the role of the raccoon dog as a vector of cdv in the carnivore community (including domestic dogs canis familiaris), but this possibility cannot be ruled out. alveolar echinococcosis caused by echinococcus multilocularis is a dangerous emerging zoonosis in europe. the parasite can even cause lethal diseases in humans (eckert et al., 2000; kern et al., 2003; moks et al., 2005) . the red fox has been the definite host in central europe, but recently cases have been detected in raccoon dogs too (thiess et al., 2001; machnicka-rowinska et al., 2002; deplazes et al., 2004; kapel et al., 2005) . prevalence in foxes is high (35%−65%) in the core area of this zoonosis . small mammals, mainly rodents, are the intermediate hosts of the parasite (schantz et al., 1995) . e. multilocularis is spreading in europe and new endemic areas have been detected in recent years (eckert et al., 2000) . e. multilocularis has even invaded cities (hofer et al., 2000; deplazes et al., 2004) . the parasite is found in denmark, the netherlands, belgium, france, germany, poland, czech republic, slovakia, switzerland, austria, northern italy, slovenia and lithuania (romig et al., 2006; vergles rataj et al., 2010) . e. multilocularis has reached as far as latvia and estonia in the north: prevalence in foxes was 35.6% in latvia and 29.4% in estonia (moks et al., 2005; bagrade et al., 2008) . the parasite is so far absent in finland (oksanen and lavikainen, 2004) . the reason behind the spread of e. multilocularis in europe is probably the growing fox and raccoon dog populations romig et al., 2006) . the raccoon dog is highly susceptible to e. multilocularis infection and may provide an additional pool of definitive hosts in europe (romig et al., 2006) . in poland prevalence in raccoon dogs was 8% (machnicka-rowińska et al., 2002) . due to high densities of the species in some areas of the country, it is a serious source of infection. in northern brandenburg, germany, prevalence in raccoon dogs was 6.3%-12.0% (s. schwarz et al., unpubl. data). the parasite is an increasing public health concern, because efficient control measures are not available (eckert et al., 2000) . however, baiting foxes in an urban area of zurich using baits with praziquantel (an antihelminthic) has decreased the prevalence of e. multilocularis in the city (hegglin et al., 2003 . sarcoptic mange, a zoonosis caused by a parasite sarcoptes scabiei, is an important mortality factor of raccoon dogs both in native and introduced ranges (kauhala, 1996b; shibata and kawamichi, 1999; kowalczyk et al., 2009) . raccoon dogs may also transmit the parasite to other animals including foxes, lynx and even brown bears (mörner et al., 2005) . mange has caused significant declines in red fox populations in, for example, sweden, in the city of bristol, uk (lindström et al., 1994; harris and baker, 2001) and also temporarily in finland (k. kauhala, pers. obs.) . the occurrence of infected raccoon dogs in the area may increase the risk of serious epizootics among foxes, because both species may use badger setts as den sites kowalczyk et al., 2008) . also, badgers may be infected on rare occasions (collins et al., 2010) . trichinella spp. are parasitic nematodes that cause trichinellosis (gottstein et al., 1997) . the disease is common in carnivores, especially scavengers, all over the world. foxes are the most common reservoirs of sylvatic trichinellosis in europe, although in finland the raccoon dog is another important reservoir (pozio, 1998; oivanen et al., 2002) . sylvatic trichinosis is more common in northern europe than in central and southern parts of the continent, because the human impact on natural ecosystems is less intense in the north (pozio, 1998) . domestic trichinellosis occurred only in two countries in the european union, finland and spain, in the 1990s (pozio, 1998) . the raccoon dog may be an important reservoir species in finland, because it carries the most intense infections and is the only species that hosts all four trichinella spp. (t. spiralis, t. nativa, t. pseudospiralis and t. britovi) that occur in finland (oivanen et al., 2002) . the prevalence and risk of infection in wild animals (e.g., foxes) is lower in northern than in southern finland, probably due to the sparse raccoon dog population in the north. the prevalence in foxes has increased simultaneously since the 1960s along with the increase in the raccoon dog population in finland (oivanen et al., 2002) . prevalence in foxes is much higher (44%) in estonia where raccoon dogs are more common than in sweden (10%) where raccoon dogs are sparse (oivanen et al., 2002) . these facts point to the conclusion that the role of the raccoon dog as a reservoir of trichinella spp. is remarkable (oksanen et al., 1998; oivanen et al., 2002) . the raccoon dog is one of those invasive species which extended its range quickly after introductions and invaded neighboring areas. by the 1980s, raccoon dogs had colonized over 1.4 million km 2 of europe (nowak, 1973; novak and pielowski, 1964; kauhala and saeki, 2004b) and in many areas became the most numerous of carnivores (jędrzejewska and jędrzejewski, 1998; sidorovich et al., 2000; . the success of the raccoon dog invasion in europe was enabled thanks to an exceptional combination of factors including: widely distributed and multiple introductions, great migratory ability and the high reproductive capacity of the species, plasticity of food habits, hibernation in areas where climate is harsh and its general adaptability to different climatic and environmental conditions, and the admixture of individuals from divergent matrilineages (e.g., lavrov, 1971; helle and kauhala, 1995; kauhala 1996a kauhala , 1996b kauhala et al., 2007 , kowalczyk et al. 2008 , 2009 pitra et al., 2010; sutor et al., 2010) . few projects have been conducted in europe on the ecology of the species, so little is still known on the impact of raccoon dogs on native fauna. it seems that in many areas raccoon dogs fit very well into the local communities and successfully coexist with native medium-sized carnivores. locally, the 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procyonoides (gray, 1834) in switzerland rabies in finland and its control 1988-90 historical perspective of rabies in europe and the mediterranean basin. paris: the world organisation for animal health distribution of rabies in europe raccoon dog found dead in sör-varanger : a new norwegian mammal biology and behavior of raccoon dogs nyctereutes procyonoides in poland acclimatization of the raccoon dog in southern ukraine infectious disease: infectious disease in the management and conservation of wild canids male parental care in the raccoon dog nyctereutes procyonoides during the early rearing period we are grateful to dr. amy eycott for her linguistic revision of our manuscript. we thank four anonymous reviewers for their critical comments on the manuscript. key: cord-330204-guhrtz1h authors: cleaveland, sarah; hampson, katie title: rabies elimination research: juxtaposing optimism, pragmatism and realism date: 2017-12-20 journal: proc biol sci doi: 10.1098/rspb.2017.1880 sha: doc_id: 330204 cord_uid: guhrtz1h more than 100 years of research has now been conducted into the prevention, control and elimination of rabies with safe and highly efficacious vaccines developed for use in human and animal populations. domestic dogs are a major reservoir for rabies, and although considerable advances have been made towards the elimination and control of canine rabies in many parts of the world, the disease continues to kill tens of thousands of people every year in africa and asia. policy efforts are now being directed towards a global target of zero human deaths from dog-mediated rabies by 2030 and the global elimination of canine rabies. here we demonstrate how research provides a cause for optimism as to the feasibility of these goals through strategies based around mass dog vaccination. we summarize some of the pragmatic insights generated from rabies epidemiology and dog ecology research that can improve the design of dog vaccination strategies in lowand middle-income countries and which should encourage implementation without further delay. we also highlight the need for realism in reaching the feasible, although technically more difficult and longer-term goal of global elimination of canine rabies. finally, we discuss how research on rabies has broader relevance to the control and elimination of a suite of diseases of current concern to human and animal health, providing an exemplar of the value of a ‘one health’ approach. for thousands of years, people have lived in fear of rabies transmitted from domestic dogs, and more than half of the world's population still do so today. from the time of the first written reference to rabies in the 23rd century bc, the link between the bite of a mad dog and the risk of human death has been well recognized [1, 2] . although many mammalian hosts can be infected with the rabies virus, the domestic dog remains to this day by far the most important species causing human rabies deaths and tens of thousands of people die from canine-mediated rabies each year [3, 4] , mostly in asia and africa where the disease is maintained in domestic dog reservoirs. in developing the first vaccines against rabies, louis pasteur recognized the potential for eliminating human rabies deaths, and considered that 'to solve the problem of rabies would be a blessing for humanity' [5] . the need for and feasibility of rabies elimination through interventions in the dog population has also been recognized for more than a century. since the first large-scale implementation of canine vaccination in the 1920s, canine rabies has now been eliminated in several parts of the world, for example in island and peninsula states of asia (e.g. japan, taiwan), in the usa, western europe and across parts of latin america [1, 6, 7] . in this review, we address the reasons why, despite the long history of rabies research and earlier successes in canine rabies elimination, new research has been needed to tackle the problem of rabies in low-and middle-income countries (lmics) of africa and asia. we demonstrate how research has generated optimism about the feasibility of achieving global targets of zero human deaths from dogmediated rabies, guided pragmatism in the design of dog vaccination strategies in lmics, and instilled realism in the path towards global canine rabies elimination. while the first decades of rabies research focused on the problem in domestic dogs, the successful control of canine rabies in many of the world's richer countries shifted emphasis towards the growing problem of wildlife rabies. during world war ii, the red fox (vulpes vulpes) emerged as the main rabies reservoir in europe, and the disease spread rapidly affecting most of western and southeastern europe by the mid-1970s [8] . in response, rabies research efforts focused on development of oral rabies vaccines and vaccination strategies for wildlife (figure 1), with large-scale distribution of oral bait vaccines across western europe in the 1980s and 1990s [8] . over 25 years, oral vaccination of foxes has resulted in the elimination of the rabies virus from western europe, with rapid progress being made towards elimination in eastern europe [9] . over this same time period, canine rabies was being brought under control in north america, and research efforts independently became directed to the emerging problem of wildlife rabies focusing on control of rabies in terrestrial carnivore reservoirs [10] . a further concern in north america related to bat-transmitted rabies [11] , coinciding with a growing interest in bats as hosts of a wider range of lyssaviruses, [12] and other emerging pathogens of global concern, such as sars coronavirus, ebola virus and mers coronavirus. it is not surprising that set against this backdrop, research into the control of canine rabies in lmics received only limited attention during the latter part of the twentieth century (figure 1). however, this resulted in a deficit of data and understanding of the burden and scale of the disease in poorer parts of the world and limited interest in potential solutions, reinforcing a cycle of neglect [13] . it has always been known that dog bites are an important source of human rabies exposures worldwide, but reliable data have been lacking on the number of dog-mediated human rabies deaths [14] , with the few hundred deaths officially reported in the african region [15] widely recognized to be a major underestimate. an initial approach to estimating human rabies deaths in africa used a probability decision tree model that incorporated data on the incidence of bite injuries from suspected rabid dogs and availability of post-exposure prophylaxis (pep) [16] . this was first applied in tanzania and then used to generate country-and regional-level estimates of human deaths across africa and asia [17] [18] [19] [20] [21] and to assess the economic impacts of canine rabies [22] . further refinements resulted in more detailed and comprehensive estimates of global disease burden by country [4] . these studies indicated that more than 99% of canine-mediated rabies deaths occurred in africa and asia, with a global estimate of 59 000 (95% confidence interval (ci) 25 000-159 000) deaths in 2010 [4] . other approaches have been adopted by the global burden of disease (gbd) collaborators, including a cause of death ensemble modelling approach, which have generated estimates ranging from 54 100 deaths (95% ci 32 400-103 400) in 1990, 26 400 (95% in 2010, 23 500 (95% ci 17 300-28 600) in 2013, and 13 300 in 2016 (95% ci 7200-19 100) [3, 23, 24] . it is well recognized that these modelling approaches all have limitations, particularly in the degree of extrapolation from data that is of variable quality, from a limited geographical area or that has been generated indirectly [25] . for several neglected tropical diseases (ntds), gbd figures are thought likely to represent an underestimate of current disease burden [26] . for rabies, there is no evidence that control measures have been implemented on a scale that would explain the dramatic recent decline in deaths indicated by the gbd estimates [23, 24] . gbd estimates rely on vital registration and verbal autopsy data and these are very limited or absent in many of the countries where rabies and other ntds are most prevalent [24] . another critical issue is the appropriate modelling of pathways from infection to disease and death [26] . while the rabies probability tree study [4] was also limited by data quality and availability, this analysis incorporated detailed data from disease-specific research in rabies-endemic countries and was based on a well-defined series of steps from rabies exposure to death. we draw further the pep data used in the probability tree model also provided important information for demonstrating the economic burden of canine rabies, indicating that $1.7 billion direct costs were incurred annually in providing pep for 29 million dog-bite victims in canine-endemic countries [4] (figure 2). regionally, the highest expenditure is seen in asia ($1.4 billion annually) reflecting a continuing high demand for pep in areas where canine rabies has not been brought under control, and contrasting with latin america where, despite much lower annual expenditure on pep ($129 million), the region is on the brink of eliminating canine-mediated human rabies as a result of relatively modest investments in mass dog vaccination ($61 million) [4] . these data contribute to a growing body of evidence that the most cost-effective preventive strategies are those underpinned by mass dog vaccination rather than reliance on pep alone [28] [29] [30] . compiling data for the global burden study also highlighted pep availability as a major determinant of human rabies deaths, with cases occurring disproportionately in impoverished rural communities. detailed contact tracing studies reveal the extent to which people have struggled to obtain pep and the consequences of the resulting delays [31, 32] , which invariably include intense anxiety as bite victims await an uncertain outcome and, in some cases, the development of a horrifying and fatal disease. while human deaths and high pep costs dominate in burden of disease studies, several other components of disease burden are also of concern, including livestock losses, which still remain poorly quantified but can have important impacts [22, 33] and wildlife conservation, with canine rabies threatening several endangered wildlife populations including the ethiopian wolf (canis simensis) and african wild dog (lycaon pictus) [34] (figure 2). a considerable body of research now exists to demonstrate the feasibility of canine rabies elimination. the basic reproductive number, r 0 , a key parameter used to understand the effectiveness of control interventions, is usually measured from the growth rates of epidemics. applying this approach to canine rabies demonstrates that r 0 is typically between 1 and 2 in populations that differ in density by an order of magnitude [35] [36] [37] . alternative approaches to estimating transmission are all consistent with this low value of r 0 [29, 36, 38, 39] suggesting that rabies should be easily controlled through mass dog vaccination and, conversely, that approaches based on reducing dog density are likely to be ineffective [40] . theoretical and empirical research has demonstrated that rabies can be eliminated where 70% coverage is sustained [35, 36] . by contrast, attempts to reduce dog population density through indiscriminate culling have consistently failed to control rabies outbreaks [41] and, in some cases, have increased disease spread through humanmediated dog movements [37] . muzzling, restriction of dog movements, and selective removal or euthanasia of unowned dogs have historically been part of successful dog rabies control, including in the uk and usa [1, 42] , but these measures are distinguished from indiscriminate culling operations in being specifically targeted to reduce rabies transmission risk rather than to reduce dog population size or density. the question of rabies reservoir dynamics has long been debated [6, [43] [44] [45] [46] [47] , and is of major importance in sub-saharan africa where the abundance of wildlife has been seen as an obstacle for canine rabies control that would render elimination efforts futile [15] . however, despite the fact that rabies can infect all mammalian species, only a few hosts are capable of maintaining infection as reservoirs, with ecological and genetic factors both likely to be important determinants of rabies reservoirs [46, 47] . while rabies virus variants are typically maintained by only a single mammalian host species, multiple variants may circulate in an area [47] . however, this need not be an insurmountable obstacle to canine rabies elimination, as shown by countries in latin america and in the usa, where canine rabies has been brought under control or eliminated even though rabies variants circulate in wild mammal populations. the overlapping circulation of multiple variants does, however, introduce different surveillance requirements for verifying the elimination of the canine rabies variant. establishing the reservoir of multi host pathogens is not easy and typically requires integration of multiple lines of evidence [44, 48] . in the serengeti ecosystem, tanzania, inference from both epidemiological and genetic data supports the idea of rabies being maintained in domestic dogs not wildlife, with occasional spillover from domestic dogs into wildlife resulting in short-lived chains of infection that are not sustained [49] [50] [51] . the conclusion from these studies is that control of canine rabies should eliminate infection in dogs, wildlife and people. it is unclear the extent to which the serengeti scenario is generalizable more globally, but currently there is no clear evidence that, in areas with domestic dog reservoirs, the canine rabies virus variants circulating in dogs are maintained independently in wildlife. in south africa, a canine variant circulates in jackals in the limpopo region [52] , but it is still unknown whether this cycle will be sustained in the absence of canine rabies, which has now been well controlled in the area. if so, vaccination of jackals may be needed to achieve canine rabies elimination, but this is likely to be feasible given the demonstration of the safety, efficacy and feasibility of oral vaccination in jackals from earlier work in zimbabwe and israel [53] [54] [55] [56] . demonstration of the operational feasibility of mass dog vaccination provides a further cause for optimism. evidence now exists to show that, contrary to widely held perceptions, the vast majority of dogs in africa have owners, dog accessibility is higher than often recognized, and achieving target levels of vaccination coverage is feasible [57, 58] . in south and southeast asia, the situation may be more challenging as a result of a larger population of less accessible community or 'street' dogs, but target levels of vaccination coverage have also been achieved in these communities where campaigns are well organized [37, 59, 60] . in summary, the last decade has seen a rapid expansion of research into canine rabies vaccination and canine rabies elimination (figure 1) generating optimism that canine rabies can be effectively controlled, and ultimately eliminated, through mass dog vaccination and that this is the underpinning strategy needed to reach the 2030 target for elimination of human deaths from canine-mediated rabies [61] . the health and economic benefits would be substantial [22] (figure 2). while it is often recommended that a detailed understanding of dog ecology is needed for effective canine rabies control, the consistency of research findings generated over the past 30 years allows us to be confident in concluding that mass dog vaccination is feasible across a wide range of settings and campaigns can and should be initiated without delay. in some cases, more nuanced understanding may be required to improve coverage, but these insights can be often be gained through implementation of control measures and used to progressively improve the design and delivery of subsequent interventions. key considerations include the nature and degree of community engagement, timing of campaigns, placement of vaccination stations and whether or not to charge owner fees [62] [63] [64] . the costs of implementing campaigns free of charge may exceed those readily available to government veterinary services [65] , but many approaches can still be explored to improve affordability, acceptability and cost-effectiveness [66] . while there is widespread agreement about the central importance of mass dog vaccination in canine rabies control and elimination, the role of dog population management remains the subject of debate [67] . there is a rich literature around fertility control for management of roaming dog and wildlife populations [68, 69] . however, as rabies transmission varies little with dog density, reproductive control measures carried out with the aim of reducing dog density are not likely to be effective for rabies control. in theory, reducing population turnover (e.g. through improving life expectancy and/or reducing fecundity) could help sustain population immunity between campaigns and improve cost-effectiveness. however, there is little empirical evidence that dog population management tools have been able to achieve this [67] . furthermore, even in populations with a high turnover, achieving a 70% coverage during annual campaigns has been sufficient to sustain population immunity above critical thresholds determined by r 0 [70] . the relatively high cost of sterilization also means that strategies which combine vaccination and sterilization are less cost-effective in terms of achieving human health outcomes than strategies based on dog vaccination alone, even in populations with a large proportion of roaming dogs [39] . improved dog population management is undoubtedly a desirable longer-term goal for animal health and welfare and may have important secondary benefits for rabies control, for example by enhancing community or political support [67] . however, a focus on mass dog vaccination currently remains the most pragmatic and cost-effective approach to canine rabies control and elimination. the limited availability and quality of routine animal rabies surveillance data in lmics [14] has been an obstacle to the application of the analytical approaches from which we have learned so much about wildlife rabies. 'gold standard' surveillance data based on laboratory-confirmed diagnosis is hampered not only by limited laboratory infrastructure but also by the practical challenges of locating, sampling and submitting specimens [71] . however, pragmatic approaches to improving rabies rspb.royalsocietypublishing.org proc. r. soc. b 284: 20171880 surveillance have yielded rich insights. in addition to providing a foundation for burden of disease estimates, data on animalbite injuries have been a used as a reliable indicator of canine rabies incidence, revealing new understanding of rabies metapopulation dynamics [50] , as well as improving detection of animal rabies cases, the management of animal bites and the cost-effectiveness of pep [36, 72] . pragmatic solutions are also being found to improve rabies diagnosis in settings with limited laboratory infrastructure, including techniques to support decentralized laboratory testing (e.g. direct rapid immunohistochemical test, drit) [73] [74] [75] [76] and field diagnosis (e.g. immunochromatographic tests) [77] [78] [79] . these have great potential for empowering field staff to engage in rabies surveillance and respond more effectively to surveillance data, but standardization and quality control of field diagnostic kits still needs improvement [80] . given the rapid advances in metagenomic sequencing methods [81] , future approaches may include real-time genomic surveillance. however, even simple technologies such as mobile phones can serve as leapfrogging technology that can dramatically improve the extent and resolution of rabies surveillance data [82] . while operational research on dog vaccination provides grounds for optimism, awareness is growing about the challenges, complexities and time scales of moving from control to elimination (figure 3). given the low r 0 for rabies, deterministic models of transmission predict that rabies should be eliminated very rapidly [29, 30, [83] [84] [85] . but, these dynamic models typically assume that dog vaccination campaigns consistently achieve high and uniform levels of coverage. by contrast, analyses of rabies surveillance and control data indicate that vaccination coverage implemented during campaigns is often patchy and that time to rabies elimination is prolonged [37, 86, 87] . once assumptions about the implementation of vaccination campaigns are more realistic, and rabies is considered on a spatial landscape, predictions about the time scale to elimination are tempered [86] . the disparities between theory and practice demand approaches that capture realism. it may be argued that the feasibility and effectiveness of mass dog vaccination should have been self-evident given the successes in latin america but the road to elimination has been accompanied by substantial challenges [88] . progress in latin america has required decades of investment in large-scale dog vaccination programmes and builds on effective regional coordination. sustaining such coverage, particularly across large geographical areas, is difficult and requires an investment in rabies control that focuses on the dog population and is over and above levels seen to date in africa and asia [65] . local leadership is also an important factor. for example, canine rabies in north america was primarily controlled at the municipal level through dog licensure. legislation and by-laws relating to rabies control and dog vaccination exist in many canine rabies-endemic countries, but there is still a need for greater engagement of local authorities to ensure appropriate and sensitive enforcement of relevant legislation. empirical evidence from wildlife rabies elimination programmes show that once controlled to less than 10% of endemic incidence, the time required to eliminate rabies is as long again [9] , a lesson that should be heeded for canine rabies. once rabies has been reduced to low levels, the remaining foci by their nature are persistent and in 'hard-to-reach' communities, socially, economically and geographically, and new challenges come to the fore [89] . increasingly the importance of metapopulations has been recognized for the persistence of rabies [38, 50] and genomic signatures in rabies-endemic countries highlight the frequent human-mediated movement of dogs [90] [91] [92] . the implications of this movement are evident when rabies invades previously uninfected areas [37] , and without maintained vigilance, rabies can re-emerge rapidly if control measures are no longer implemented effectively [93] . the long-term implications of figure 3 . hypothetical timeline of rabies control and elimination highlighting policy targets and epidemiological milestones, illustrating relative rapid progress to zero human deaths but the need for sustained effort to reach elimination of canine rabies and sustained surveillance to identify the causes of cases. in this example, drawn from a scenario typical of latin america, human cases following declaration of zero human deaths from dog-mediated rabies might occur as a result of (i) an incursion of canine rabies (in which case the rabies-free status of the country would be reset); and (ii) vampire bat rabies and (iii) an imported human case (in which cases the status of the country as being free of dog-mediated rabies would not change). cases of canine rabies are shown in grey and human cases in red. rspb.royalsocietypublishing.org proc. r. soc. b 284: 20171880 these incursions to the persistence of rabies are not yet fully understood but will, undoubtedly prolong elimination efforts, and highlight the need for coordinated control at scale and across international boundaries as well as realistic projections of the investment required to eliminate rabies [94] . recent research has contributed pivotal evidence in making the case for rabies to be considered a priority ntd and, in 2012, rabies was included within the world health organization (who) accelerated roadmap for ntds [95] . in 2016, the tripartite partnership (who, the world organization for animal health (oie), and the food and agriculture organization of the united nations (fao), together with the global alliance for rabies control, declared a goal of zero human deaths from dog-mediated rabies by 2030 [61] , underpinned by an investment case incorporating data on the human health and economic burden of canine rabies [4, 22] . this purposely sets dog-mediated human rabies deaths as the first target, both because of its public health importance, but also its shorterterm feasibility (figure 3) through a combination of mass dog vaccination and improved pep provision to under-served communities. the longer-term goal of disrupting transmission and eliminating canine rabies will require more time. nonetheless, the example of latin america demonstrates that it is within reach [88] . the control and elimination of canine rabies provides an exemplar of 'one health' interventions, that is, interventions in animal populations that generate human health benefits. although challenges remain in the operationalization of one health [96] , these approaches not only provide the most cost-effective strategy for preventing human rabies deaths but also offer a more equitable approach than relying only on interventions directed at humans only (i.e. pep) [97] . interventions that effectively reduce the force of infection from the animal reservoir convey benefits to all without regard to socioeconomic status. by contrast, under a strategy of reliance on pep, the social, political and economic factors constraining access to healthcare are likely to prevail, with rabies deaths continuing to affect the most disadvantaged communities well beyond 2030. it is perhaps understandable that the medical sector emphasizes prevention of human rabies through pep. but this approach can lead to neglect of the problem at sourcein the dogs-and impede progress towards large-scale mass dog vaccination programmes. this is true even in 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global governance challenges one health contributions towards more effective and equitable approaches to health in low-and middle-income countries acknowledgements. the views expressed here have been developed through many years of collaborative research with colleagues working on rabies around the world, and with international organizations, including the world health organization, the world organization for animal health, the food and agriculture organization of the united nations and the global alliance for rabies control. for s.c., this includes over 25 years of interactions with colleagues at the institute of zoology, london, the london school of hygiene and tropical medicine, the university of edinburgh and the university of glasgow; and, for k.h., 15 years of interactions with colleagues at princeton university and the university of glasgow. we are very grateful to support from tanzanian institutions, particularly the tanzania wildlife research institute, tanzania national parks, ifakara health institute and sokoine university of agriculture as well as national and local governments. we thank many colleagues for support, stimulating research and debate, but particularly acknowledge chris dye, dan haydon, tiziana lembo, jonathan dushoff, magai kaare, felix lankester, rudovick kazwala, darryn knobel, michelle morters and louise taylor. key: cord-263811-w0983x19 authors: decaro, nicola; martella, vito; buonavoglia, canio title: canine adenoviruses and herpesvirus date: 2008-05-22 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2008.02.006 sha: doc_id: 263811 cord_uid: w0983x19 canine adenoviruses (cavs) and canine herpesvirus (chv) are pathogens of dogs that have been known for several decades. the two distinct types of cavs, type 1 and type 2, are responsible for infectious canine hepatitis and infectious tracheobronchitis, respectively. in the present article, the currently available literature on cavs and chv is reviewed, providing a meaningful update on the epidemiologic, pathogenetic, clinical, diagnostic, and prophylactic aspects of the infections caused by these important pathogens. hybridization [19] . they also exhibit different hemagglutination patterns and cell tropism. cav-1 recognizes the vascular endothelial cells and hepatic and renal parenchymal cells as targets for viral replication, whereas cav-2 replicates efficiently in the respiratory tract and, to a limited extent, in the intestinal epithelia [20] [21] [22] . infection by cavs has been described worldwide in several mammalian species. dogs, red foxes, wolves, and coyotes are highly susceptible to cav infection [3] . the overall prevalence of antibodies to cavs in european red foxes (vulpes vulpes) in australia was 23.2%, with marked geographic, seasonal, and age differences [23] , whereas the prevalence of antibody was 97% in island foxes (urocyon littoralis) in the channel islands, california [24] . antibodies to cavs were also detected in free-ranging terrestrial carnivores and marine mammals in alaska and canada, including black bears (ursus americanus), fishers (martes pennanti), polar bears (ursus maritimus), wolves (canis lupus), walruses (odobenus rosmarus), and steller sea lions (eumetopias jubatus) [25, 26] . recently, a fatal cav-1 infection has been reported in a eurasian river otter (lutra lutra) [27] . canine ich is a systemic disease described in canidae and ursidae. cav-1 replication in vascular endothelial cells and hepatocytes produces acute necrohemorrhagic hepatitis, and the disease is more severe in young animals [28, 29] . transmission occurs through animal-to-animal contact or indirectly through exposure to infectious saliva, feces, urine, or respiratory secretions. cav-1 is shed in urine up to 6 to 9 months after infection [30] . the incubation period in dogs is 4 to 6 days after ingestion of infectious material and 6 to 9 days after direct contact with infected dogs [31] . the mortality rate is 10% to 30% [32] . coinfections with canine coronavirus (ccov) [33, 34] , canine distemper virus (cdv) [34] [35] [36] [37] , or canine parvovirus [34] can exacerbate the disease, increasing the mortality rates. fever (>40 c) is the earliest clinical sign and displays a biphasic course. after the first febrile peak (1-2 days), some dogs recover from the infection. dogs displaying a second peak of hyperthermia frequently undergo a more severe form of ich. commonly observed symptoms are depression, loss of appetite, increased heart rate, hyperventilation, vomiting, and diarrhea. abdominal pain and distention can occur as a result of accumulation of serosanguineous or hemorrhagic fluid and enlargement of the liver. frequently, hemorrhagic diathesis is observed with epistaxis, congestion, or hemorrhage of the mucous membranes and skin. respiratory distress can also be observed as a consequence of laryngitis, tracheitis, and, less frequently, pneumonia. neurologic signs (hypersalivation, ataxia, and seizures) are rare in dogs and are associated with vascular damage in the central nervous system (cns) [28, 38] . corneal opacity (''blue eye''; fig. 1 ) and interstitial nephritis may occur 1 to 3 weeks after recovery because of deposition of immune complexes [39] [40] [41] . hematologic findings include leukopenia (<2000 cells/ll of blood; mainly attributable to a decrease in neutrophil count), increase in the serum transaminases (only in the severe forms of disease) [42] , and coagulation disorders associated with disseminated intravascular coagulation (dic; thrombocytopenia, altered platelet formation, and prolonged prothrombin time) [43] . proteinuria (albuminuria) can easily reach values greater than 50 mg/dl because of immunomediated glomerulonephritis [29] . at necropsy, the dogs that die during the acute phase of the disease often appear in good nutritional state. external examination can reveal ecchymoses and petechial hemorrhages, whereas the abdominal cavity contains abundant clear or serosanguineous fluid. the liver is enlarged, yellowish brown, congested, and spotted with small rounding areas of necrosis; the gallbladder appears thickened, edematous, and grayish or bluish white opaque in color (fig. 2) . edema of the gallbladder wall is a constant finding. congestion and hemorrhagic lesions are observed in the spleen, lymph nodes ( fig. 3) , thymus, pancreas, and kidneys. lungs show patchy areas of consolidation because of bronchopneumonia. hemorrhagic enteritis can also be observed (fig. 4) [3, 28] . histologic changes in the liver are characterized by centrolobular necrosis, along with neutrophilic and mononuclear cell infiltration and intranuclear inclusions in the kupffer's cells and hepatocytes. multifocal areas of congestion, hemorrhage, and leukocyte infiltration can be observed in several organs, mainly in the liver and kidneys, because of vascular damage and inflammation. interstitial nephritis and iridocyclitis with corneal edema are also present in dogs recovering from ich [44] . the route of infection by cav-2 is oronasal. respiratory signs are consistent with damage of bronchial epithelial cells. cav-2 infections rarely result in overt clinical signs, however, despite the presence of extensive lung lesions. clinical signs typical of itb are observed when cav-2 infection is complicated by other viral or bacterial pathogens of dogs, including canine parainfluenza 3 virus [45] , cdv [46] [47] [48] , bordetella bronchiseptica [49] , mycoplasmas [50, 51] , and streptococcus equi subsp. zooepidemicus [52] [53] [54] . in addition, other viruses with tropism for the respiratory tract have been recently identified and associated with itb-like forms in dogs, such as influenza a virus [54, 55] , a pantropic variant of ccov [56] , and the canine respiratory coronavirus (crcov) [57, 58] . chv and mammalian reoviruses have rarely been reported from dogs with itb and likely do not play a major role in the disease complex [59, 60] . itb (kennel cough) is an acute and highly contagious respiratory disease of dogs affecting the larynx, trachea, bronchi, and, occasionally, lower respiratory tract [61] . kennel cough is typically a complex of diseases caused by viral pathogens (eg, cavs, chv, canine parainfluenza virus, reoviruses) in association with bacteria, mainly b bronchiseptica and mycoplasma spp. most frequently, a dry hacking cough is observed as a consequence of an uncomplicated, self-limiting, and primarily viral infection of the trachea and bronchi. in complicated forms, which are more common in pups and immunocompromised dogs, secondary bacterial infections and involvement of pulmonary tissue overlap the viral infection. cough is usually associated with mucoid discharges. the condition may progress to bronchopneumonia and, in the most severe instances, death [61] . usually, cns involvement is not seen, although death in pups with neurologic disease associated with cav-2 infection has been reported [62] . at postmortem examination, red areas of consolidation can be observed in the lungs, especially in the complicated forms. histologically, necrotizing bronchitis and bronchiolitis obliterans may be observed. infection of type 2 alveolar cells is associated with interstitial pneumonia and the presence of viral inclusion bodies in their nuclei [63] [64] [65] [66] [67] [68] . hematologic findings (eg, leukopenia, prolonged blood clotting, increased activities of alanine aminotransferase [alt] and aspartate aminotransferase [ast]) may be indicative of cav-1 infection, although the increase of transaminases is commonly observed only in severely affected or moribund dogs. postmortem findings and histopathologic changes are highly suggestive of cav-1 infection. confirmation of a diagnosis of ich is obtained by virus isolation on permissive cell lines, such as madin darby canine kidney (mdck) cells. a polymerase chain reaction (pcr) protocol has recently been developed for molecular diagnosis [69] . ocular swabs, feces, and urine can be collected in vivo for virus isolation and pcr. postmortem samples can be withdrawn from the kidney, lung, and lymphoid tissues. the liver is rich in arginase, which inhibits viral growth in cell cultures [70] , but it represents the most important organ for histopathologic examination [28, 29] . viral growth in cells is revealed by rounding cells that form clusters and detach from the monolayers [34] . immunofluorescence (if) can detect viral antigens in infected cell cultures and in acetone-fixed tissue sections or smears. viral replication can also be demonstrated by detection of nuclear inclusion bodies in the cells after hematoxylin-eosin staining. neither virus isolation nor if is able to distinguish between the two adenovirus types. because cav-2 can also be detected in the internal organs and feces of vaccinated or acutely infected dogs [46] and cav-1 is also frequently isolated from respiratory secretions, trachea, and lungs, distinction between cav-1 and cav-2 necessarily deserves laboratory examination. restriction fragment length polymorphism analysis on viral genomes using the endonucleases psti and hpaii generates differential patterns [17, 18] . detection and differentiation of cav-1 and cav-2 by pcr with a single primer pair are also possible [69] . although cavs agglutinate erythrocytes of several species, hemagglutination is not used in routine diagnosis [71] . because most dogs are vaccinated and cav-2 infection is frequent in dogs, serology has low diagnostic relevance [21, 39] . treatment of ich is primarily symptomatic and supportive. dehydration and dic require administration of fluids, plasma, or whole-blood transfusions and anticoagulants. hyperammonemia attributable to hepatic and renal damages can be corrected by oral administration of nonabsorbable antibiotics and lactulose and by oral or parenteral administration of potassium and urinary acidificants (ascorbic acid). supportive therapy may facilitate the clinical recovery of infected dogs, provided that there is time for hepatocellular regeneration [29] . uncomplicated forms of cav-2-associated itb can be treated with glucocorticoids, antitussives, and bronchodilators as cough suppressants. aerosol therapy can be effective in dogs displaying excessive accumulation of tracheal and bronchial secretions. antimicrobial therapy is recommended in the complicated forms and when the lower respiratory tract seems to be involved [29] . use of vaccines has greatly reduced the burden of ich in canine populations. initial attempts were made with cav-1 inactivated vaccines, which require repeated inoculations [72] . cav-1-based modified-live virus (mlv) vaccines proved to be highly effective but were associated with interstitial nephritis and corneal opacity [22] . administration of cav-1 in conjunction with cdv vaccines was also associated with postvaccinal encephalitis [73] . because cav-1 and cav-2 are able to confer cross-protection, the current vaccines contain mlv cav-2, which is not able to induce renal or ocular damage. the cav-2 attenuated strain toronto a26/61 is contained in most vaccine formulations [22, 74] . in the absence of maternally derived antibodies (mdas), a single dose administered subcutaneously or intramuscularly is protective against ich and itb. because of the possible interference of mdas, however, the vaccination schedule requires administration of at least two vaccine doses at a 3-to 4-week interval, starting when pups are 8 to 10 weeks old. intranasal administration of an mlv cav-2 vaccine has been proposed to overcome mda interference, but it may be associated with the onset of mild respiratory disease [29] . vaccination is usually repeated yearly, although after administration of two doses of cav-2 vaccine, immunity seems to persist for more than 3 years [75, 76] . although extensive vaccination has greatly reduced the incidence of cav infections, re-emergence of ich has been described in italy, likely as the result of parallel trading of pups with uncertain sanitary status from eastern european countries [34] . at the moment, there are few data on the molecular epidemiology of cavs, but it is commonly accepted that vaccine breaks occur rarely with cav vaccines, because the viruses are genetically stable. accordingly, cav infection in vaccinated dogs has been associated with mda interference in the early life of the pups rather than with emergence of variants genetically distant from the prototype strains contained in cav-2 vaccines [34] . cause chv was first described in the mid-1960s as the causative agent of a fatal septicemic disease of puppies [77] . chv is included in the alphaherpesvirinae subfamily, herpesviridae family [78] . the virus is sensitive to lipid solvents, is readily inactivated at temperatures greater than 40 c, and is rapidly inactivated by common disinfectants. chv seems to be a monotypic virus, as defined by antigenic comparison of various isolates [77, 79] . the genome structure of chv resembles that of other members of the alphaherpesvirus subfamily [80] [81] [82] [83] . southern blot hybridization and sequence analysis of various genes have shown a close genetic relatedness to feline herpesvirus (fhv-1), to phocid herpesvirus 1, and to the equid herpesviruses 1 and 4 [84] [85] [86] . the host range of chv is restricted to dogs [87] . antibodies to chv have been detected in sera of european red foxes (v vulpes) in australia [23] and germany [88] , however, and in sera of north american river otters (lontra canadensis) from new york [89] , whereas a chv-like virus has been isolated from captive coyote pups [90] . the virus seems to be present worldwide in domestic and wild dogs. serologic surveys have shown a relatively high prevalence of chv in household and colony-bred dogs. the prevalence of antibodies in dogs was 88% in england, 45.8% in belgium, and 39.3% in the netherlands [91] [92] [93] . serologic studies in italy have revealed a high prevalence in kenneled dogs (27.9%), whereas the prevalence was lower in pets (3.1%) [94] . in the united states, fulton and colleagues [95] studied the prevalence of antibodies against chv in washington and found only a 6% seroprevalence. transmission occurs by direct contact with oronasal or genital secretions, because chv is quickly inactivated in the environment. the age of the pups at the time of infection is critical for the outcome of the disease. infection of susceptible puppies at 1 to 2 weeks of age may be associated with fatal generalized necrotizing and hemorrhagic disease, whereas infection of pups older than 2 weeks of age and adult dogs is often asymptomatic [77] . infection in older dogs seems to be restricted to the upper respiratory tract [96] . also, chv has been identified in corneal swabs of adult dogs with corneal ulcerations [97] . transplacental transmission of chv and fetal death may also occur [98] , and chv infection is suspected in dogs with fertility disorders. the high susceptibility of newborn pups to fatal acute chv-induced disease is likely related to the fact that pups have low and poorly regulated body temperature and chv growth is optimal at lower than normal body temperature [99] . neonatal mortality chv infection is generally fatal in neonatal pups lacking maternally derived immunity. death of 1-to 4-week-old pups is most common. neonatal pups may be infected during passage through the birth canal or by contact with oronasal secretions of other dogs. the duration of illness in newborn pups is 1 to 3 days. signs include vocalization, anorexia, dyspnea, abdominal pain, incoordination, and soft feces, whereas the rectal temperature is not elevated and may be low. serous or hemorrhagic nasal discharge and petechial hemorrhage on the mucous membranes may also be observed. in pups less than 1 week of age at the time of infection, chv replicates in the nasal mucosa, pharynx, and tonsils before spreading by means of the blood (in macrophages) to the liver, kidneys, lymphatic tissues, lungs, and cns. the incubation period is approximately 6 to 10 days. death in affected litters usually occurs over a period of a few days to a week. litter mortality can reach a peak of 100%. in pups older than 2 to 3 weeks of age at the time of infection, chv infection is generally asymptomatic, although cns signs, including blindness and deafness, have been described [100] . reproductive disorders chv can cause occasional in utero infections that result in death of the fetus or pup shortly after birth [77, 98] . pregnant dogs infected at midgestation or later may abort weak or stillborn pups. pups may seem normal at parturition but die within a few days of birth. the infected dams develop protective immunity, and chv-related diseases are not observed in subsequent litters because maternally derived immunity protects the pups during the first week of life when they are most susceptible. primary genital infections in susceptible adult animals may be associated with lymphofollicular lesions and vaginal hyperemia (fig. 5) . male animals may have similar lesions over the base of the penis and the prepuce. respiratory disease chv has been detected in dogs with itb [101] , but its role remains controversial. experimental infection has been shown to cause mild clinical symptoms of rhinitis and pharyngitis [96] or tracheobronchitis [102] . experimental infection by the intravenous route in adult foxes resulted in fever, lethargy, and respiratory signs, although peroral infection did not [103] . a long-term survey in a population of dogs in a shelter has demonstrated chv in 9.6% of lung and 12.8% of tracheal samples. chv infections occurred later than other viral infections. chv was detected more frequently at weeks 3 and 4 after a dog's introduction in the kennel, whereas crcov and canine parainfluenza were detected more frequently within the first and second weeks, respectively. interestingly, chv infection was apparently related to more severe respiratory signs [53] . in a 1-year study in training centers for working dogs, however, seroconversion to chv seemed to be more frequent in dogs infected with crcov [104] , suggesting virus reactivation after disease-induced stress. after symptomatic and asymptomatic infections, dogs remain latently infected and virus may be excreted at unpredictable intervals over periods of several months or years. reactivation of latent virus may be provoked by environmental or social stress or, experimentally, by immunosuppressive drugs (corticosteroids) or antilymphocyte serum. latent virus persists in the trigeminal ganglia and other sites, such as lumbosacral ganglia, tonsils, and parotid salivary glands [3, [105] [106] [107] . latently infected dogs represent a source of infection for susceptible animals, and this is of particular concern in breeding dogs that can ensure chv transmission through genital secretions. multifocal areas of necrosis and hemorrhage may be observed in most organs, including the lungs, liver, brain, and intestine, with the kidneys being the most classic organ affected. circumscribed areas of hemorrhage and necrosis on a pale gray cortex give the organs a spotted appearance (fig. 6) . lymph nodes and spleens appear enlarged. meningoencephalitis also is common. necrosis in the placenta is observed in infected pregnant animals. fetal lesions are similar to those seen in affected puppies. diagnosis of chv infection may be achieved by isolation of the virus on permissive cell lines. the virus can be adapted for growth on canine primary or secondary kidney or testicular cells and in canine cell lines. growth is optimal at 34 c to 35 c, with diminished virus yields at temperatures higher than 36 c. in cell cultures, virus growth is revealed by formation of typical clusters of rounded cells that tend to detach, and for certain isolates, by formation of syncytia with type a intranuclear inclusions. pcr assays are available, significantly increasing diagnostic reliability and sensitivity [107] . serologic screenings to evaluate the neutralizing antibodies may be useful to investigate the presence of chv in kennels. because chv growth is optimal at temperatures lower than 36 c [99] , attempts were made to influence the evolution of chv-induced disease in experimentally infected pups. experimentally infected newborn pups reared at elevated temperatures that raised their body temperature to 38.5 c to 39.5 c survived chv infection but presented with permanent neurologic damage [108] . likewise, residual neurologic damage may be observed in infected dogs treated with antiviral drugs, such as vidarabine. accordingly, neither artificial temperature nor vidarabine may be applied for the therapy of chv. an inactivated subunit vaccine is available commercially in europe. the vaccine should be administered to bitches during heat or the initial stages of pregnancy and again at the sixth to seventh week of gestation. a temperature-resistant mutant of chv attenuated through serial cell passages has been proposed as an mlv vaccine [109] , but its safety and efficacy have not been evaluated and such a vaccine is not available commercially. cav infections have been satisfactorily controlled in the past decades as a consequence of the vaccination programs adopted in all developed countries. nevertheless, there are some concerns about the possible introduction of infected dogs from areas of uncertain epidemiologic conditions, in which both cav types are widespread as a result of the lack of systematic canine immunization [34] . cav vaccines have been proved to be safe and effective for prevention of ich and itb, conferring protection against more recent cav strains, albeit prepared with old cav-2 strains [28, 110] . conversely, chv is still circulating in canine populations worldwide, mainly in shelters and breeding kennels. active immunization is recommended in pregnant bitches to prevent fatal infections in newborn pups [111] . when the mdas decrease, however, pups born to vaccinated bitches become susceptible and, along with unvaccinated dogs, maintain chv infection. it is unclear whether vaccination prevents chv infection and virus shedding through secretions. in addition, control of the infection is hindered by the fact that chv is often associated with asymptomatic infections, and the real prevalence of chv infection is likely underestimated [87] . the intensification of surveillance activity using new diagnostic techniques and molecular analysis tools may help to investigate the epidemiology of cav and chv infections more thoroughly and to plan adequate measures of control. epizootic fox encephalitis. i. general description a comparison of certain intranuclear inclusions found in the livers of dogs without history of infection with intranuclear inclusions characteristic of the action of filtrable viruses 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reactivation in bitches with a medical history of herpesvirus infection detection of canine herpesvirus 1 in a wide range of tissues using the polymerase chain reaction temperature as a factor of resistance of young puppies to canine herpesvirus small-plaque variant of canine of canine herpesvirus with reduced pathogenicity for newborn pups guidelines for the vaccination of dogs and cats. compiled by the vaccination guidelines group (vgg) of the world small animal veterinary association (wsava) protection of puppies against canine herpesvirus by vaccination of the dams key: cord-002122-s2r0en6f authors: toom, marjolein lisette den; dobak, tetyda paulina; broens, els marion; valtolina, chiara title: interstitial pneumonia and pulmonary hypertension associated with suspected ehrlichiosis in a dog date: 2016-07-07 journal: acta vet scand doi: 10.1186/s13028-016-0228-1 sha: doc_id: 2122 cord_uid: s2r0en6f background: in dogs with canine monocytic ehrlichiosis (cme), respiratory signs are uncommon and clinical and radiographic signs of interstitial pneumonia are poorly described. however, in human monocytic ehrlichiosis, respiratory signs are common and signs of interstitial pneumonia are well known. pulmonary hypertension (ph) is classified based on the underlying disease and its treatment is aimed at reducing the clinical signs and, if possible, addressing the primary disease process. ph is often irreversible, but can be reversible if it is secondary to a treatable underlying etiology. cme is currently not generally recognized as one of the possible diseases leading to interstitial pneumonia and secondary ph in dogs. only one case of ph associated with cme has been reported worldwide. case presentation: a seven-year-old, male intact, mixed breed dog was presented with 2 weeks history of lethargy and dyspnea. the dog previously lived in the cape verdean islands. physical examination showed signs of right-sided congestive heart failure and poor peripheral perfusion. thoracic radiography showed moderate right-sided cardiomegaly with dilation of the main pulmonary artery and a mild diffuse interstitial lung pattern with peribronchial cuffing. echocardiography showed severe pulmonary hypertension with an estimated pressure gradient of 136 mm hg. on arterial blood gas analysis, severe hypoxemia was found and complete blood count revealed moderate regenerative anemia and severe thrombocytopenia. a severe gamma hyperglobulinemia was also documented. serology for ehrlichia canis was highly positive. treatment with oxygen supplementation, a typed packed red blood cell transfusion and medical therapy with doxycycline, pimobendan and sildenafil was initiated and the dog improved clinically. approximately 2 weeks later, there was complete resolution of all clinical signs and marked improvement of the ph. conclusion: this report illustrates that cme might be associated with significant pulmonary disease and should be considered as a possible differential diagnosis in dogs presenting with dyspnea and secondary pulmonary hypertension, especially in dogs that have been in endemic areas. this is important because cme is a treatable disease and its secondary lung and cardiac manifestations may be completely reversible. ehrlichia canis is a pleomorphic bacterium that infects circulating monocytes and can cause canine monocytic ehrlichiosis (cme). cme results in variable nonspecific clinical manifestations and clinical signs can be subclinical, acute or chronic. most dogs present with depression, lethargy, mild weight loss, anorexia, splenomegaly, and lymphadenopathy with or without hemorrhagic tendencies [1, 2] . respiratory signs are sporadically reported in dogs but are regularly described in human patients infected with human monocytic ehrlichiosis (hme) [3] . interstitial pneumonia can have an infectious or noninfectious etiology. in dogs, reported infectious agents leading to interstitial pneumonia are angiostrongylus acta veterinaria scandinavica *correspondence: m.l.dentoom@uu.nl 1 department of clinical sciences of companion animals, faculty of veterinary medicine, utrecht university, yalelaan 108, 3508 td utrecht, the netherlands full list of author information is available at the end of the article vasorum, leishmania chagasi, toxoplasma gondii, pneumocystis carinii, babesia canis, leptospira sp., mycoplasma sp, canine distemper virus and adenovirus [4] [5] [6] [7] [8] [9] [10] [11] [12] . in patients with interstitial pneumonia, gas exchange is often impaired due to ventilation-perfusion mismatching, intrapulmonary shunting, and decreased diffusion across the abnormal interstitium with arterial hypoxia as a consequence. in contrast to the systemic vasculature that responds with arterial vasodilation to better perfuse hypoxic tissue, the pulmonary vasculature constricts in response to hypoxia. besides pulmonary vasoconstriction, hypoxia also causes proliferation of the smooth muscle cells in the arterial wall. both phenomena lead to a decrease in luminal cross-sectional area and an increase in pulmonary vascular resistance index with pulmonary hypertension (ph) as a consequence. pulmonary hypertension is classified based on the underlying disease and its treatment is aimed at improving the clinical signs and addressing the primary disease process [13] . although ph is often irreversible, ph is reversible in some cases if the underlying etiology is diagnosed and treated accordingly. reversibility of ph has for instance been demonstrated in dogs after successful treatment for a. vasorum [14] . pulmonary changes consistent with interstitial pneumonia have been reported previously in humans with hme [3] and as an atypical finding in dogs with cme [15] [16] [17] [18] . however, cme is generally not recognized as one of the possible diseases leading to interstitial pneumonia and secondary ph in dogs. only one case of ph associated with e. canis infection has been reported worldwide [19] . consequently, cme might be underdiagnosed as a possible cause of interstitial pneumonia and secondary ph. this case report describes the clinical, radiographic and echocardiographic presentation of a dog with interstitial pneumonia and severe ph suspected to be associated with e. canis infection. a seven-year-old, intact male, mixed breed dog weighing 8.1 kg was presented to the emergency service of the department of clinical science of companion animals of the faculty of veterinary medicine, utrecht university with a 2 weeks history of lethargy, progressive dyspnea and coughing. the dog previously lived in the cape verdean islands for approximately 3 years and returned to the netherlands 10 months before presentation. in the past 2 years, the dog had showed chronic mild exercise intolerance and had a few episodes of diarrhea that resolved with symptomatic therapy. the dog was up-to date with his vaccinations and anthelminthic treatments. physical examination showed generalized weakness and decrease mental state. cardiovascular examination revealed tachycardia, weak peripheral pulses, pale mucous membranes, prolonged capillary refill time, jugular distensions and venous pulses, and a grade three out of six systolic murmur with the point of maximal intensity over the right cardiac apex. the dog was also severely dyspneic and demonstrated harsh lung sounds on auscultation. the abdomen was distended and positive undulation was detected. these findings were consistent with pulmonary disease, right-sided heart failure and poor peripheral perfusion. complete blood count (cbc) showed a moderate microcytic, hypochromic anemia, moderate leukocytosis with a marked left shift and a severe thrombocytopenia. biochemistry showed severe hyperproteinemia, hyperglobulinemia and a mild hypoalbuminemia. serum protein electrophoresis showed a polyclonal peak in the gamma globulin region. arterial blood gas analysis showed a severe hypoxemia with hypocapnia. urinalysis showed mild hemoglobinuria, glucosuria and proteinuria. blood samples were submitted for serological and molecular biological testing. immunofluorescence antibody test (ifat) for e. canis (megafluo ehrlichia canis ® , mega cor diagnostik gmbh, hörbranz, austria) was positive (igg titer >2560), but polymerase chain reaction (pcr) amplification for ehrlichia genus (realtime pcr, light cycler ® 2.0, roche diagnostics gmbh, mannheim, germany, primers used as described previously [20] ) was negative. serology for leishmania sp. (dog-dat ® , leishmania specific antibody detection kit, koninklijk instituut voor de tropen, amsterdam, the netherlands) and b. canis. (megafluo babesis canis ® , mega cor diagnostik gmbh, hörbranz, austria) and antigen snap tests for a. vasorum (angio detect ™ test, idexx laboratories) and dirofilaria immitis (snap ® heartworm rt test, idexx laboratories) were also negative. laboratory results are summarized in table 1 . on thoracic radiographs, a mild diffuse increase in pulmonary opacity with an interstitial lung pattern and mild peribronchial cuffing was seen, which was most accentuated in the caudodorsal lung lobes. thin pleural fissure lines were noted between all lung lobes. the cardiac silhouette showed signs of right-sided cardiomegaly (vertebral heart score (vhs): 11.0, reference interval <9.7 ± 0.5) and a main pulmonary artery knuckle was present on the dorsoventral view ( fig. 1) . echocardiography showed dilation of the right ventricle and the pulmonary artery, septal flattening and a severe tricuspid regurgitation. the left ventricle was severely under filled (fig. 2) . congenital defects and left heart disease were excluded. the maximal tricuspid systolic velocity was 5.83 m/s, indicating a peak tricuspid gradient of approximately 136 mm hg which is graded as severe ph (reference <40 mm hg, severe >75 mm hg) [13] , (fig. 3) . to address the severe hypoxemia and ph the dog was placed in an oxygen cage with an inspired concentration of oxygen between 40 and 50 %. the clinical signs of the dog did not improve markedly with the extra oxygen supplementation. because anemia could have contributed to the cardiovascular signs and the poor tissue oxygenation, a typed packed red blood cell transfusion was administered. based on the travel history, the abnormalities within the cbc and biochemical analysis and the positive serology for e. canis, an infection with cme was suspected. treatment with doxycycline (5 mg/ kg, orally twice daily) (doxoral ® , ast farma, oudewater, the netherlands), pimobendan (0.3 mg/kg, orally twice daily) (cardisure ® flavour, eurovet animal health bv, bladel, the netherlands) and sildenafil (1.5 mg/kg, orally twice daily) (viagra ® , pfizer, new york, usa) was initiated. the hematocrit increased from 20 to 36 % after the blood transfusion and the dog's clinical condition improved remarkably. however, the dog remained moderately dyspneic and severely hypoxic (pao 2 : 46.5 mm hg, reference interval: 85.0-103.3 mm hg). the dyspnea gradually improved and the dog seemed comfortable outside the oxygen cage after 6 days of treatment, although the improvement of the hypoxemia was only minimal (pao 2 : 55 mm hg, reference interval: 85-103.3 mm hg, table 1 ). because of financial limitations of the owner, the dog was discharged with the above-mentioned therapies at that time. seventeen days after initiation of treatment, the dog was admitted via the cardiology polyclinics of the same university and re-examined. at that time, there was complete resolution of all clinical signs and physical examination was completely unremarkable. cbc showed normal platelet counts and leukogram, with only a very mild microcytic hypochromic anemia. biochemical analysis again showed severe hyperproteinemia and hyperglobulinemia. the 1) . thoracic radiography showed marked improvement with resolution of cardiomegaly (vhs: 10.2, reference interval 9.7 ± 0.5) and only a very mild interstitial pattern of the caudodorsal lung lobes and a very mild dilation of the pulmonary artery (fig. 4) as remaining abnormalities. echocardiography also showed a remarkable improvement. tricuspid regurgitation was no longer present and only a very mild uniform dilation of the pulmonary artery was still present (fig. 5) . therapy with pimobendan and sildenafil were discontinued and the treatment with doxycycline was continued for another 5 weeks. the dog was reevaluated 4 weeks later and was clinically doing very well. nevertheless, the dog again developed mild anemia and the hyperproteinemia persisted. proteinuria had resolved at that time (table 1) . doxycycline therapy was continued for another 4 weeks and another re-examination was advised. unfortunately, the dog was lost to follow-up at that time. interstitial pneumonia with secondary ph was suspected in this dog based on the presentation on thoracic radiography and the severe hypoxemia on arterial blood gas, but the etiology was initially unclear. however, cme was suspected based on the travel history of the dog, the results of cbc, biochemical analysis and a positive serology result for e. canis. exposure to e. canis was demonstrated with the ifat for anti-e. canis igg antibodies. igg titers >40 are considered positive for e. canis exposure [21] . in right lateral (a) and dorsoventral (b) thoracic radiographs 2 weeks after discharge. radiographs demonstrating resolution of cardiomegaly (vertebral heart score: 10.2, reference interval <9.7 ± 0.5) and reduction of the dilation of pulmonary arteries and the diffuse interstitial pattern this case, the igg titer was high (>2560), which strengthened the suspicion of an active infection [22] . however, definite active infection at the time of presentation could not be proven, because pcr amplification for ehrlichia genus was negative and anti-ehrlichial igg antibodies persist for several months to years after elimination of the parasite [23] . a negative result from a pcr test can occur when organisms in circulation are below the level of detection, as may happen when infections are chronic. it has been demonstrated that dogs can be pcr negative on blood samples, but pcr positive on splenic aspirates [21] . it is hypothesized that the e. canis are sequestered in splenic macrophages to avoid immune elimination. unfortunately, in the present case, the owners declined fine needle aspirates of the spleen due to the potential risks and stress involved, e.g. internal bleeding, aggravation of the dyspnea. paired serology samples can also provide useful information about antibody kinetics, which may point to current status of infection. a fourfold increase in igg antibodies over time is suggested to be evidence for an active infection [24] . antibody titers will decrease gradually after appropriate treatment, but may persist for months to years even after full clinical recovery [23, 25] . in this case, the ehrlichia igg titer was still very high 17 days after initiation of doxycycline therapy, probably due to the short time between start of the treatment and retesting; ifat was unfortunately not repeated on day 50. although doxycycline therapy generally results in fast clinical improvement and improvement of most laboratory abnormalities, persistence of hyperglobulinemia is generally observed for a longer period. most studies have shown normalization of serum protein electrophoresis results after 3-9 months of therapy [1, 15] . this explains the persistence of hyperglobulinemia at the re-examinations of the dog at days 17 and 50. in human medicine, respiratory signs are commonly described as a consequence of hme infection [3] and acute respiratory distress syndrome (ards) has also been reported as a severe, although uncommon, finding in hme [26] [27] [28] . both cme and hme animal models also revealed prominent mononuclear cellular infiltration in the interalveolar septa, endothelial damage and vasculitis in the lungs [16, 29] . this could explain the pulmonary changes found in this dog and the clinical and radiographic improvement after treatment with doxycycline. however, recently, acute resolution of patchy pulmonary alveolar infiltrates has been described after sildenafil therapy in dogs with idiopathic ph and ph secondary to idiopathic lung fibrosis [30] . therefore, sildenafil therapy might also have contributed to the improvement of the radiographic changes in this case. another explanation for the radiographic abnormalities and the improvement on doxycycline therapy that cannot be excluded is that the dog suffered from a bacterial pneumonia. furthermore, an important possible factor that might have contributed to the hypoxemia and ph in this dog is pulmonary thromboembolism (pte) [31] . pte can occur as complicating sequelae in patients with ph or can be the primary cause of ph [32] . in people, it is increasingly recognized that patients with pulmonary arterial hypertension have dysregulated coagulation and antithrombotic homeostasis, which may contribute to a prothrombotic state [33] . unfortunately, we did not perform diagnostic investigations such as d-dimer concentration [34] , computed tomography pulmonary angiography [35] or thromboelastography (teg) [36, 37] to investigate if the dog suffered from pte or a prothrombotic state. however, to the author's knowledge, pte has never been associated with cme in veterinary literature. only two cases of aortic and portal vein thrombosis have been described in dogs with cme [38, 39] . treatment of ph is aimed at eliminating or improving the underlying disease process. if the ph is not controlled by primary disease therapy or if the ph is idiopathic, treatment with pulmonary arterial dilators may be implemented. in veterinary medicine currently, only phosphodiesterase inhibitors are used. sildenafil is a highly selective phosphodiesterase five inhibitor that has been used in veterinary medicine with encouraging results [30, 40] . pimobendan, a calcium-sensitizing agent with phosphodiesterase three inhibiting actions has also been used, especially when left heart disease is a contributing cause [41] . in the present case, a dual therapy with pulmonary arterial dilators was initiated, because we hoped this would ameliorate the very severe ph and clinical signs faster than a monotherapy with sildenafil. although recovery has been described in a similar case with just a monotherapy with doxycycline [19] , we believe that the symptomatic support with pulmonary vasodilators was justified in this dog. mild to moderate improvement of tricuspid regurgitation gradient by sildenafil therapy has been described in some dogs with ph [30] , but not in others [42] . consequently, we cannot rule out that the improvement of the echocardiographic changes could also be partially explained by the use of the vasodilating drugs. ideally, another echocardiogram should have been performed after discontinuation of the vasodilator therapy. however, the improvement was so dramatic, that we do not believe that this could be solely explained by the vasodilator therapy. this case report illustrates that cme might be associated with significant pulmonary disease and that it should be considered as a possible differential diagnosis in dogs presenting with dyspnea and secondary pulmonary hypertension, especially in dogs that have been in endemic areas. this is important because cme is a treatable disease and its lung and cardiac manifestations may be completely reversible. guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in europe ehrlichiosis and anaplasmosis in dogs and 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treatment of pulmonary hypertension secondary to degenerative mitral valve disease in dogs sildenafil citrate therapy in 22 dogs with pulmonary hypertension cv performed the initial clinical investigations of the dog and was responsible for the care and treatment of the dog during admittance at the intensive care unit. eb was responsible for the serological and molecular biological diagnostic testing. mdt also performed clinical investigations of the dog and performed and evaluated the echocardiograms of the dog during hospitalization and at follow-up. td performed and evaluated the initial echocardiogram of the dog at presentation and was responsible for the interpretation of the thoracic radiography. the manuscript was drafted by mdt and finalized jointly by all authors. all authors read and approved the final manuscript. the authors declare that they have no competing interests. submit your next manuscript to biomed central and we will help you at every step: key: cord-316746-toen5nvr authors: alves, f.; prata, s.; nunes, t.; gomes, j.; aguiar, s.; aires da silva, f.; tavares, l.; almeida, v.; gil, s. title: canine parvovirus: a predicting canine model for sepsis date: 2020-06-15 journal: bmc vet res doi: 10.1186/s12917-020-02417-0 sha: doc_id: 316746 cord_uid: toen5nvr background: sepsis is a severe condition associated with high prevalence and mortality rates. parvovirus enteritis is a predisposing factor for sepsis, as it promotes intestinal bacterial translocation and severe immunosuppression. this makes dogs infected by parvovirus a suitable study population as far as sepsis is concerned. the main objective of the present study was to evaluate the differences between two sets of sirs (systemic inflammatory response syndrome) criteria in outcome prediction: sirs 1991 and sirs 2001. the possibility of stratifying and classifying septic dogs was assessed using a proposed animal adapted piro (predisposition, infection, response and organ dysfunction) scoring system. results: the 72 dogs enrolled in this study were scored for each of the piro elements, except for infection, as all were considered to have the same infection score, and subjected to two sets of sirs criteria, in order to measure their correlation with the outcome. concerning sirs criteria, it was found that the proposed alterations on sirs 2001 (capillary refill time or mucous membrane colour alteration) were significantly associated with the outcome (or = 4.09, p < 0.05), contrasting with the 1991 sirs criteria (p = 0.352) that did not correlate with the outcome. no significant statistical association was found between predisposition (p = 1), response (p = 0.1135), organ dysfunction (p = 0.1135), total piro score (p = 0.093) and outcome. to explore the possibility of using the sirs criteria as a fast decision-making tool, a fast-and-frugal tree (fft) was created with a sensitivity of 92% and a specificity of 29%. conclusion: these results suggest that increasing the sirs criteria specificity may improve their prognostic value and their clinical usefulness. in order to improve the proposed piro scoring system outcome prediction ability, more specific criteria should be added, mainly inflammatory and organ dysfunction biomarkers. according to the most recent scientific consensus, the term sepsis should be used to describe the organ dysfunction triggered by a deleterious inflammatory host response to infection [1] . after the release of bacteria into the bloodstream as a result of an infection, sepsis only takes place if the host immune system is overpowered resulting in clinically significant bacteraemia [2] . with the goal to establish the correlation between the systemic inflammatory response and sepsis a conference was held in 1991 in which the criteria to assess if a systemic inflammatory response syndrome (sirs) is taking place were defined [3] . since the first adaptation of these criteria to animals [4] they have been subjected to a series of modifications and the cut off values slightly vary among investigations. to increase the sensitivity (se) and specificity (sp) of these parameters, it is important to use them in association with the clinical judgement when screening animals for sepsis, as they are not sufficiently accurate to establish a definitive diagnosis [5] . for a dog to be diagnosed with sirs at least two of the following four criteria need to be met: body temperature < 37.8°c or > 39.4°c, hr > 140 bpm, rr > 30 breaths/min or pco2 < 32 mmhg (venous or arterial), wbc < 6000 or > 16,000 cells/μl, or > 3% band neutrophils. cats need to meet three of the four criteria for sirs to be diagnosed: body temperature < 37.8°c or > 39.7°c, hr < 140 or > 225 bpm, rr > 40 breaths/min, wbc > 19,500 or < 5000 cells/μl, or > 5% band neutrophils [2] . when the circulatory and cellular/metabolic abnormalities are severe enough to increase mortality, septic shock should be considered [1] . this translates into a sepsis associated hypotension non-responsive to intravascular volume expansion, that is, dogs with systolic blood pressure < 90 mmhg or mean arterial pressure < 70 mmhg that only respond to vasopressor therapy [2] . the definition of multiple organ dysfunction syndrome (mods) has remained unchanged since the first sepsis consensus conference in 1991 [3] , where it was defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention". during sepsis mods can be considered when at least two organ systems, distant from the infection site, become dysfunctional [2] . in the suspicion of sepsis, vital parameters including temperature (temp.), heart rate (hr) and respiratory rate (rr) should be measured and compared with the sirs criteria, as sepsis is diagnosed when the sirs criteria are fulfilled and an infection is confirmed. the clinical signs tend to be unspecific as they correlate not only with the organ system originally affected by the infectious agent but also with secondary organ dysfunctions. some dogs may already be in septic shock upon hospital admission. in the initial phase of shock patients´present with pale mucous membranes, prolonged capillary refill time (crt) and weak pulses. later, in the hyperdynamic phase of shock, vasodilation subsists with resulting hyperaemic mucous membranes, a decreased crt (< 1 s), and strong or bounding pulses. blood pressure should always be part of physical examination of a suspected sirs patient as hypotension may be present [5, 6] . the diagnostic approach to a septic animal should include a complete blood count, biochemistry profile and coagulation tests. the hemogram may reveal abnormalities in different cellular lineages. the hematocrit most frequently reveals anaemia secondary to blood loss, haemolysis, oxidative damage and reduced erythrocyte production. polycythaemia can also be present in hypovolemic animals due to hemoconcentration and splenic contraction. most animals present leucocytosis and band neutrophils and the blood smear reveal toxic changes to the neutrophils. due to the immunosuppression and lymphocyte apoptosis, it is also possible for lymphopenia and leukopenia to persist. platelet consumption and disseminated intravascular coagulation (dic) resulting in thrombocytopenia is a usual finding. biochemical abnormalities vary and reflect the organ dysfunctions taking place, either primarily affected by the infection or secondary to the inflammatory state. common findings include hypoalbuminemia, glycaemia alterations, hypocalcaemia and hyperbilirubinemia [2, 6, 7] . multiple factors contribute to the development of sepsis in canine parvovirus infections. cellular destruction, intestinal hypomotility, dysbiosis, gut inflammation and tissue necrosis all contribute to cause disruption of the gastrointestinal mucosal barrier, allowing gram-negative and anaerobic bacteria translocation from the intestinal lumen to the bloodstream developing bacteraemia [8, 9] . along with the mucosal barrier disruption, impaired immunity develops increasing the susceptibility to secondary infections. marked leukopenia (mostly neutropenia and lymphopenia) is often observed in cpv infected dogs, as the virus also targets the mitotically active precursors of leukocytes and lymphoid cells of the bone marrow and lymphoid tissue. neutropenia and bacteria overload impair the elimination of luminal bacteria from the bloodstream in contrast to healthy animals [8, 10, 11] . with the sirs progression and the release of inflammatory mediators, the gastrointestinal barrier is compromised again, contributing for the cycle of bacterial translocation [9] . in order to stage septic patients by their risk of mortality/adverse outcome and their potential to respond to treatment, a new stratification system, acronym piro, was introduced in 2001. this system allows to stratify patients based on their predisposing conditions, the nature and characteristics of the insult/infection, the extent of the host immune response to it, and the associated organ dysfunction [12] . the purpose of piro is to help in the enrolment of individuals in clinical studies and prognosis of septic patients, allowing adapting the therapy offered and improving survival. the main objective of the current study was to assess the prognostic value of the presenting vital signs as well as to evaluate the possibility of stratifying and classifying septic animals according to a proposed piro classification system, using parvovirus infection as a natural model for sepsis study [10] . in addition developing a fast-and-frugal tree to reinforce and speed the decisionmaking process. this methodology could help to assess prognosis and be part of the clinical decision making, as well as helping in the enrolment of study populations in future sepsis studies. for that purpose, parvovirus naturally infected dogs hospitalized in the infectious disease isolation unit (idiu) of the veterinary teaching hospital (vth) of the faculty of veterinary medicine (fmv) of the university of lisbon (ulisboa), were subjected to two sets of sirs criteria. the first set, named sirs 1991, considered sirs criteria as they were originally proposed [2] . the second set, named sirs 2001, keeps the same criteria of sirs 1991 plus capillary refill time or mucous membrane colour alteration [12] to attempt to improve the criteria specificity. then patients were classified by a piro classification system adapted from humans to dogs. finally, all individual variables of piro, the total piro score and both sirs criteria were correlated with the outcome. all dogs included in this study were evaluated by a veterinarian from the vth. the target population included dogs hospitalized in the idiu from november 2013 until june 2019, with a positive laboratory diagnosis of canine parvovirosis either by elisa or pcr faecal antigen detection, with clinical exam, haemogram, biochemistry records and known outcome, discharge or death. all dogs that did not fulfil the previous inclusion criteria or had concomitant diseases able to induce gastrointestinal signs were excluded. the sample size was 72 dogs. concerning the outcome 59 (81.9%) dogs were discharged and 13 (18.1%) dogs died. regarding gender 42 (58.3%) were male and 30 (41.7%) female. the majority of the dogs, 52 (72.2%), fell under the described susceptible age group of over 6 weeks and under 6 months, 12 (16.7%) dogs were over 6 months old, 5 (6.9%) were under 6 weeks old and 3 (4.2%) were of unknown age. as far as vaccination status is concerned, most dogs, 37 (51.3%) had no vaccination history, 29 (40.3%) an incomplete vaccination programme, 4 (5.6%) an unknown vaccination history and only 2 (2.8%) were considered to have a complete vaccination status for parvovirus infection. most dogs, 31(43.1%) had no defined breed. table 1 gathers all leucocyte counts, a selection of clinical examination parameters (temperature, heart rate and respiratory rate), all individual variables of piro (p=predisposition, i=infection, r = response, o=organ dysfunction), the total piro score and both sirs criteria for survivors and non-survivors dogs. no significant statistical association was found between the fulfilment of the sirs 1991 criteria and the outcome (p = 0.352). however, when considering the new criteria sirs 2001 requiring crt or mucous membrane colour alteration, a statistical association was found between sirs 2001 criteria and the outcome (or = 4.09, p = 0.0242). this suggests that dogs fulfilling the sirs 2001 criteria upon admission were approximately 4 times more likely to die than those who did not ( table 2) . for the current study, the predisposing factors included were age, breed and vaccination status ( table 3 ). the sample was composed mainly by undefined breed dogs (43.1%) and 14 dogs were considered to have a breed predisposition for parvovirus enteritis (10 labrador, 2 german shepherd, 1 rottweiler and 1 alaskan malamute) [17] . most dogs (72.2%) were aged between 6 weeks and 6 months and the majority of the animals included had no vaccination history (51.3%) or an incomplete/incorrect vaccination (40.3%). as far as age and vaccination status are concerned, this sample reflects descriptions in the literature about parvovirus infection predisposition, with young unvaccinated dogs being the most susceptible to infection. in the present study no significant statistical association was found between predisposition and outcome (p = 1) ( table 4 ). in our study parvovirus was considered to be the only infection inducing element and, since all the animals were considered to have the same infection score of "1", its' correlation with the outcome was not statistically evaluated. to explore the possibility of using the sirs criteria as a fast decision-making tool, a fast-and-frugal tree (fft) ( fig. 1 ) was created using the criteria considered to characterize the response (r) element. this fft revealed a sensitivity of 92% and a specificity of 29%. factors considered for the characterization of the host inflammatory response were the sirs diagnosis criteria (hr, rr, t and leucocytes count) ( table 5 ). no significant statistical association was found between response (r) and outcome (p = 0.1135) ( table 4 ). regarding organ dysfunction from the 72 dogs enrolled in this study, only 13 were considered to have some kind of organ dysfunction, each scoring "1" (table 6 ). hepatic dysfunction was the most frequent cause of organ dysfunction with 8 dogs falling under this classification, 6 due to hypoalbuminemia and 2 with elevated alp. regarding these 8 dogs 4 were deceased and the other 4 survived. from the remaining dogs two were considered to have renal dysfunction, one with a creatinine increment and the other with both creatinine and urea elevation. only the first of these dogs died. three dogs with low platelet count were included on the coagulation dysfunction group and they all survived. none of the animals included showed signs of cardiovascular or respiratory dysfunction. since it was first adapted for veterinary medicine, sirs has been widely used by clinicians and researchers to diagnose sepsis in animals. on a 2006 sepsis survey, 80% of veterinarians acknowledged to use sirs criteria [19] . a pioneer study to propose sirs classification criteria for animals and to assess their ability to diagnose sepsis accurately was carried out by hauptman et al. [5] . the meeting of at least two of the four criteria method revealed a se of 97% and sp of 64%. even though the high sensitivity found indicates that almost all septic animals could be detected, the low specificity implies an over diagnosing of sepsis due to 36% false positives (fp). in an attempt to enhance the criteria specificity in sepsis diagnosis, the 2001 sccm/esicm/accp/ats/sis international sepsis definitions conference task force proposed a list of possible signs of inflammatory response to infection that could be added to the existing sirs criteria, hence augmenting their specificity [12] . in the present study one of the proposed variables was added to the existing sirs criteria in order to create a new set of criteria denominated sirs 2001. accordingly, the sirs criteria would only be applied upon an increased capillary refill time or a mucous membrane colour alteration. in our study, when the new classification criteria were applied, a significant statistical association with the outcome was found (or = 4.09, p < 0.05). in fact, according to our results, dogs that met the sirs 2001 criteria on alves et al. bmc veterinary research (2020) 16:199 admission were about 4 times more likely to die than those who did not ( table 2 ). this improved mortality forecast in medical emergency ward canine patients may be due to an increased specificity of the criteria applied, as they might be indicators of hemodynamic instability and tissue perfusion compromise. even though none of the proposed parameters are specific for sepsis, they can be indicators of an onset of organ dysfunction, which is consistent with the most recent sepsis definition, as a "life-threatening organ dysfunction caused by a dysregulated host response to infection", thus helping to increase the specificity of the diagnosing criteria [1] . sepsis predisposition takes into account all the factors that are present before the onset of sepsis and that may influence the outcome upon an infectious insult. apart from genetic factors, both age and medical co-morbidities were reported in various studies to be associated with hospital mortality [20] [21] [22] . further studies are needed to increase knowledge about the influence of predisposing factors in the systemic inflammatory response of animal species. one study concluded that geriatric dogs had a weaker il-10 production upon bacterial lps stimulation, which may turn into an exacerbated inflammatory response and greater risk of mortality when compared to younger dogs [23] . breed has also been shown to influence the inflammatory response. nemzek et al. [17] found that cpv highly susceptible breeds such as rottweiler and doberman pinscher showed an increase in tumour necrosis factor-α (tnf-α) production in response to lps stimulation when compared to mixed breeds. in the current study, the predisposing factors investigated were age, breed and vaccination status ( table 3) . the sample was composed mainly by undefined breed dogs (43.1%) and 14 dogs were considered to have a breed predisposition for cpv enteritis (10 labrador, 2 german shepherd, 1 rottweiler and 1 alaskan malamute) [17] . most dogs (72.2%) were aged between 6 weeks and 6 months and most of the animals had no vaccination history (51.3%) or an incomplete/incorrect vaccination (40.3%). as far as age and vaccination status are concerned, this sample reflects what has been previously reported about cpv epidemiology, with young unvaccinated dogs being the most susceptible to infection [8] . no significant statistical association was found between predisposition and outcome (p = 1) ( table 4 ). this result differs from most human medicine studies that have shown a significant statistical association between predisposition and outcome, with granja et al. [20] and howell et al. [21] reporting a strong correlation with mortality (p < 0.001). when considering the predisposition (p) discriminatory ability for predicting outcome, a poor to fair accuracy was described by rathour et al. [22] reporting an area under the receiver operating characteristics curve (auc-roc) of 0.79, while granja et al. [20] reported an auc-roc of just 0.66. these results suggest that predisposition alone is not a good outcome prediction element but, since it is associated with the outcome, it should be included in the total piro classification in order to improve its overall accuracy. population characteristics including an overrepresentation of animals within the susceptible age group and underrepresentation of death among the susceptible breeds may also have contributed for the discrepancy between the results obtained in this study and the ones previously cited. on the other hand, our findings come into agreement with the results observed by kalli et al. [24] on cpv infected dogs, where no correlation between any particular breed or age group and the outcome was found. only purebreds were 2.5 times more likely to develop the disease. it might be the case that the parameters chosen to characterize the dogs´predisposition in this study, for example breed, were not the most suitable to evaluate the predisposition influence on sepsis mortality. for the current study cpv was considered to be the only infection inducing element and, since all the dogs were considered to have the same infection score of "1", its' correlation with the outcome could not be statistically evaluated. moreover, this was a limitation of the study, because whenever a dog exhibited clinical examination results, haematological and serum biochemistry compatible with parvovirosis, other agents of acute gastroenteritis of infectious or parasitic origin were not investigated. yet when the pattern of clinical signs or haematological and serum biochemistry suggested the possibility of another disease, these dogs were always tested and rejected according to the exclusion criteria described in methods. one interesting use of sirs criteria is the possibility to construct a fast-and-frugal tree (fft) to predict sepsis outcome, thus helping to speed decision-making. figure 1 represents one fft that may be helpful to characterize and score the response (r) element of the piro system. this fft was not tested on another sample and so the results may only reflect the tree's performance for this particular sample [25] . this fft has a sensitivity of 92% and a specificity of 29%. sensitivity was primed over specificity in the making of this fft, as it is more important to have a higher sensitivity when considering life-threatening conditions like sepsis, in order to reduce the number of false negatives and the risk of missing critically ill animals. the high sensitivity obtained means a good prediction might be expected 92% of the times, when attributing a good prognosis. the low specificity observed implies that 71% of the dogs would be wrongly given a poor prognosis but would end up surviving. using this fft for prognosis attribution may imply a high number of false alarms. however, while the consequences of a low specificity would be a closer monitoring of not so critically ill animals, if specificity was privileged over sensitivity, we would risk attributing a good prognosis to critically ill dogs. this could lead to a less rigorous clinical monitoring of these animals, reducing their survival chances. decision aids, like this fft, may contribute to define clinical parameters and to establish cut off values for them, making them a valuable tool in sepsis research. the inflammatory response is also accountable for the clinical changes observed during sepsis and can have predictive value. variables proven to be related with mortality include heart and respiratory rate, leucocyte and band neutrophils count [10, [19] [20] [21] . in this study the factors used for the characterization of the host inflammatory response were the sirs diagnosis criteria: hr, rr, t and leucocytes count (table 5) . no significant statistical association was found between response (r) and outcome (p = 0.1135) ( table 4 ). these results differ from what has been reported in human medicine studies, in which response was positively associated with mortality. granja et al. [20] and howell et al. [21] both observed a strong correlation between response and outcome, with a p = 0.002 and a p < 0.001 being reported respectively. on another study the only two response variables related with hospital mortality were increased respiratory rate (> 20 breaths/min) and bandemia (> 5% immature band neutrophils). in that study a fair outcome prediction accuracy was reported for the response element, with an auc-roc = 0.74 [22] . the same limitations described above for the sirs criteria may be pointed (higher respiratory and heart rate in puppies and lower leukocyte count due to viral destruction) as the same criteria were used to characterize both [26, 27] . to improve the outcome prediction ability of the response element, biochemical markers of inflammation such as inflammatory cytokines (il-6 and tnf), c-reactive protein (crp) or coagulation proteins should be included in future investigations [21, 22] . the retrospective nature of the present study made it impossible to include biochemical markers of inflammation, as they are not part of routine biochemical analyses. the presence of organ systems dysfunction caused by a deleterious inflammatory response is what differentiates sepsis from an infection, and the presence of organ failure has been shown to correlate with the outcome. yet in this study, no statistical association was found between organ dysfunction and outcome (p = 0.1135) ( table 4) , diverging from the results reported in other studies. kenney et al. [28] reported that cardiovascular dysfunction, coagulation dysfunction, renal dysfunction (p < 0.001) and respiratory dysfunction (p < 0.01) in dogs, were all independently associated with the outcome. they reported that mortality rate rose as the number of organ systems affected increased. for the overall organ dysfunction score and its' correlation with the outcome, rathour et al. [22] described a good outcome prediction accuracy, with an auc-roc = 0.81. a statistical association between overall organ dysfunction score and outcome was also reported by granja et al. [20] . from the 72 dogs enrolled in this study, only 13 were considered to have some kind of organ dysfunction, each scoring "1" (table 1) . hepatic dysfunction was the most frequent cause of organ dysfunction with 8 dogs falling under this classification, 6 due to hypoalbuminemia and 2 with elevated alp. of these eight dogs, four died and the other four survived. hypoalbuminemia is a rather nonspecific parameter to access hepatic function especially during sepsis, as increased vascular permeability and shifting to acute phase proteins production also contribute to the albumin decrease [6, 13] . considering that our study population was composed of cpv infected dogs, hypoalbuminemia may even be less specific as an hepatic function marker, with the most probable cause for hypoalbuminemia being gastrointestinal protein loss. alkaline phosphatase (alp) is not directly related with impaired liver function, even though it can be increased during sepsis due to cholestasis [13] . the alp increment was slight in both dogs suggesting it could result from individual variation. the low specificity of the variables chosen to evaluate liver function and the reported inconsistency of hepatic dysfunction as a mortality predictor, may have affected the results of the present study. two remaining dogs were considered to have renal dysfunction, one with a creatinine increment and the other with both creatinine and urea raise. only the first of these dogs died. based on renal dysfunction consensus the criteria that should be included in the definition of renal dysfunction include serum creatinine concentration, glomerular filtration rate and urine output. in the present study only creatinine and urea were considered as renal dysfunction markers, which may have impaired the ability to identify the presence of renal dysfunction on this sample [29] . nonetheless, kenney et al. [28] reported a strong association between renal dysfunction and outcome even when using serum creatinine concentration as the only renal dysfunction marker. finally, three dogs with low platelet count were included on the coagulation dysfunction group and they all survived. even though coagulation dysfunction has been independently associated with mortality, there are still conflicting results when it comes to platelets count. while hauptman et al. [5] reported thrombocytopenia as a good sepsis marker, laforcade et al. [30] found no significant difference on platelet counts between dogs with sepsis and the control group. the measurement of other coagulation markers like prothrombin time (pt), partial thromboplastin time (ptt), fibrin degradation products (fdp) and d-dimer (dd) concentrations, could have helped on the detection of more animals suffering from coagulation disorders. none of the animals investigated showed signs of cardiovascular or respiratory dysfunction so it was impossible to assess their influence on the outcome. the fact that the criteria proposed to characterize both of these organ systems dysfunctions were rather subjective, as they require clinical judgment, may contribute for the results obtained. future studies should consider other cardiovascular and respiratory function markers. as far as the respiratory function is concerned there are, since 2007, veterinary medicine criteria to assess the presence of acute lung injury and acute respiratory distress syndrome [31] . helpful clinical exams for the characterization of cardiac dysfunction should include echocardiography as it would identify the presence of biventricular dilatation or a decreased ejection fraction [32] . other classification systems like the sequential organ failure assessment (sofa) may be used to improve the scoring of organ dysfunction (o) and contribute to a more accurate overall piro scoring system. in the present study no significant statistical association was found between the total piro score and the outcome (p = 0.093). different studies described a fair to good outcome prediction capacity. rubulotta et al. [33] , reported an area under the curve of 0.696. even though this result defines only a fair mortality prediction ability for piro, it was equivalent to the auc published for other scoring systems at the time, which ranged from 0.6 to 0.7 [34] . howell et al. [21] conducted a study that validated piro's utility. the proposed classification system was created based on the variables found to be independently statistically significant associated with mortality, which increased the outcome prediction accuracy. the scoring system was applied to a sample group and to two validation cohorts. results revealed that mortality was strongly related to an increased piro score in all groups, with an auc-roc of 0.9, 0.86 and 0.83, respectively. on another study carried out by nguyen et al. [35] piro performed better (auc-roc = 0.71) than meds (mortality in emergency department sepsis) and was comparable to the apache ii (acute physiology and chronic health evaluation). li [36] conducted a study on community-acquired sepsis in which an auc of 0.90 for piro 28-day mortality prediction was reported, outperforming the apache ii. recently, results reported by songsangjinda and khwannimit [37] on septic patients admitted over a 9 year period, showed that the moreno piro had the best discriminating capacity with an auc-roc of 0.835, outperforming all the other classification systems and only closely followed by sofa (auc-roc = 0.828). the best discriminative capacity for piro mortality prediction was described by rathour et al. [22] with an auc of 0.94. the mortality rate on this study was 18.1%, which may indicate an underrepresentation of the death group thus contributing to the results obtained, as sepsis mortality rates have been reported to go up to 68% and reaching 90% in the presence of septic shock [28, 38] . the relatively narrow range of the total piro score may also have contributed to a worse outcome prediction capacity. future studies should include more parameters to better characterize the systemic inflammatory response and distinguish animals based on severity of illness. the individual assessment of the parameters and how they independently affect the outcome, as well as the addition of other biomarkers [39, 40] , that may help to characterize the inflammatory response, could contribute to a better outcome prediction accuracy of the proposed piro scoring system. even though no significant statistical association was found between the total piro score and the outcome in this study, this is the first version of the dog piro model and may serve as reference for future studies. the present study stands as a contribution for the development of a robust and validated classification system for sepsis in dogs. to our knowledge, this is the first study to propose and assess the implementation of a piro classification system in dogs, adapting it from what has been used in human medicine and testing it on a sepsis predisposed population of parvovirus naturally infected dogs. concerning the sirs criteria, this study confirms the potential of including animals with altered mucous membrane colour or prolonged capillary refill time. this increased the system specificity, allowing for the identification of a statistical association with the outcome. this was an attempt to address the need for more specific sepsis related systemic inflammatory criteria. to explore the possibility of using the sirs criteria as a fast decision-making tool, a fast-and-frugal tree (fft) was created. in our study no significant statistical association was found between piro's elements and outcome. nevertheless, it demonstrates that defining and applying a classification system for sepsis suspected canine patients is possible. future studies should include more inflammation and organ dysfunction biomarkers, that may help to characterize each of the piro's components and consequently its´overall performance. further adjustments to overcome the weakness reported in this study are necessary to improve its´outcome prediction capacity and to turn it into a useful tool in the clinical management of sepsis. all patients also diagnosed with other viral infections compatible with acute gastroenteritis, namely, distemper (n = 1), infectious canine hepatitis (n = 2) and canine coronavirus (n = 3), were excluded from the study, amounting to six dogs with dual viral infectious diseases. the same criteria applied to all dogs with concomitant internal parasitic infections (n = 9). the remaining 24 dogs excluded from the study population (n = 102) missed either the hemogram, biochemistry results, some clinical examination parameters or the outcome. in order to evaluate if a sirs was taking place, the animals were subjected to two sets of criteria. the first set was denominated sirs 1991 based upon the originally proposed sirs criteria, still currently used in clinical practice. the values considered for each variable are those described above in the background. the second set of criteria, designated sirs 2001, is a combination of sirs 1991 plus crt or mucous membrane colour alteration. sirs 2001 was created in an attempt to increase the specificity of said criteria, as suggested by levy et al. [12] . the proposed criteria applied for the piro scoring, and for each of its' individual components -p, r and o -were extrapolated from an array of bibliographic references, from human and veterinary medicine studies. these criteria were already compiled in a previous study [14] . the parameters proposed for each of the piro's components are described in tables 3, 5 and 6. for predisposition (p) age, breed and vaccination status were considered. response (r) was characterized by temperature, heart rate, respiratory rate and leucocytes count. organ dysfunction (o) by biochemical and clinical markers of renal, cardiovascular, respiratory, hepatic and coagulation system dysfunction. since dogs naturally infected with canine parvovirus were enrolled in this study, all animals had the same infection (i) score (equal to 1). the total piro score was obtained by adding all piro's components score. all dogs that participated in this study were clientowned animals and joined the study after owner's written consent and ethical committee approval (cebea). to evaluate the correlation between the fulfilment of the sirs criteria and the outcome, all dogs were classified according to the original sirs 1991 and to the proposed sirs 2001 criteria. fisher's exact test was used. a 95% confidence interval was considered. to assess the correlation between the piro scoring and the outcome, the scores for total piro and each of its' components were split into two groups, a fisher's exact test requirement. for each variable, one of the groups contained the dogs in the lower half of the values and the other comprised the animals in the upper half. a 95% confidence interval was used in fisher's exact test. the third international consensus definitions for sepsis and septic shock (sepsis-3) definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis systemic inflammatory response syndrome: septic shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of small animal emergency medicine apoptosis-induced lymphopenia in sepsis and other severe injuries canine parvoviral enteritis : an update on the clinical diagnosis , treatment, and prevention bacterial translocation in critical illness characterisation of lipid profiles in dogs with parvoviral enteritis sccm/esicm/accp/ats/sis international sepsis definitions conference infectious diseases of the dog and cat modelo canino para sépsis : contribuição para a classificação e estratificação em doentes sépticosno title breed-related risk factors for canine parvovirus enteritis risk factors associated with parvovirus entiritis in dogs: 283 cases (1982-1991) breed-specific pro-inflammatory cytokine production as a predisposing factor for susceptibility to sepsis in the dog microbial infection and the septic response in critical surgical illness sepsis in veterinary patients: what do we know and where can we go? the predisposition, infection, response and organ failure (piro) sepsis classification system: results of hospital mortality using a novel concept and methodological approach proof of principle: the predisposition, infection, response, organ failure sepsis staging system piro concept: staging of sepsis age-associated changes to pathogen-associated molecular pattern-induced inflammatory mediator production in dogs research in veterinary science factors affecting the occurrence , duration of hospitalization and final outcome in canine parvovirus infection fftrees: a toolbox to create, visualize, and evaluate fast-and-frugal decision trees c-reactive protein , tumor necrosis factor a , and interleukin-6 in dogs with pyometra and sirs use of oseltamivir in the treatment of canine parvoviral enteritis association between outcome and organ system dysfunction in dogs with sepsis: 114 cases acute renal failuredefinition , outcome measures , animal models , fluid therapy and information technology needs hemostatic changes in dogs with naturally occurring sepsis acute lung injury and acute respiratory distress syndromes in veterinary medicine: consensus definitions : the dorothy russell havemeyer working group on ali and ards in veterinary medicine multiple organ dysfunction syndrome in humans and animals predisposition, insult/infection, response, and organ dysfunction: a new model for staging severe sepsis promoting global research excellence in severe sepsis (progress): lessons from an international sepsis registry comparison of predisposition, insult / infection, response, and organ dysfunction, acute physiology and chronic health evaluation ii , and mortality in emergency department sepsis in patients meeting criteria for early goal-directed therapy and the se evaluation of community-acquired sepsis by piro system in the emergency department comparison of severity score models based on different sepsis definitions to predict in-hospital mortality among sepsis patients in the intensive care comparison of dogs with septic peritonitis : 1988^1993 versus changes in salivary analytes in canine parvovirus: a highresolution quantitative proteomic study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions fa and sp performed the experiments and analyzed the data. tn performed the statistical analysis and helped drafting and revising the manuscript. jg, sa and fas helped to perform the experiments and to analyze the data. lt and va contributed to the analysis and interpretation of data. sg conceived the study and participated in its coordination, helped to draft the manuscript and supervised throughout. all authors read and approved the final manuscript. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. all dogs that participated in this study were client-owned animals and joined the study after owner's written consent and ethical committee approval approved by the committee for ethics and animal welfare (cebea) of the faculty of veterinary medicine, university of lisbon. not applicable. the authors declare that they have no competing interests. key: cord-279026-s3yx62u6 authors: tizard, ian r. title: adverse consequences of vaccination date: 2020-07-10 journal: vaccines for veterinarians doi: 10.1016/b978-0-323-68299-2.00019-8 sha: doc_id: 279026 cord_uid: s3yx62u6 the importance of adverse effects from vaccination must not be overstated. vaccine benefits greatly exceed any risks from the procedure. neither must they be minimized. unnecessary vaccination must be discouraged. hypersensitivity reactions to vaccine components are real and must be guarded against. residual virulence, although a concern tends to be more a hypothetical than a real problem. progressive improvements in animal vaccines have significantly reduced the chances of adverse effects occurring, although some issues persist. one such example is injection-site sarcomas in cats. another issue is the influence of animal size on the prevalence of adverse events in dogs. vaccination is the only safe, reliable, and effective way of protecting animals against the major infectious diseases. society does not remember the devastating toll taken by infectious diseases before the development of modern vaccines. exaggerated fear of negative side effects has discouraged owners from having their pets (and themselves) from being vaccinated. the rise of the internet and the development of social media have enabled those who oppose vaccination to spread their opinions. those who resist vaccination for themselves or their children are unlikely to be enthusiastic about vaccinating their pets. much of this resistance is a result of adverse events and controversy regarding effectiveness associated with the earliest vaccines. in spite of the fact that these problems have long been solved, it takes a considerable time before confidence is restored. there is a lack of awareness of the rigorous safety tests that modern vaccines must undergo before they are marketed. good manufacturing practices and the quality control procedures used by the biologics industry, together with rigorous regulatory controls, serve to minimize the occurrence of these events. past issues have been corrected and vaccine safety has steadily improved. modern vaccines are safe to use and overwhelmingly beneficial. adverse events associated with vaccination that might compromise the health of an animal are usually rare, mild, and transient. hypothetical, speculative, or historical adverse effects sometimes dominate perceptions. nevertheless, it has been truly said, "the most dangerous vaccine is the one not given." in reading this chapter the reader should be aware that the events described here are rare, somewhat historical, and relatively unimportant when compared with the benefits of vaccination. drivers of vaccine usage differ significantly between companion animals and commercial livestock. owners of companion animals are concerned for the health and well-being of their pets and are intolerant of any adverse events that cause discomfort, pain, or sickness. livestock producers in contrast vaccinate to maintain livestock health, prevent disease spread, maximize economic return, and to minimize zoonotic disease risks. vaccines that cause a drop in milk production, decreased feed conversion, increased time to market, or a decline in carcass quality may have significant economic consequences and will not be used. in determining whether a vaccine causes an adverse effect, the following three principles should apply. first, is the effect consistent? the clinical responses should be the same if the vaccine is given to a different group of animals, by different investigators, and irrespective of the method of investigation. second, is the effect specific? the association should be distinctive and the adverse event linked specifically to the vaccine concerned. it is important to remember that an adverse event may be caused by vaccine adjuvants and components other than the major antigens. finally, there must be a temporal relationship. administration of the vaccine should precede the earliest manifestations of the event or a clear exacerbation of a continuing condition. the us centers for disease control and prevention (cdc) has classified adverse events as follows: 1. vaccine-induced events: these are events that would not occur in the absence of vaccination and are therefore attributed to the vaccine. an example would be an allergic response to a vaccine component such as egg protein. vaccine potentiated reactions: these are events that might have occurred anyway but may have been precipitated by the vaccine. one possible example is purpura hemorrhagica in horses. 3. programmatic error: events that occur in response to technical errors in vaccine storage, preparation, handling, and administration. 4. coincidental events: these are simply events that happen by chance or result from some underlying illness. the use of vaccines is not free of risk, and an owner has reason to be upset if their healthy animal is sickened by the administration of a vaccine. residual virulence and toxicity, allergic responses, disease in immunodeficient hosts, neurological complications, and harmful effects on the fetus are potential risks associated with the use of vaccines (table 10 .1). veterinarians should use only licensed vaccines, and the manufacturer's recommendations must be carefully followed. before using a vaccine, the veterinarian should consider the likelihood that an adverse event will happen, and also the possible consequences or severity of this event. these factors must be weighed against the benefits to the animal. a common but mild complication requires a very different consideration than a rare, severe complication (table 10 .2). the issue of the risk associated with vaccination remains in large part a philosophical one because the advantages of vaccination are well documented and extensive, whereas the risk for adverse effects is poorly documented, and in many cases, largely speculative. nevertheless, established facts should be recognized, unsubstantiated allegations rebutted by sound data, and uncertainties acknowledged. for example, there is absolutely no evidence that vaccination itself leads to ill health. although difficult to prove, a negative, competent statistical analysis has consistently failed to demonstrate any general adverse effect of vaccination. identification of an adverse event is based on the clinical judgment of the attending veterinarian and is therefore subject to bias. standard case definitions of a vaccine-associated adverse event are not yet available. it still is often difficult to distinguish association from causality (box 10.1). traditionally, adverse events resulting from vaccine administration have been reported by veterinarians to manufacturers or government agencies. the resulting numbers have been difficult to analyze satisfactorily for two major reasons. first, reporting is voluntary, so significant underreporting occurs. adverse events are often regarded as insignificant, or it may be inconvenient to report them. second, very little data has been available on the number of animals vaccinated. although manufacturers know the number of doses of vaccine sold, they are unable to measure the number of animals vaccinated. it has, however, proved possible by examining the electronic medical records of a very large small animal general practice, to determine the prevalence of vaccine-associated adverse events in over a million dogs. the use of a standardized reporting system within a very large population has permitted objective analysis of the prevalence of adverse events occurring within three days of vaccine administration. out of 1,226,159 dogs receiving 3,439,576 vaccine doses, 4678 adverse events were recorded (38.2/10,000 dogs); 72.8% of these events occurred on the same day the vaccine was administered, 31.7% were considered to be allergic reactions, 1.7% were classified as anaphylaxis, and 65.8% were considered "vaccine reactions" and were likely caused by innate immune responses. three dogs died. the lowest rate of such events was associated with bordetella more than 1 in 10 animals showing adverse reactions (.10%) common greater than 1 but less than 10 animals per 100 animals vaccinated (1%-10%) uncommon more than 1 but less than 10 animals per 1000 animals vaccinated (0.1%-1%) rare more than 1 but less than 10 animals per 10,000 animals vaccinated (0.01%-0.1%) very rare less than 1 animal in 10,000 reported (,0.01%) autism spectrum disorder is a chronic developmental disorder in children. its causes are largely unknown. it usually becomes apparent in young children over one year of age at around the same time they receive their initial vaccinations. in a paper published in 1998, a physician studied 12 children with autism. he asked the parents if the children had been vaccinated, with the measles, mumps, and rubella vaccine, within the previous two weeks. eight said yes, so the author went on to assert in his paper that this vaccine caused autism. he postulated that autism resulted from measles infection. the paper was eventually retracted and the author lost his medical license. subsequent population-based studies have failed to demonstrate any link between vaccination and autism. thousands of children are vaccinated every year and large amounts of data are available for analysis. all these show the same thing. there is no link between vaccination and autism risk. however, the word was out. the internet and twitter spread the word. additionally, pet owners began to claim that their dog's behavior had changed after vaccinationcanine autism. the british veterinary association felt obliged to issue a statement regarding these claims. "there is currently no reliable scientific evidence to indicate autism in dogs or a link between vaccination and autism. vaccinations save lives and are an important tool in keeping our pets healthy. all medicines have potential side-effects but in the case of vaccines, these are rare and the benefits of vaccination in protecting against disease far outweigh the potential for an adverse reaction." vaccination and the highest rate with lyme disease vaccine. additional analysis indicated that the risk of adverse events was significantly greater for small dogs than for large dogs ( fig. 10 .1); for neutered than for sexually intact dogs; and for dogs that received multiple vaccines on one occasion. each additional vaccine dose administered increased the risk of an adverse event occurring by 27% in dogs under 10 kg and by 12% in dogs heavier than 12 kg ( fig. 10 .2). highrisk breeds included dachshunds, pugs, boston terriers, miniature pinschers, and chihuahuas. overall, the increased prevalence of adverse events in young adult, small-breed, neutered dogs and their relationship to multiple dosing suggests that veterinarians should look carefully at the practice of giving the same vaccine dose to all dogs irrespective of their size. in another report, from japan 351 dogs showed an adverse event out of 57,300 vaccinated (62.7/10,000 doses). (vaccines used included canine parvovirus, canine distemper, canine adenovirus 2, canine coronavirus, and leptospirosis.) of these 351 dogs, 1 died, 41 had anaphylaxis, 244 developed dermatological signs, and 160 showed gastrointestinal signs. about half the anaphylaxis events occurred within 5 minutes of vaccination. additional analysis of these anaphylaxis cases reported 87% collapse, 77% cyanosis, and both collapse and cyanosis in 71% of affected dogs. breeds affected included miniature dachshunds (50%; these accounted for about 30% of all the anaphylaxis cases), chihuahuas (10%), mixed breeds (5%), and toy poodles (5%). miniature schnauzers also appeared to be unusually prone to anaphylaxis. the highest frequency of adverse reactions occurred in dogs under 5 kg. most adverse events were observed within 12 hours after vaccination. the adverse event rate in japan as reported here (62.7/10,000 doses) is much higher than in the united kingdom (0.093/10,000 doses), or in the united states (38.2/10,000 dogs). vaccines may elicit mild transient injection site reactions as a result of inflammation. these inflammatory responses may manifest themselves within two to three days. as pointed out in chapter 2, some degree of inflammation is required for the efficient induction of protection. this may cause pain or pruritus. the sting produced by some vaccines may present problems, not only to the animal being vaccinated, but also to the vaccinator, if the animal reacts violently. lethargy, anorexia, soreness, minor behavioral changes, and tenderness at the vaccine site are normal postvaccinal responses and should resolve within 12 to 24 hours. swellings may develop at the reaction site less commonly. these may be firm or edematous and may be warm to the touch. they appear within 24 hours and can last for about a week. unless an injection-site abscess develops, these swellings leave little trace. vaccines containing killed gram-negative bacteria may be intrinsically toxic owing to the presence of pathogen-associated molecular patterns such as endotoxins, lipids, muramyl peptides, and porins that can bind to pattern recognition receptors and provoke cytokine release. in extreme cases this may lead to anorexia, and fever. although such reactions are usually only a temporary inconvenience to male animals, they may be sufficient to provoke early embryonic deaths in pregnant females. it may be prudent to avoid vaccinating pregnant animals unless the risks of not giving the vaccine are considered to be too great. vaccination with either immunestimulating complex (iscom) vaccines or live recombinant vectored vaccines against influenza and tetanus may induce an acute-phase response in horses. innate immune responses may reduce an animal's growth rate and diminish its feed efficiency. this growth suppression can be mimicked by injection of interleukin (il)-1 and tumor necrosis factor (tnf)-a. these cytokines act on the brain to reduce appetite while at the same time, causing degradation of skeletal muscle. intranasal vaccines such as those containing bordetella bronchiseptica and some viruses may cause transient cough or sneezing. this simply reflects the mild innate response triggered as the vaccine organisms invade the upper respiratory tract. vaccines have the potential to cause rare but serious allergic reactions (type i hypersensitivity). for example, allergic responses may occur when an animal produces immunoglobulin (ig)e in response, not only to the immunizing antigen, but also to other components in vaccines. the most significant allergens are often vaccine excipients. for example, reactions are most likely to occur after injection of vaccines that contain trace amounts of fetal calf serum (specifically bovine serum albumin), egg proteins (ovalbumin), or gelatin. (gelatin and serum albumin are added to vaccines as stabilizers to protect the vaccine antigens during the freeze-drying process.) some vaccines may also contain antibiotics such as neomycin to which an animal may be sensitized. severe allergic responses have been associated with the use of killed foot-and-mouth disease, rabies, and contagious bovine pleuropneumonia vaccines in cattle. signs include angioedema, affecting mainly the head and ears, urticaria, pruritus, acute-onset diarrhea, vomiting, dyspnea, and collapse. all forms of hypersensitivity are more commonly associated with multiple injections of antigens and therefore tend to be associated with the use of killed vaccines. it is important to emphasize that a type i hypersensitivity reaction is an immediate response to an antigen and occurs within a few minutes after exposure to an antigen ( fig. 10 .3). it is good practice to keep an animal in the clinic for 15 to 25 minutes after vaccination to ensure that any immediate problems can be promptly recognized and treated (box 10.2). reactions occurring more than two or three hours after administration of a vaccine are likely not type i hypersensitivity reactions. in type ii hypersensitivity reactions, antibodies directed against an animal's own cells act together with complement to cause cell lysis. these antibodies are usually induced by the presence of animal cells in the vaccine. natural hemolytic disease of the newborn (hdn) in calves is very rare, but it has resulted from vaccination against anaplasmosis or babesiosis. these vaccines contain pooled red cells from infected calves. in the case of anaplasma vaccines, for example, the blood from infected donors is pooled, freeze-dried, and mixed with adjuvant before being administered to cattle. the vaccine against babesiosis consists of fresh, infected calf blood. both vaccines cause infection, and consequently, the development of immunity in recipients. they also stimulate the production of antibodies against the injected red cells. if cows sensitized by these vaccines are then mated with bulls carrying the same blood groups, they can transmit these antibodies to their calves through colostrum. the calves that drink this colostrum may then develop hemolytic disease. hdn in piglets had a similar pathogenesis when sows were immunized with a hog cholera vaccine containing pig blood. beginning in 2007, multiple outbreaks of an unexplained hemorrhagic disease in newborn beef calves were reported from many countries in western europe. affected calves showed sudden onset bleeding including nasal hemorrhage, petechiation on mucus membranes, and excessive bleeding from minor wounds such as injection, or ear-tag sites. the disease appeared 7 to 8 days after birth and affected calves could die within 48 hours. it is now called bovine neonatal pancytopenia (bnp). investigation showed an early drop in platelets, monocytes, and neutrophils was followed by drops in erythrocyte and lymphocyte numbers. the net result was a profound pancytopenia. the bone marrow could be completely aplastic. mortality was as high as 90% in severely affected calves, but there were also many subclinical cases. because this disease only occurred in suckled calves and developed within hours of first suckling, it appeared to result from the consumption of colostrum. further investigations showed that the colostrum from these cows contained antibodies directed against the major histocompatibility complex (mhc) class i molecules expressed on neonatal leukocytes and bone marrow stem cells. cells of the thrombocyte, lymphocyte and monocyte lineages, and precursors of neutrophil, erythrocyte, and eosinophil lineages were affected. further investigations showed that the disease was triggered by administration of a specific vaccine against bovine virus diarrhea (bvd). this vaccine-pregshure-contained inactivated bovine viral diarrhea virus (bvdv) grown in bovine kidney cells. a potent, oil-in-water emulsion adjuvant containing quil a, cholesterol, and mineral oil was then added. immunization with this anaphylaxis is a life-threatening medical emergency. deterioration can occur very rapidly and time is of the essence. initiate treatment immediately. if an animal is undergoing acute anaphylaxis take the following steps: 1. stop administering the vaccine. in the case of dogs and cats, administer epinephrine 1:1000 at 0.01 mg/kg intramuscularly. repeat every 5-15 minutes if necessary. if very severe and shock has developed, place an intravenous catheter and administer 0.1 mg/kg of 1:10,000 epinephrine by slow intravenous infusion and monitor blood pressure and perfusion. alternative routes of administration include intracardiac or intratracheal. avoid subcutaneous administration because epinephrine is a potent vasoconstrictor and absorption is delayed. in the case of foals administer epinephrine 1:1000 at 0.01 to 0.02 mg/kg (0.5-1ml for a 50 kg foal) given slowly intravenously or intramuscularly. in adult horses administer epinephrine at 0.01 mg/kg (3-8 ml for a 450 kg horse) slowly intravenously. if the condition is mild, this dose may be administered intramuscularly. repeat every 10-20 minutes if necessary. 3. secure the airway, intubate if necessary, and administer oxygen to animals showing respiratory symptoms. 4. provide isotonic shock crystalloid fluids (normal saline or lactated ringer solution) intravenously to help restore adequate blood pressure in hypotensive animals. the volume required depends on the animal's response, but may be as high as 90ml/kg for dogs and 60ml/kg for cats. administer an h1-antihistamine such as diphenhydramine every 8-12 hours if necessary. 6. once the animal is stabilized consider administering a fast acting glucocorticosteroid by the slow intravenous route to prevent late-phase responses. vaccine induced antibodies against the bovine kidney cells in some cows. these antibodies, when transferred to calves via colostrum, bound to their leukocytes and bone marrow stem cells, killed them, and so induced pancytopenia (fig. 10.4) . not all calves born from cows that received this specific vaccine developed clinical disease. the quantity and specificity of their antibody response determined the risk to their calves. antibody levels remained high in some cows for many years and were boosted by each pregnancy. as a result, bnp cases occurred for many years after pregshure was removed from the market in 2010. type iii hypersensitivity reactions (immune-complex-mediated) may be induced by vaccination. the deposition of immune-complexes in tissues may cause local inflammation or cause a generalized vasculitis such as purpura. some rabies vaccines may also induce a local complementmediated vasculitis in the skin resulting in ischemic dermatitis and local alopecia. this may occur at the injection site or at remote locations such as the ear tips, footpad, tail, or scrotum. this vasculitis is most often seen in small dogs such as dachshunds, miniature poodles, bichon frises, and terriers. in dogs infected with canine adenovirus-1 (cav-1, infectious canine hepatitis), an immunecomplex-mediated uveitis and a focal glomerulonephritis both develop. the uveitis, commonly called "blue-eye," is seen both in dogs with natural infections and in those vaccinated with live attenuated cav-1 vaccine (fig. 10 .5). the uveitis results from the formation of virus-antibody complexes in the anterior chamber of the eye and in the cornea with complement activation and consequent neutrophil accumulation. the neutrophils release enzymes and oxidants that damage corneal epithelial cells, leading to edema and opacity. the condition resolves spontaneously in about 90% of affected dogs. replacing cav-1 with cav-2 in vaccines has largely eliminated this problem. see chapter 15. type iv hypersensitivity (delayed) reactions are t-cell-mediated inflammatory responses. they may occur at the injection site in response to vaccination, but a more common reaction is local granuloma formation. this may be in response to persistent adjuvants containing alum or oil. vaccines containing a water-in-oil adjuvant produce larger and more persistent lesions at injection sites than vaccines containing alum or aluminum hydroxide. these lesions may develop into sterile abscesses and if the injection site is dirty, these abscesses may become infected. injection site lesions are of major concern in the meat industries. modified live vaccines must be able to establish themselves transiently in a vaccinated animal yet at the same time not cause disease. they must be safe in animals and their human companions. they must be as stable as possible to enable long-term storage. they must be environmentally safe. it may be possible to achieve minimal virulence with maximal immunogenicity, but this may be unattainable in animals with any defects in their immune function. the normal distribution of immunological competence in an outbred population is such that some animals will inevitably be susceptible to an otherwise avirulent organism. this immunosuppression may result from minor stresses, but equally important some common viral infections such as canine distemper, feline pancytopenia, or feline leukemia also cause immunosuppression to a degree that an animal may become susceptible to otherwise avirulent vaccinal agents. it is also appropriate to point out that modified live vaccines are attenuated for a specific target species for administration by a specified route. if administered to the wrong species or in the wrong way residual virulence may cause disease. thus some modified live vaccines may retain the ability to cause disease. a good example is brucella abortus strain 19. although highly immunogenic in cattle, s19 can cause severe reactions in vaccinated cows. swelling, fever, anorexia, depression, and a drop in milk yield have been reported. s19 can also cause abortion in pregnant cows and orchitis in bulls and humans. safer attenuated brucella vaccines are now available. similar residual virulence hazards are associated with the soremouth vaccine and the sheep toxoplasmosis vaccine. some modified live herpes vaccines or calicivirus vaccines given intranasally may spread to the oropharynx and result in persistent infection. in these cases, the vaccine virus may infect (and protect) other animals in contact. because live vaccine strains may be released into the environment, safety issues involving not only the animal but also its environment must be addressed. are there changes in the tissue tropism of the virus? are there changes in the carrier through the incorporation of new foreign genes? is there reversion to virulence through the incorporation of complementation genes? is there exchange of genetic information with other wild type or vaccine strains of the carrier? will the carrier spread unwanted genes such as antibiotic resistance into the environment? these questions are highly relevant in the aquaculture industry where modified live vaccine viruses may escape into the aquatic environment (chapter 21). postvaccinal canine distemper encephalitis is a rare complication that may develop in dogs and ferrets after administration of modified live canine distemper vaccines. affected animals may show neurologic signs such as aggression, incoordination, and seizures, or die suddenly. the pathogenesis of this condition is unclear. it may be the result of residual virulence, increased susceptibility, or triggering of a latent paramyxovirus by the vaccine. vaccination during pregnancy carries uncertain risks, especially when live vaccines are used. the fetal immune system may not have developed sufficiently to defend itself against the vaccine strain of the virus. mlv bluetongue virus vaccine has been reported to cause malformations in the offspring of ewes vaccinated while pregnant. the severity of the lesions depends upon the stage of pregnancy at vaccination. for example, mlv bluetongue administered to ewes between 50 and 100 days of gestation has caused hydranencephaly and retinal dysplasia in lambs. live erysipelothrix rhusiopathiae vaccines have been reported to cause abortions in sows. the stress from this type of vaccination may also be sufficient to reactivate latent infections; for example, reactivation of equine herpesviruses has been triggered by vaccination against african horse sickness. a modified live virus (mlv) parvovirus vaccine administered during pregnancy has been reported to cause hydranencephaly and cerebellar hypoplasia in kittens. many viruses promote their own survival by suppressing their host's immune system. although immunosuppression is greatest in virulent strains, some mlvs may remain somewhat immunosuppressive. for example, some mlv canine parvovirus strains may depress t cell responses to mitogens in puppies for two to five weeks following administration, or even cause a lymphopenia. similarly, mlv canine distemper may cause immunosuppression and thrombocytopenia. in view of this it may be best to avoid performing elective surgery on dogs for at least one week postvaccination. mlv bovine viral diarrhea (mlv-bcd) vaccines may suppress neutrophil functions and lymphocyte blastogenesis in vaccinated calves. as a result, they may potentiate intercurrent infections. mlv-bvd may also induce mucosal disease 7 to 20 days after vaccination. vaccination with an mlv-bhv1 vaccine has been shown to exacerbate the lesions of experimental moraxella-induced pinkeye (chapter 16). several vaccine combinations may also result in transient immunosuppression. for example, a combination of distemper and adenovirus vaccines can reduce canine lymphocyte counts and their responsiveness to mitogens, although the individual components are not detectably immunosuppressive. this t cell suppression may be accompanied by simultaneous enhancement of b cell responses and raised immunoglobulin levels. many of these cases of "immunosuppression" attributed to vaccines may however simply reflect alterations in the th1/th2 balance or transient alterations in lymphocyte recirculation patterns. they are rarely of clinical significance. as pointed out in an earlier chapter, older vaccine viruses were attenuated by prolonged passage in tissue culture or eggs. in some cases, it is possible to reverse the attenuation process by backpassage through their natural hosts. for example, attenuated distemper strains cannot grow in canine lung macrophages. back-passage of the canine distemper virus (cdv) rockborn strain for as few as three passages in puppies resulted in the virus regaining this ability. by four passages the virus could cause weight loss. by five passages, immunosuppression returned. the virus that had been back-passaged six to seven times had regained its virulence. the use of genetically defined, gene deleted attenuated vaccines has largely eliminated this type of problem. louis pasteur's first rabies vaccine contained dried rabbit brain tissue. when injected into patients it induced antibodies against myelin basic protein and an acute demyelinating encephalomyelitis developed in about 0.1% of recipients. rabies vaccines have had an undeserved bad reputation ever since. in 2011, it was proposed that a new syndrome existed that linked diverse human autoimmune diseases with the use of adjuvanted vaccines. it was called autoimmune/ autoinflammatory syndrome induced by adjuvants (asia). this syndrome has been investigated to determine whether it is an insignificant clinical term or whether there is an underlying mechanism that links adjuvants to autoimmunity. aluminum-containing adjuvants were claimed to be the "cause" of asia. however, patients receiving allergen-specific immunotherapy receive up to 500 times more injected aluminum than regular vaccine recipients and have a lower incidence of autoimmune disease. current data does not support the causation of asia by vaccine adjuvants. there is a lack of any reproducible evidence for any link between adjuvants and autoimmunity. one obvious problem with this proposed syndrome is that vaccination is so commonplace whereas autoimmunity remains uncommon. after all, huge numbers of people receive influenza vaccines annually without untoward effect. there is a single animal study that appears to show that a link might exist between vaccination and the development of autoimmunity. a retrospective analysis of the history of dogs presenting with immune-mediated hemolytic anemia (imha) showed that 15 of 58 (26%) dogs with imha had been vaccinated within the previous month, compared with a randomly selected control group of 70 dogs in which 5% had been vaccinated. dogs with imha that developed within a month of vaccination differed in some clinical features from dogs with imha unassociated with prior vaccination. some studies using very large databases have tended to confirm this effect, in that they showed an approximately three-fold increase in diagnoses of autoimmune thrombocytopenia, and a two-fold increase in diagnoses of imha in dogs in the 30 days following vaccination, compared with other time periods. other studies have failed to show any association between vaccination and imha. the overall prevalence of these diseases remains low, and they can be diagnosed at times not temporally associated with vaccination. vaccination may therefore serve as a trigger for these diseases in some dogs-a vaccine potentiated reaction. contaminating thyroglobulin found in some vaccines (usually from the presence of fetal bovine serum) may lead to the production of antithyroid antibodies in vaccinated dogs. lymphocytic thyroiditis has been found in 40% of beagles on necropsy, but there was no association detected between vaccination and the development of this thyroiditis. in the 1970s, a swine influenza vaccine induced guillain-barrã© syndrome (an autoimmune polyradiculoneuritis) in about 1 case per 100,000 human recipients. (current influenza vaccines have a risk of about 1:1 million. it appears that the older influenza vaccine was unique in this respect.) cases of this syndrome in dogs have been rarely reported. in some animals, the administration of potent, adjuvanted vaccines may stimulate the transient production of autoantibodies to connective tissue components such as fibronectin and laminin. vaccination of some weimaraner puppies may lead to the development of a severe hypertrophic osteodystrophy. the disease appears within 10 days of administration of mlv canine distemper vaccine. systemic signs include anorexia, depression, fever, and gastrointestinal, nervous, and respiratory symptoms, in addition to symmetrical metaphyseal lesions with painful swollen metaphyses. radiological examination shows radiolucent zones in the metaphyses, flared diaphyses, and formation of new periosteal bone. it is possible that the condition is triggered by the vaccine in genetically susceptible animals. these dogs may have a preexisting immune dysfunction with low concentrations of one or more immunoglobulin classes, recurrent infections, and inflammatory disease. it has been suggested that weimaraners are especially susceptible to this condition and that they therefore receive only killed virus vaccines. a mild transient polyarthritis has been reported in some dogs following vaccination. the dogs show a sudden onset of lameness with swollen and painful joints within two weeks of vaccination. the dogs recover within two days. no specific breed or vaccine has been associated with this problem. vaccination against calicivirus has been associated with polyarthritis and a postvaccination limping syndrome in cats. a search of web sites regarding vaccination of pets reveals that a large number express great concern regarding the practice of overvaccination. by this is meant the use of unnecessary vaccines and by implication a significant threat to the health of pets. conversely a search of pubmed, the ncbi web site, reveals only a single scientific paper regarding this subject. the paper describes renal disease in a spaniel that received seven doses of vaccine from its owner, one vaccine per month, in the absence of any veterinary supervision. as a result, the dog developed immune-complex lesions in its kidney glomeruli. this was very likely a type iii hypersensitivity nephropathy. clearly administration of excessive and unneeded vaccines is inappropriate. there are no health benefits and each additional dose of vaccine carries with it the chances of an untoward event. as pointed out throughout this text, the risk/benefit assessment of any vaccination procedure must be a subject for discussion between a veterinarian and the pet owner. there are many reasons why a veterinarian may suggest that it may be beneficial to vaccinate an animal and it is inappropriate to blame those vets who choose to vaccinate animals more frequently than currently recommended without a full knowledge of each specific case. this is called clinical judgment. these are discussed in chapter 14. errors in manufacture and administration modified live vaccines cannot contain preservatives (except antibiotics in viral vaccines). as a result, occasional cases of vaccine contamination have occurred. these have been a major issue in the past when viral identification required culturing. modern identification techniques such as the polymerase chain reaction have made such contamination a thing of the past. there are numerous examples of such contamination. for example, mycoplasma contamination was a feature of many live virus veterinary vaccines. the pestivirus of border disease contaminated some soremouth and pseudorabies vaccines; bovine leukemia virus has contaminated bovine blood vaccines such as those against babesiosis and anaplasmosis. bluetongue virus has contaminated some canine vaccines. injection site selection should include consideration of potential adverse reactions in addition to the hypersensitivity reactions described earlier. for example, injection in the gluteal muscles/hip region of cattle should be discouraged because gravitational drainage along fascial planes can occur. should an abscess develop, considerable tissue damage may occur and result in eruptions in undesirable locations with lesions that require prolonged time to heal. they may result in unacceptable blemishes in meat destined for human consumption (chapter 16). veterinarians and other vaccine users may be inadvertently exposed to animal vaccines as a result of unintended inoculation or spraying. some of these vaccines may cause sickness. veterinarians, their assistants, and other animal handlers should be especially careful when administering injectable vaccines to avoid needle-stick and eye injuries. if an individual is accidentally self-injected with a mineral oil-adjuvanted vaccine, seek immediate medical treatment regardless of the dose injected. with the notable exception of brucellosis, these events are rarely reported. nevertheless, accidents do occur and veterinarians should be fully aware of these risks. brucellosis is an existential hazard to veterinarians. the cdc has established a passive surveillance registry. in the two years 1998 to 1999, 21 individuals reported needlestick injury related exposure to the brucella vaccine strain rb51, five were splashed in the eye, and one was splashed into an open wound. although most received antibiotics, 19 reported clinical disease. approximately 4 to 5 million doses of brucella vaccines were administered annually in 1997 to 2000. it is estimated these would have resulted in at least 8000 needle-stick injuries, suggesting that exposure to rb51 is substantially under-reported. a vaccinia recombinant rabies vaccine bait has been air-dropped across many states in the united states to vaccinate wildlife. several instances of human exposure to these baits have been reported. (the vaccine baits have toll-free numbers printed on them.) in ohio, there were 160 reports of bait contact and 20 of these involved contacts with the vaccine. one individual developed a severe vaccinia infection and had to be hospitalized. bordetella bronchiseptica causes respiratory disease in dogs and atrophic rhinitis in pigs. infection of humans is rare but has been documented. in at least one case a young boy was inadvertently sprayed in the face with a "kennel cough vaccine." he had been holding his dog but the dog moved. he developed a pertussis-like respiratory disease that lasted several months despite antibiotic treatment. there have been reports of clients experiencing respiratory difficulty following administration of an intranasal vaccine to their dogs. needle-stick injuries are not uncommon and many involve vaccines. a woman was inadvertently inoculated with the sterne anthrax vaccine while vaccinating her horse. she did not develop anthrax but did develop a local reaction within 24 hours. serious inflammatory reactions are associated with injected mycobacterium paratuberculosis vaccine. self-injections appear to be a major issue in the aquaculture industry where workers have to work fast to vaccinate slippery fish. veterinarians are encouraged to report all adverse reactions to the vaccine's manufacturer and the regulatory authorities. this provides both with the critical information that is used to evaluate and monitor vaccine safety in the field. in this way vaccine safety can be progressively improved. adverse reactions should be reported to the vaccine manufacturer first. after that, they should be reported to the appropriate regulatory authorities. in the united states, adverse vaccine events should also be reported to the us department of agriculture aphis center for veterinary biologics at 1-800-752-6255. they have an online electronic report form. reports can also be made by fax or mail. vaccine lot and serial numbers should be noted in vaccination records because this will facilitate an investigation. the use of standardized reporting systems is encouraged. web: http://www.aphis.usda.gov/animal_health/vet_biologics/vb_adverse_event.shtml fax or mail: download the pdf form at http://www.aphis.usda.gov/animal_health/vet_ biologics/publications/adverseeventreportform.pdf and fax to (515) 337-6120 or mail to the center for veterinary biologics (cvb), 1920 dayton avenue, po box 844, ames, iowa 50010, usa. telephone: (800) 752-6255 in canada, suspected adverse events (sae) should be reported to the canadian center for veterinary biologics (ccvb) in ottawa at 1-855-212-7695. as stipulated by the health of animals regulations, all reports that indicate "serious expected" or "serious unexpected" adverse events related to the use of a veterinary biologic, including lack of efficacy, must be reported to ccvb within 15 days of that information becoming known to the permit or license holder. follow-up reports, including case conclusions, must be submitted to ccvb in a timely manner. all other reports should be investigated by the license/permit holder, summarized in a summary update report, and submitted to ccvb every six months. summary update reports should be submitted within 60 days of the end of the reporting period. sae related to veterinary biologics are categorized as one of the following: adverse event (ae), serious ae, unexpected ae, and lack of efficacy. a causality assessment should also be assigned to each sae. each case should be classified as probable, possible, unlikely, or unknown. form cfia/acia 2205, notification of suspected adverse events to veterinary biologics, may be found at http://inspection.gc.ca/english/for/pdf/c2205e.pdf. in the united kingdom adverse events should be reported to the veterinary medicines directorate. forms can be obtained at their website at www.vmd.defra.gov.uk or by calling their pharmacovigilance team at 01932 338427. the veterinary medicines directorate (vmd), an agency of the department for environment, food, and rural affairs, is responsible for the suspected adverse reaction surveillance scheme (sarss) for veterinary medicines. adverse reactions in animals in the united kingdom should be reported at http://www.vmd.defra.gov.uk/ adversereactionreporting/default.aspx. human reactions to veterinary medicines in the united kingdom should be re new zealand in new zealand adverse event reports should be made to the ministry for primary industries adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine rb51 human illness associated with use of veterinary vaccines evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period pharmacovigilance: suspected adverse events vaccination and ill-health in dogs: a lack of temporal association and evidence of equivalence vaccine hypersensitivity-update and overview adverse reactions to vaccination: from anaphylaxis to autoimmunity revisiting adverse reactions to vaccines: a critical appraisal of autoimmune syndrome induced by adjuvants (asia) vaccine-induced enhancement of viral infections rabies vaccine is associated with decreased all-cause mortality in dogs vaccine-associated adverse events large-scale survey of adverse reactions to canine non-rabies combined vaccines in japan adverse events after vaccine administration in cats: 2,560 cases adverse events diagnosed within three days of vaccine administration in dogs adverse vaccinal reactions in dogs and cats a space-time cluster of adverse events associated with canine rabies vaccine aa amyloidosis in vaccinated growing chickens ige reactivity to vaccine components in dogs that developed immediate-type allergic reactions after vaccination membranoproliferative glomerulonephritis possibly associated with over-vaccination in a cocker spaniel fatal adverse pulmonary reaction in calves after inadvertent intravenous vaccination anaphylaxis in dogs and cats comparative vaccine-specific and other injectable-specific risks of injection-site sarcomas in cats immune modulation following immunization with polyvalent vaccines in dogs key: cord-285493-eg2ltip6 authors: schwab, s.; herden, c.; seeliger, f.; papaioannou, n.; psalla, d.; polizopulou, z.; baumgärtner, w. title: non-suppurative meningoencephalitis of unknown origin in cats and dogs: an immunohistochemical study date: 2007-02-01 journal: j comp pathol doi: 10.1016/j.jcpa.2006.11.006 sha: doc_id: 285493 cord_uid: eg2ltip6 non-suppurative meningoencephalitis of unknown cause is a frequent finding in dogs and cats. fifty-three dogs and 33 cats with non-suppurative meningoencephalitis of unknown aetiology were examined immunohistochemically for 18 different infectious agents, including viruses, bacteria and prion protein(sc). in 14 (26%) of the dogs and 13 (39%) of the cats a causative agent was identified in the central nervous system (cns), two dogs and one cat giving positive results for two infectious agents simultaneously. the study revealed infections with known causative agents (porcine herpes virus 1, feline infectious peritonitis virus, escherichia coli) and a new disease pattern of parvovirus infection in the cns of dogs and cats. infection of the cns with feline leukaemia virus was found in a cat. five dogs and four cats gave positive results for west nile virus (wnv) antigen. in one dog, canine parainfluenza virus antigen was detected in the brain. four dogs and four cats gave positive results for encephalomyocarditis virus (emcv). the significance of the detection of wnv and emcv antigen requires further study. the aetiology remained undetermined in 39 dogs (74%) and 20 cats (61%). although it is possible that non-infectious causes play a more important role than previously thought, infections with hitherto unrecognized agents cannot be ruled out. non-suppurative meningoencephalitis in cats and dogs is usually considered to be caused by viral infections (summers et al., 1995) , and the histopathological ¢ndings consist mainly of perivascular and parenchymal in¢ltration with lymphocytes, macrophages and plasma cells, usually associated with meningitis and occasionally with in£ammation of the plexus chorioideus and the ependyma (braund, 1980; luttgen, 1988) . viruses, due to cell tropism and route of infection, often induce a typical pattern of in£ammation in the brain and spinal cord (braund, 2001) . in newborn animals, infections of the central nervous system (cns) with bacteria, including various strains of escherichia coli, are a common sequel to septicaemia, whereas in adult dogs and cats bacteria may follow a neurogenic, otogenic or rhinogenic route to enter the cns (meric,1988; summers et al.,1995) . whilst most bacterial cns infections induce a purulent in-£ammation, some bacteria, particularly listeria spp., as well as protozoa, fungi and algae, cause a non-suppurative, mainly granulomatous in£ammation of the cns (rand et al., 1994; summers et al., 1995; tipold, 1995; quesnel et al., 1997) . in the brains of beagles with granulomatous leptomeningitis, maeda et al. www.elsevier.com/locate/jcpa (1993) demonstrated e. coli and considered it to be the causative agent. if parasites enter the cns, they often induce a granulomatous in£ammation (summers et al., 1995) . in dogs, the most common parasite to cause infection of the cns is toxoplasma gondii, followed by neospora caninum and, albeit rarely, cuterebra spp. in cats, cuterebra spp. are the most common parasites to be found in the cns, followed by t. gondii in rare cases (braund, 2001) . there would also seem to be non-infectious causes of non-suppurative meningoencephalitis. thus, autoimmune and hereditary mechanisms are suspected to be the underlying cause of cns disorders such as granulomatous meningoencephalitis (gme) in dogs (harris et al., 1988; kipar et al., 1998) , pug dog encephalitis and non-suppurative meningoencephalitis in greyhounds (callanan et al., 2002) . so far no aetiological agent has been detected for feline ''staggering disease'' or so-called feline polioencephalomyelitis (vandevelde and braund, 1979; nowotny and weissenbo º ck, 1995; braund, 2001) , although some suspect staggering disease to be due to borna disease virus (bdv) infection (lundgren et al.,1995) . paraneoplastic disorders of the cns, well known in man, have not been reported in cats and dogs (braund et al.,1987) . recent studies on a limited range of infectious agents failed to identify the cause of non-suppurative meningoencephalitis in 47% and 23% of dogs and cats, respectively (rand et al., 1994; tipold, 1995; quesnel et al., 1997; k.melzer, personal communication) . to provide further information, the present study sought to determine the cause of 86 cases of non-suppurative meningoencephalitis in dogs and cats by the immunohistochemical examination of formalin-¢xed, para⁄n wax-embedded tissue. in addition to examination for known viral and bacterial pathogens and prion protein sc ,west nile virus (wnv), a recently emerging cause of disease, was included in the present study (komar, 2000; lichtensteiger et al., 2003; austgen et al., 2004) , together with encephalomyocarditis virus (emcv), an agent so far demonstrated only in the cns of pigs and rodents (nowotny, 1996; sue et al., 2003) . fifty-three canine and 33 feline cases of non-suppurative meningoencephalitis of unknown origin, collected during the period 1998^2003 by the department of pathology of the university of veterinary medicine hannover, germany, were selected. the case selection was made in the light of a preliminary investigation which revealed that 274 of 1209 (23%) dogs and 146 of 741 (20%) cats examined in a 5-year-period (1998^2002) displayed changes in the cns. non-suppurative meningoencephalitis accounted for 22% and 49% of the cns changes in dogs and cats, respectively. the cause of 49 canine and 32 feline cases remained undetermined after examination by routine diagnostic procedures. these 81 animals, together with four dogs and one cat with undetermined non-suppurative meningoencephalitis encountered in 2003, were included in this study. due to the retrospective nature of the present study, clinical data were restricted to a short statement from the records of the department of pathology in most cases (46 dogs and 30 cats). in seven dogs and three cats, no clinical history was available. archived cns sections of each case, stained with haematoxylin and eosin (he), were re-examined for in-£ammatory changes. from each animal, one section of the cerebral cortex, the hippocampus (missing in three dogs and four cats), the midbrain (missing in ¢ve dogs and ¢ve cats), the cerebellum (missing in two dogs and two cats), the brain stem (missing in 17 dogs and 13 cats) and the spinal cord (available in only seven dogs and two cats) were examined. in one canine section, a spinal cord ganglion was present. the distribution of the lesions (e.g., polio-, leuco-, and panencephalitis) was categorized according to summers et al. (1995) . the type of in£ammation was de¢ned as follows: lymphohistiocytic, if in¢ltrates consisted mainly of lymphocytes and macrophages; granulomatous, if macrophages predominated; pyogranulomatous, if neutrophils and macrophages were the main cell types; and mixed, if macrophages, lymphocytes, plasma cells and neutrophils appeared in equal quantities. the degree of histological changes were determined semiquantitatively. perivascular in¢ltrates were described as: mild (1^15 perivascular cells per â100 ¢eld, or 1^2 cell layers per vessel, or both); moderate (16^30 perivascular cells per â100 ¢eld or 2^3 cell layers per vessel, or both); and severe (430 perivascular cells per â 100 ¢eld or x3 cell layers per vessel, or both). meningeal in¢ltrates were described as mild (one or no more than a few mild in¢ltrates), moderate (one moderate in¢ltrate or several mild in¢ltrates), or severe (several moderate in¢ltrates or con£uent in¢ltrates). if suggested by histological results, slides were additionally stained with periodic acid^schi¡ (16 dogs, six cats), ziehl^neelsen (11dogs, one cat), grocott (10 dogs, four cats), luxol-fast blue (nine dogs, one cat), cresylecht-violet (two dogs, one cat), alcian-blue (one cat) or congo-red (one cat), as described by romeis (1994) . for the detection of 18 infectious agents (table 1) , the avidin^biotin-peroxidase complex (abc) method was performed, as previously described (hsu et al., 1981; wu º nschmann et al.,1997) . due to their known cross-reactivity for the detection of canine and feline herpesvirus, parvovirus and coronavirus antigen, the same antibodies were used for both species. feline tissue was not examined for canine adenovirus 1and canine tissue was not examined for feline leukaemia virus (felv). ihc for the detection of felvantigen was additionally performed on lymphoid tissue; however, such tissue was available from only 20 of the 33 cats. in one dog that showed immunoreactivity for porcine herpesvirus 1, tissue from the gastrointestinal tract was also examined, and in one dog and two cats showing immunoreactivity for emcv, myocardial tissue was examined. brie£y, specimens were dewaxed in graded alcohols and endogenous peroxidase was quenched with h 2 o 2 0.5% in ethanol.the pre-treatments applied to sections (table 2 ) depended on the primary antibody and were performed as previously described (see below). the various pre-treatments were as follows: pronase e (merck, darmstadt, germany; bahn, 1988) ; trypsin (fluka, neu ulm, germany; nietfeld et al., 1989) ; kovacevic et al., 1997) ; pepsin (dako, hamburg, germany; bahn, 1988) , proteinase k and formic acid (merck, darmstadt, germany; hardt et al., 2000) ; and microwave pre-treatment (kahveci et al., 2003) . goat serum, diluted 1 in 5 in phosphate-bu¡ered saline (pbs), served as a blocking serum. primary antibodies were, unless stated otherwise, diluted in pbs containing bovine serum albumin (bsa) 1%, and incubated overnight (12^16 h) at 4 1c ( table 2) . biotinylated goat-anti-mouse (gam-b), goatanti-rabbit (gar-b) and goat-anti-£uorescein-isothiocyanate (ga-fitc-b; directed against the fitcligated anti-feline infectious peritonitis [fip] virus antibody) antibodies served as secondary antibodies (vector laboratories, burlingame, ca, usa) and were all diluted 1 in 200 in pbs. finally, sections were incubated with abc (vector) for 30 min at room temperature. between the incubation steps, sections were thoroughly rinsed with pbs. positive antigenâ ntibody reactions were ''visualized'' by incubation with 3,3 0 -diaminobenzidine-tetrahydrochloride (dab) with h 2 o 2 0.5% for 10 min followed by mild counterstaining with haematoxylin. sections were mounted with roti-histokit (carl roth kg, karlsruhe, germany) under coverslips. in control sections the primary and secondary antibodies were replaced by pbs, control serum or isotype-speci¢c antibodies directed against irrelevant antigens. toy breeds constituted 27% (14) ). animals aged less than 1 year accounted for 38% of a¡ected dogs,37% were1^5 years old, and 21% were aged45 years. in 4% of dogs the age was unknown. the domestic shorthair breed accounted for 52% of affected cats. in 11 cats the breed was unknown. cats less than 1 year of age accounted for 27%, 26% were aged 1^5 years, and 26% were45 years old. in 21%, the age was unknown. the sexes were represented equally in both species. neurological signs, including ataxia, seizures and central blindness, were reported in 35 dogs (68%). nine dogs (17%) showed other symptoms such as vomiting and diarrhoea but lacked neurological signs. one dog died spontaneously and one under anaesthesia during routine surgery, no clinical signs having been reported before death. in seven dogs no clinical data were available. neurological signs were reported in 17 cats (52%). thirteen (39%) showed gastrointestinal or respiratory signs but not neurological signs. in three cats, one of which died spontaneously, no clinical data were available. lymphohistiocytic, perivascular in¢ltrates were found in 40 dogs (75%) and 23 cats (70%). predominantly granulomatous in¢ltrates were found in 10 dogs and two cats. mixed in£ammatory in¢ltrates appeared more often in cats (six animals) than in dogs (two animals). two cats and one dog exhibited pyogranulomatous in£ammation. in 39 dogs (74%), encephalitis was the most prominent ¢nding. in 13 dogs (25%), in¢ltrates were restricted to the meninx and a ganglioneuritis without encephalitis or meningitis was found in one animal. in 26 cats (79%), encephalitis was observed. in six cats (18%), in¢ltrates were restricted to the meninx and a chorioiditis without encephalitis was found in one animal. six dogs showed, in addition to the in£ammatory changes in the cns, mild-to-moderate vacuolation of the white matter of the cerebrum (three animals) or cerebellum (one animal) or both (two animals). luxol-fast-blue staining revealed demyelination in two of these dogs. only one of these six dogs showed an in-£ammatory in¢ltrate within the areas of vacuolation. two cats showed in addition to the in£ammatory changes in the cns, mild vacuolation of the white matter of the cerebellum (one animal; severe) or both cerebrum and cerebellum (one animal; mild). luxol-fastblue staining did not reveal demyelination. of the nine dogs without neurological signs, one showed mild, focal meningitis and two showed mild, multifocal meningitis. moderate in£ammation, consisting of multifocal or focal in¢ltrations in various brain regions (midbrain, cerebellum, meninges, choroid plexus) was observed in six dogs. in£ammatory changes were lymphohistiocytic (six cases), mixed (two cases) or granulomatous (one case). the 13 cats without neurological signs showed mild, multifocal in£ammation of the meninx (two cases), meninx and cerebellum (two), meninx and cerebrum (one), meninx, cerebrum and brainstem (one), meninx 8000 (pbs contained 20% nss but no bsa), adsorbed to rat brain powder microwave gam-b anti-prion protein sc 400 (pbs contained 10% goat serum but no bsa); no further blocking with goat serum necessary formic acid, proteinase k, microwave anti-e. coli 1600 none gar-b nss, normal swine serum; tuf, target unmasking £uid; gam-b, biotinylated goat-anti-mouse antibody; gar-b, biotinylated goat-anti-rabbit antibody; ga-fitc-b, biotinylated goat-anti-fitc antibody (see materials and methods). ã against agents listed intable 1. y in phosphate-bu¡ered saline (pbs) containing bovine serum albumin (bsa) 1%. and brainstem (one) or choroid plexus (one). also seen were mild-to-moderate in£ammation of the brainstem (one) and of the meninx and cerebral cortex in addition (one). one cat showed moderate, focal meningitis, one a moderate multifocal meningoencephalitis and one a severe, generalized choroiditis. the type of in£ammation was lymphohistiocytic in six cases, mixed in three, granulomatous in two, and pyogranulomatous in two. the gastrointestinal tissue examined in one dog, and the myocardial tissue examined in one dog and two cats, showed no signi¢cant histological changes. in 14 dogs (26%) and 13 cats (39%), viral or bacterial antigens were detected by ihc (table 3) , two dogs and one cat giving positive results for two infectious agents simultaneously. control sections containing the appropriate antigens gave positive signals, in contrast to negative controls. special histochemical stains invariably gave negative results. in12 of the14 positive dogs and nine of the13 positive cats, reactions were found only in cns regions in¢ltrated with in£ammatory cells. the remaining two dogs and four cats showed immunoreactivity only in cns regions without in£ammatory in¢ltration. of the 14 positive dogs,12 (94%), had shown neurological signs, almost always said to be severe. it was not possible, however, to relate the histological or ihc results to the clinical signs. one dog positive for emcv antigen did not show neurological signs, and for one dog positive for wnv antigen no clinical data were available. of the 13 positive cats, eight had shown neurological signs (severe in three cases). in the remaining ¢ve cats, no statement regarding the severity of signs was recorded. four cats with a positive immunoreaction showed no neurological signs. for one positive cat, no clinical data were available. in a 2-year-old male canadian shepherd dog with diarrhoea and progressive convulsions, porcine herpesvirus-1 (phv-1) antigen was demonstrated in neuronal perikarya of the gyrus parahippocampalis. no phv-1 antigen was detected in the hippocampus, cerebellum, brain stem, spinal cord, remaining part of the cerebrum, or the gastrointestinal tract. histologically, there was mild, lymphohistiocytic polioencephalitis in the gyrus parahippocampalis and occasional neuronal necrosis in the ca1 and ca2 regions of the hippocampus. parvovirus antigen was demonstrated in the cns of ¢ve greek hunting dog puppies from two litters originating from the same breeder, and in a 2-week-old cat. clinically, the dogs had shown generalized tremor and jumping movements in the hind limbs, and the neurological signs in the cat were characterized by severe ataxia, opisthotonus and convulsions. neither in the dogs nor in the cat were cerebellar hypoplasia or necrotic purkinje cells observed. histopathologically, all affected dogs showed mild to moderate lymphohistiocytic meningitis or leucoencephalitis (or both) as well as mild to moderate, and in one case severe, vacuolation in the white matter of cerebrum and cerebellum. parvovirus antigen was demonstrated in periventricular cells resembling spongioblasts, in macrophages, microglia and astrocytes, and in cells of the outer granular layer of the cerebellum (fig. 1) . the histopathological changes in the cat consisted of mild, lymphohistiocytic in£ammation of the meninges and cerebellum, without vacuolation. parvovirus antigen was detected in the cytoplasm and nucleus of numerous small and large neurons, mainly in the granular layer and outer granular layer of the cerebellum, in the grey matter of the spinal cord, and multifocally in most other brain regions (fig. 2) . in addition, parvovirus antigen was observed in macrophages, microglia, astrocytes and ependymal cells. fip virus antigen was detected in three cats aged 6,18 and 36 months.two a¡ected cats had shown increasing apathy and recurrent fever attacks (one animal) or diarrhoea and dyspnoea (one). no clinical data were available for the third cat. viral antigen was demonstrated in the cytoplasm of macrophages within the in-£amed brain regions in all three cases. histologically, lesions consisted of pyogranulomatous and mixed in¢ltrates in the plexus chorioideus and the meninges. the third cat showed moderate lymphohistiocytic leucoencephalitis in the caudal regions of the cerebrum. weak immunolabelling for feline leukaemia virus antigen was seen in microglia and astrocytes in the hypothalamus of a 9.5-year-old european shorthair cat with progressive apathy and increased salivation (fig. 3) . histopathological changes consisted of mild multifocal lymphohistiocytic meningitis and severe satellitosis in the cerebral cortex. no lymphoid tissues or other organs were available for further investigations. five dogs (8.9%) and four cats (11.8%) showed immunoreactivity for west nile virus (wnv) antigen. in one dog, canine parain£uenza virus (cpiv) antigen was also detected in the same brain region (see also below). one of these dogs, and one cat also gave positive reactions with emcv-speci¢c antibodies (see also below). in four of the ¢ve dogs, severe neurological signs, including ataxia, convulsions and central blindness, had been recorded. no clinical data were avail-able for the ¢fth dog. all four cats had shown neurological signs, reported in one case to consist of severe circling, hypermetria and blindness. in four of the ¢ve dogs and two of the four cats a moderate to severe meningoencephalitis a¡ecting the grey and white matter was observed; this varied from granulomatous, pyogranulomatous, lymphohistiocytic to ¢brinopurulent, occasionally associated with neuronal necrosis or malacia. in the remaining dog, in£ammation was restricted to the grey matter of the cerebrum and cerebellum, being associated with severe malacia in the hippocampus. one cat showed mild focal, lymphocytic polioencephalitis and moderate focal, lymphohistiocytic meningitis, as well as severe vacuolation of the cerebral white matter and severe generalized neuronal necrosis. in another cat, severe, focal, ¢brinopurulent meningitis and severe, acute neuronal necrosis in the hippocampus were observed. wnv immunolabelling was seen in the cytoplasm of various types of neurons, macrophages, astrocytes and microglia (fig. 4) . strong immunolabelling of neutrophils was observed in two cats with pyogranulomatous in£ammation (fig. 5) . these immunohistochemical reactions were in general more intense with the anti-nsp-1wnvantibody than with the anti-mep-e wnvantibody. in some cases, immunoreactivity was observed with only one of these two wnv monoclonal antibodies. one dog showed immunoreactivity in pyramidal cells of the ca1 and ca2 sector of the hippocampus, and another in the frontal cortex, respectively. a positive reaction in numerous macrophages and microglial cells in the midbrain, pons and frontal cerebrum was detected only with the anti-nsp-1 antibody. one dog showed wnv immunolabelling in numerous macrophages and microglial cells, and pyramidal cells in the cerebrum, hippocampus and midbrain, but only with the anti-nsp-1 antibody. another dog, in contrast, showed immunoreactivity in a few neurons and macrophages in an in£amed part of the midbrain, but only with the anti-mep-e antibody. one cat showed immunolabelling with both wnv antibodies in numerous neutrophils and occasional macrophages in the plexus chorioideus and laterally to the third ventricle. in this cat, small neurons, macrophages and microglial cells in the same brain region reacted only with the anti-nsp-1 antibody. in another cat, astrocytes and perivascular macrophages in the lobus parietalis reacted with both antibodies, whereas the anti-nsp-1 antibody additionally labelled pyramidal cells in the same brain region. in one cat, astrocytes and oligodendrocytes of the medulla oblongata displayed more abundant positive signals with the anti-nsp-1 antibody than with anti-mep-e. additionally, in this animal, the anti-nsp-1 antibody labelled large neurons in the medulla oblongata which failed to react with the anti-mep-e antibody. canine parain£uenza virus (cpiv) antigen was detected in the brain of a 9-year-old male collie that was also positive forwest nile virus antigen (see alsownv, above). this dog had exhibited changed behaviour and was generally weak. histopathologically, severe periventricular granulomatous meningoencephalitis was found. viral antigen was demonstrated in the cytoplasm of occasional macrophages in the hypothalamus (fig. 6) . ihc revealed encephalomyocarditis virus (emcv) antigen in the brains of four dogs (7.1%) and four cats (11.8%). in one dog and one cat, wnv-antigen was detected in addition (see also wnv, above). the dogs were aged 0.3^8 years and the cats 0.2^10 years. two domestic shorthairs, one persian and one cat of unknown breed as well as a landseer, a west highland white terrier, a bordeaux dogge and an entlebucher sennenhund were a¡ected. a granulomatous immune response was found in three of the four dogs. the remaining dog displayed a lymphohistiocytic chorioiditis and meningitis. in¢ltration with macrophages, neutrophils, lymphocytes and plasma cells or pyogranulomatous in£ammation was present in two of the four cats, and lymphohistiocytic in£ammation in the remaining two cats. in three of these four dogs, severe neurological signs had been recorded; the fourth dog had shown dyspnoea and anaemia but no neurological signs. in three of the cats, neurological signs were recorded without reference to their severity. the fourth cat showed respiratory signs only. the eight animals showed di¡erent patterns of immunoreactivity. dab precipitates were observed in the cytoplasm of perivascular and parenchymal macrophages, in the perikarya and processes of various neurons distant non-suppurative meningoencephalitis from or within the in£ammatory lesions (fig.7) , and in periventricular and submeningeal astrocytic foot processes (fig. 8) . in one dog and two cats, vessel-associated cells, probably pericytes or endothelial cells, showed immunolabelling (fig. 9) . seven of the eight animals showed immunolabelling with the emcv-speci-¢c monoclonal antibody (mab) 4f3; mab 3e5 gave results in selected cases only. one cat which was negative with the 4f3 antibody gave emcv-speci¢c reactions with mab 3e5. in one dog the results with mab 4f3 were con¢rmed with the mab 3e5. the mab 4e4, which was applied only in one cat, con¢rmed the reactions observed with the 4f3 antibody in numerous cells of the medulla oblongata and in perivascular macrophages and occasional neurons of the mediocaudal thalamus. the histologically intact myocardial tissue available from one dog and two cats was not immunolabelled with the mab 4f3. e. coli antigen was demonstrated in the brain of a 5-month old male cat. histopathologically, mild to moderate, multifocal, perivascular in¢ltration with lymphocytes, plasma cells, macrophages and neutrophilic granulocytes was observed in the cerebellum and meninges. moderate numbers of bacterial emboli in various vessels were visible in he-stained sections. e. coli antigen was demonstrated immunohistochemically in the cytoplasm of numerous perivascular, meningeal and periventricular macrophages, and in intravascular bacteria. cases remaining unclear included 39 (74%) dogs and 20 (61%) cats, no infectious agent being identi¢ed as the underlying cause of the observed non-suppurative meningoencephalitis. in the present study, cns tissue of 53 dogs and 33 cats with non-suppurative meningoencephalitis of unknown cause was investigated for the presence of 18 different infectious agents. former studies either investigated at most six agents or were based on cases selected only on the presence of neurological signs (tipold, 1995; quesnel et al., 1997; bradshaw et al., 2004; k. melzer, personal communication) . as in other studies (sorjonen, 1992; tipold, 1995) , no speci¢c canine breed predisposition was observed. cases were seen more often in toy breeds than in other breeds, but this may merely have re£ected the normal distribution of dog breeds in germany. so far, no breed predisposition for non-suppurative meningoencephalitis has been described in respect of the cat (sorjonen, 1992) . in the present study, domestic shorthair cats accounted for more than half of the feline cases, probably due to the normal feline breed distribution in germany. the cns of the majority of dogs (40/53) and cats (23/ 33) examined showed lymphohistiocytic perivascular in¢ltrates. this is generally suggestive of a viral aetiology (summers et al., 1995) but has also been reported following hypoxic or toxic damage, which also sometimes produces demyelinating lesions (braund, 1980; lee et al., 2002) . granulomatous cns in¢ltrates, observed in 10 dogs and two cats, are often considered to be associated with fungal, parasitic or bacterial infections. fungal infections occur mainly in warmer climates (gerds-grogan and dayrell-hart, 1997 ) and, like parasitic infections, were not detected in the present study. in the 10 dogs referred to above, granulomatous meningoencephalitis (gme) was suggested by the histopathological ¢ndings and distribution of lesions. in six of these cases, toy breeds were a¡ected (twowest highland white terriers, two chihuahuas, a shih tzu and a rehpinscher). interestingly, three of the a¡ected dogs belonged to large breeds (landseer, bordeaux dogge and collie), in which gme is only rarely reported (braund, 1985) . gme has been thought to be associated with viral infections including canine distemper, but no viral agent has been demonstrated in the cns so far (thomas and eger,1989; kipar et al.,1998) . e. coli antigen, as found in the brains of beagles with granulomatous leptomeningitis (maeda et al., 1993) or in association with human cases of granulomatous encephalitis (rickert et al., 2000) , was not observed in the present cases with suspected gme. the cat with abundant e. coli antigen in the brain in the present study showed perivascular in-¢ltrations, indicating septicaemia. pyogranulomatous and mixed in£ammatory in¢ltration in the cns occurred less often in dogs than in cats. in cats, this type of change is reminiscent of fip (rand et al.,1994) . in the present study, fip virus antigen was also detected in a cat showing lymphohistiocytic leucoencephalitis, a rare consequence of fip (summers et al.,1995; braund, 2001) . in dogs, pyogranulomatous or mixed in£ammatory changes in the cns are occasionally described, but no infectious agents have so far been detected (meric,1988) . it cannot be ruled out that aetiologically undetermined feline cases were due to feline immunode¢ciency virus (fiv), which leads to lymphocytic encephalitis in 30% of naturally infected cats (gunn-moore et al., 1996) . only ¢ve of the 33 cats had been examined serologically for fiv, a positive result having being obtained in one animal. in a canadian shepherd dog, porcine herpesvirus 1 (phv 1) antigen was detected in neurons of the gyrus parahippocampalis. due to a retrograde neural route of infection, phv 1 antigen usually occurs in the brain stem or spinal cord ganglions (quiroga et al., 1998; honavar and meldrum, 2002) . the dog in this study showed diarrhoea for 3 weeks, reminiscent of the rare alimentary form of aujeszky disease (pensaert and maes, 1987) ; however, no phv 1 antigen was detected in the gastrointestinal tract. only later did the animal develop neurological signs, and an unusual portal of entry of the virus to the brain remains a possibility. neither the ¢ve dogs nor the cat with parvovirus infection of the brain showed typical cns manifestations, such as cerebellar hypoplasia or death of purkinje cells (sharp et al., 1999) , or encephalomalacia (johnson and castro, 1984; agungpriyono et al., 1999) . the ¢ndings indicated, however, a potential association between parvovirus and spongiform lesions in the cns. in chronic feline leukaemia virus (felv) infection, lymphocytic in¢ltration of peripheral nerves of the spinal cord has been reported occasionally (pedersen, 1987) . in the brain, however, felv infection has been associated only with degenerative cns disease (carmichael et al., 2002) . in one cat, felv infection of microglia and astrocytes was detected. histologically, this animal showed mild, multifocal, lymphohistiocytic meningitis. since felv regularly persists for years in lymphocytes and macrophages (rohn and overbaugh,1999) , a persistent infection of glial cells seemed possible but unfortunately no lymphoid tissue, including bone marrow (in which felv-antigen can typically be demonstrated), was available. surprisingly, ¢ve (9.4%) dogs and four (12%) cats showed immunolabelling for wnv. histopathologically, these animals showed mild to moderate meningoencephalitis in the grey and white matter, mostly with granulomatous or pyogranulomatous in£ammation. in contrast, previous reports of wnv infection in birds and in mammals indicated that the grey matter was mainly a¡ected and that the histopathology was characterized by lymphoplasmahistiocytic in£ammation with small numbers of neutrophils (steele et al., 2000; cantile et al., 2001; buchweitz et al., 2003; kelly et al., 2003; lichtensteiger et al., 2003) . polyclonal wnv-speci¢c antibodies used for immunolabelling often cross-react with other £aviviruses (steele et al., 2000; kelly et al., 2003; chvala et al., 2004) . the monoclonal antibodies speci¢c for the major envelope protein e (anti-mep) and non-structural protein 1 (anti-nsp-1) used in this study (tables 1 and 2 ) crossreact with kunjin virus, also a member of the flaviviridae (scherret et al., 2001) . presumably, the anti-nsp-1 antibody is more speci¢c than the anti-mep antibody (hall, 2000) . this might explain its stronger immunoreactivity in the present study and might support the identi¢cation of a wnv infection. the a¡ected animals did not react positively for tick-borne encephalitis (tbe) virus antigen with a polyclonal speci¢c antibody that may cross-react with other £aviviruses (h. weissenbo º ck, personal communication). however, it is still possible that the positive wnv reaction was due to infection with other cross-reactive agents or represented molecular mimicry of host-derived antigens (oldstone, 1998) . the latter possibility receives some support from studies indicating that dogs and cats can be infected with wnv but usually develop only a low viraemia, without clinical signs or histopathological changes in the brain (blackburn et al.,1989; lichtensteiger et al., 2003; austgen et al., 2004) . furthermore, no evidence of wnv infections in domestic animals has so far been detected in germany. therefore, the signi¢cance of the present ¢ndings requires further studies. natural infections of dogs with canine parain£uenza virus (cpiv) are typically associated with hydrocephalus and necrotizing periventricular encephalitis, but neither viral antigen nor rna has been detected so far in spontaneous cases (baumgìrtner et al., 1982b; vieler et al.,1994; cantile et al.,1997) . after experimental infection with cpiv, viral antigen was detected in ependymal cells and neurons and in macrophages, in ferrets and dogs respectively (baumgìrtner et al., 1982a (baumgìrtner et al., , b, 1989a w. baumgìrtner, unpublished) . cpiv infection of brain macrophages was also observed in the present study in a collie with granulomatous encephalitis characteristic of gme. interestingly, this dog also showed immunolabelling of wnv antigen. it remains unclear, therefore, whether the detection of cpivantigen represented a true infection, or whether immunoreactivity was due to a cross-reaction of the antibody with degraded cellular antigens, or to molecular mimicry (oldstone,1998) . a further surprising ¢nding was the detection of emcvantigen in the cns of four dogs and four cats. emcv-speci¢c antibodies have been detected in primates, mice, rats, horses and elephants in various parts of the world, the rat being the natural host (nowotny et al.,1993; nowotny,1996; psalla et al., 2006) . in austria, a low seroprevalence has been reported in cats (nowotny,1996) , but not dogs. in germany, emcv-associated non-suppurative meningoencephalitis is reported occasionally in man, but the virus has been isolated from the cns in only a few cases (nowotny et al., 1993; larue et al., 2003) . the histopathological changes reported here varied (neuronal necrosis, mild lymphohistiocytic meningitis, severe generalized granulomatous or pyogranulomatous meningoencephalitis). in pigs with natural emcv infections, lymphohistiocytic in¢ltrates in the cns have been described (gainer et al., 1968) . experimentally infected mice and pigs also develop lymphohistiocytic, perivascular in¢ltrates in the cns, poliomeningoencephalomyelitis being the predominant feature in mice (topham et al., 1991; larue et al., 2003) . in the present study, emcv immunolabelling was observed in perikarya and processes of neurons in some animals, indicating neurotropism similar to that observed in experimental emcv infection. the emcv-positive cells, associated with cerebral vessels, may have been endothelial cells or pericytes. neither cell type has been reported to be susceptible to emcv, but endothelial and mesenchymal cells in organs other than the cns are regularly infected with emcv, as shown by papaioannou et al. (2003) in pigs. the detection of emcv antigen in macrophages in the cns of dogs and cats may support their role in viral replication and distribution, as proposed by papaioannou et al. (2003) . in natural emcv infection of pigs, interstitial myocarditis and myocardial degeneration represent the main features (papaioannou et al., 2003) . neither change was observed in any of the present cases, but myocardial tissue was available from only one dog and two cats. despite the broad spectrum of antibodies used, the aetiology of the cns lesions could be determined in only 26% and 39% of the cases in dogs and cats, respectively. due to the retrospective character of the study, a standardized clinical and neurological investigation and serum and csf laboratory procedures were not possible. in the remaining cases, infection cannot be ruled out. other agents that might have caused the observed histopathological changes in dogs include ehrlichia spp. (glaus and jaggy, 1992; maretzki et al., 1994) , leptospira spp. (rentko et al., 1992) and borrelia spp. (mandel et al.,1993; chang et al., 2001) , and in cats bartonella henselae (breitschwerdt et al., 1999) cannot be excluded. however, the high percentage of cases in which no infectious agent was identi¢ed suggests that non-infectious causes are more common than anticipated. various factors, ranging from autoimmune processes to intoxication, have already been suggested as causes of non-suppurative changes in the cns (kipar et al.,1998; , lee et al., 2002 . the immunohistochemical reactions for wnv, cpivand emcv, which were unexpected, were possibly due to molecular mimicry, a phenomenon reported for various paramyxoviruses and forjapanese encephalitis virus (jpev; shesberadaran and norrby, 1984) , which belongs to the flaviviridae. in most of the cases showing these unexpected reactions the histopathological changes were severe and often resembled gme. autoimmune mechanisms have already been discussed as a possible cause of canine gme and pug dog encephalitis (kipar et al., 1998; . other factors that may have played a role in the ¢ndings described include the following: epitope spreading vanderlugt et al., 1998) ; hereditary factors, as already suspected in pug dog encephalitis and non-suppurative meningoencephalitis in greyhounds (hinrichs et al., 1996; callanan et al., 2002) ; intoxication (lee et al., 2002) ; increased permeability of the blood-brain barrier in systemic illness, due to prostaglandins and interleukin (il)-1, il-6 or tumour necrosis factor (tnf)-a, leading to in¢ltration of the cns by in£ammatory cells (reiss et al., 2000) . in addition, it should be mentioned that, unlike paraneoplastic encephalitis, which has been described in man but not in dogs or cats (scaravilli et al., 1999) , paraneoplastic polyneuropathy has been described in dogs (braund, 1990; wagner, 2002) . as tumours in the cns or other organs were present in only one of the cases examined, paraneoplastic encephalitis seemed unlikely to have played any role in the ¢ndings. in conclusion, in a high proportion of dogs and cats with non-suppurative meningoencephalitis, immunohistochemical examination with 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practicing veterinarian granulocytic ehrlichiosis and meningitis in a dog canine meningitisça changing emphasis rabies-like inclusions in dogs serological studies of domestic cats for potential human pathogenic virus infections from wild rodents encephalomyocarditis-virusinfektion beim german.). wiener tierìrztliche monatsschrift description of feline meningoencephalomyelitis (''staggering disease'') and studies of its aetiology molecular mimicry and immunemediated diseases pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study feline leukaemia virus pseudorabies virus (aujeszky 0 s disease) pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats diagnostic evaluation of cats with seizure disorders: 30 cases diagnosis of aujeszky 0 s disease virus infections in dogs by use of immunohistochemistry and 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dogs: a retrospective study indirect role of tcells in development of polioencephalitis and encephalomyelitis induced by encephalomyocarditis virus the functional signi¢cance of epitope spreading and its regulation by co-stimulatory molecules polioencephalomyelitis in cats isolation of parain£uenzavirus type 2 from the prostatic £uid of a dog immunohistochemical detection of encephalomyocarditis virus (emcv) both in formalin and ethanol-¢xed, para⁄n-embedded tissues of pigs identi¢cation of cd4+ and cd8+ t cell subsets and b cells in the brain of dogs with spontaneous acute, subacute, and chronic-demyelinating distemper encephalitis glycoprotein speci¢c immune response in canine herpesvirus infection for providng antibodies, the authors thank: prof. haas, institute of virology, university of veterinary medicine hannover, germany, prof. richt, national animal disease center, ames, iowa, usa; dr eskens, veterinìr-untersuchungsamt mittelhessen; prof. holzmann, institute of virology, university of vienna; prof. groschup, bundesforschungsanstalt fu º rviruskrankheiten dertiere, insel riems; dr randall, department of biochemistry and microbiology, university of st andrews, uk; and dr o º rvell, huddinge hospital, huddinge, sweden. thanks are also due to mrs petra gru º nig and mrs danuta waschke for excellent technical support. key: cord-303641-v2kl62c8 authors: cruz-chan, julio vladimir; carmen aguilar-cetina, amarú del; estefanía villanueva-lizama, liliana; pablo martínez-vega, pedro; jesús ramírez-sierra, maria; enrique rosado-vallado, miguel; leonardo guillermo-cordero, josé; dumonteil, eric title: a canine model of experimental infection with leishmania (l.) mexicana date: 2014-08-09 journal: parasit vectors doi: 10.1186/1756-3305-7-361 sha: doc_id: 303641 cord_uid: v2kl62c8 background: cutaneous leishmaniasis is a tropical disease affecting over one million patients annually and leishmania (l.) mexicana is one of the major etiological agents in the americas. here we established the first experimental infection of l. (l.) mexicana in canids. methods: beagle dogs were infected intradermally with culture-derived l. (l.) mexicana. we followed skin ulcer development, histopathological signs, parasite burden and the immune status of the infected dogs. results: all infected dogs developed uniform oval-craterform ulcers similar to those observed in humans, associated with mixed t helper 1/t helper 2 immune responses. parasites were detected in the healed lesions 15 weeks post-infection. higher anti-leishmania igg levels correlated with larger lesions and high igg1/igg2 ratio was associated with some level of splenomegaly. conclusions: the canine model described in this work will be of use for further understanding of l. (l.) mexicana immunopathogenensis, and for drug and vaccine development. the leishmaniases are a group of parasitic disease affecting up to 1.4 millions people annually [1] . cutaneous (chiclero's ulcer), mucocutaneous and visceral leishmaniasis (kala-azar) are the different clinical forms of the disease, which is caused by parasites from two subgenus: leishmania (leishmania) and leishmania (viannia). the parasite is transmitted by sand flies of the phlebotomus and lutzomyia genus in the old and new world, respectively. cutaneous leishmaniasis (cl) ranks ninth of infectious neglected-tropical diseases, with an estimated burden of 1.2 million new cases per year, and of 770,000 dalys (disability-adjusted life years) [1] [2] [3] . l. (l.) mexicana is one of the major species responsible for cl in the americas. it can be found in argentina, brazil, costa rica, guatemala, and as far north as mexico [1, 4, 5] . in mexico, the ministry of health reports an incidence of 500-900 cases annually, although the disease is likely to be under-reported [5, 6] . drug treatment for infected patients is complicated, with only few drugs available with limited efficacy, and a vaccine is still in early experimental stages [1, 5, 7, 8] . dogs are believed to play an important role as a domestic reservoir of leishmania parasites, as demonstrated for l. (l.) chagasi [9] . this is due to relatively high natural infection rates in many regions, ranging from 4 to 60% [10] [11] [12] [13] [14] [15] and the life-long persistence of parasites, particularly in the skin, with or without clinical signs of disease [9] . drug treatment of infected dogs is not recommended, to prevent the emergence of drug resistance of the parasite [16] . as a consequence, some countries such as brazil are recommending the culling of infected dogs to prevent spreading of the disease, although the efficacy of such measure has been criticized [17] . an important priority is thus to develop veterinary vaccines, which require appropriate animal models for their evaluation. rodent models have been extensively used for the study of leishmania infections, including inbred and outbred mouse strains and hamsters [18, 19] , and have provided key information on the immunopathology of the disease. however, studies in dogs are also warranted for a further understanding of their role in leishmania transmission and for testing drug and vaccine efficacy [20, 21] . most studies on canine leishmaniasis have focused on the visceral form caused by l. (l.) chagasi or l. (l.) infantum, and are based on observations of both naturally and experimentally infected animals [21] [22] [23] [24] [25] [26] [27] . unexpectedly, canine cutaneous leishmaniasis has been little studied in domestic dogs and is poorly described. a high prevalence of infection by l. (v.) braziliensis, l. (v.) guayanensis, and l. (l.) panamensis has been reported in dogs from colombia [27, 28] . in mexico, cases of canine leishmaniasis have been reported, possibly caused by l. (l.) infantum [29] , although the parasite species was not determined. cases of natural infection with l. (l.) mexicana have also been described [30] . dogs naturally infected with l. (v.) braziliensis were found to present thrombocytopenia, anemia and skin lesions [31] . parasites could be observed in 35% of the lesions, which also presented diffuse chronic inflammation of the dermis and fibrinoid degeneration, and less frequently some vasculitis [32] . however, experimental models of canine cutaneous leishmaniasis are scarce. the experimental infection with l. (v.) brasiliensis was found to induce a cutaneous lesion in three of four mongrel dogs, 4-8 months after infection, and the lesions tended to heal after 3-5 months [33] . histologically, these lesions had similar characteristics as those observed in naturally infected animals [33] , but additional studies are needed to further characterize this dog model. in this study, we developed a canine model of cutaneous leishmaniasis caused by experimental infection with l. (l.) mexicana parasites, the main species circulating in southern mexico and central america. we described the clinical, parasitological and immunological aspects of the experimental infection in beagle dogs, which provide a good model for the future testing of novel drugs or vaccines against cutaneous leishmaniasis, as well as for further studies on the immunopathogenensis of this canine host. six beagle dogs from three litters and aged between two and three months were used. animals were acclimated for three/four months in the animal facility. temperature, light and food were controlled. they received treatment against helminths and were vaccinated against rabies virus, canine distemper virus, type 2 adenovirus, coronavirus, parainfluenza, parvovirus and leptospira. all animal procedures were performed according to national and international guidelines approved by the institutional bioethics committee from the autonomous university of yucatan (authorization number cbi-cir-11-04). leishmania (l.) mexicana mhet/mx/97/hd18 strain was cultured in 199 media with 15% of fetal bovine serum, 5 μm mercaptoethanol, 20 mm sodium pyruvate, 100 iu penicillin and 100 mg/ml streptomycin, and 5% of filtered human urine. under general anesthesia (ketamine/xilazine, 8:1 mg/kg, iv), beagle dogs received 7 × 10 7 promastigotes resuspended in 50 μl of pbs solution intradermally on the shaved back [34] . beagle dogs were under daily examination for clinical signs including rectal temperature, breath and heart rate. additionally, feces color, presence of polydipsia, polyuria and/or any abnormal excretions were observed. skin lesions were photographed and measured weekly using a vernier caliper over a period of 15 weeks. sybr green-based real time-pcr was optimized to quantify l. (l.) mexicana parasite burden in prescapular lymph nodes and skin lesions. first, 25 mg of tissue was ground with a morter and pestle and dna was obtained with a wizard® genomic dna purification kit (promega madison wi) following the manufacturer's instructions. we used primers targeting a 140 bp sequence from l. (l.) mexicana minicircle: forward primer: 5′-aatgc gagtgttgcccttttg-3′ and reverse primer: 5′-gccgaacaacgccatattaacc-3′ [35] . reactions contained 50 ng of dna in a reaction volume of 20 μl with 500 nm (each) forward and reverse primers, and 1× kappa sybr fast universal qpcr mix. reactions consisted of a 10 min activation at 95°c followed by 40 cycles of 15 s at 95°c and 1 min 60°c, and a high resolution melt curve analysis at the end of the reaction. a standard curve was prepared with uninfected dog dna spiked with serial dilutions of l. (l.) mexicana dna covering a dynamic range of 150 to 1.5 × 10 6 parasite equivalent/reaction. all samples and standards were run in triplicates and the standard deviation among triplicates was less than 0.6 cq. dogs were euthanized 15 weeks post-infection with a barbiturate overdose and necropsies were performed immediately registering detail of cavities, organs and fluids. spleen and liver were extracted, weighted and measured [36] . biopsies from the skin, spleen, prescapular lymph node and liver tissues were embedded in paraffin and sections were stained with hematoxylin and eosin to evaluate lesions and/or parasites. the humoral immune response induced by l. (l.) mexicana infection was evaluated by measuring levels of total igg, igg1 and igg2 subtypes, against parasite lysate and nh36 recombinant antigen [37, 38] . soluble l. (l.) mexicana antigen (sla) was prepared from parasites cultured as described previously following 6 days culture in 199 hanks® medium (life technologies carlsbad ca). briefly, 4 × 10 5 /ml parasites were washed three times with phosphate buffer solution (pbs) ph 7.2 and sonicated by three ultrasound cycles (sonics vibra cell 130, newtown ca) of 1 min at 45 w at 4°c. the lysed suspension was centrifuged at 5000 × g for 30 min at 4°c. the supernatant was recollected and stored in aliquots at 70°c until used [39] . recombinant nh36 (rnh36) was generously provided by dr. peter hotez (baylor college of medicine, houston, tx, usa). ninety six-well plates were coated overnight with soluble l. (l.) mexicana mhet/mx/97/ hd18 antigen (20 μg/ml) or rnh36 recombinant protein (2.5 μg/ml). after blocking, plasma samples (1:100 and 1:50 dilutions for total igg and igg isotypes, respectively) were incubated for 1 h at 37°c, and then washed. phosphatase-conjugated rabbit anti-dog igg (1:4000 dilution, sigma, usa), peroxidase-conjugated goat antidog igg1 or sheep anti-dog igg2 (1:1000 dilution, serotec, uk) were added and after washing, the plates were revealed with pnpp or o-phenylenediamine substrates, respectively, and absorbance was measured on a bio-rad 550 microplate reader. cytokines ifnγ and il-10 levels were quantified in plasma as well as spleen and liver extract supernatants. for this, 1 g of fresh tissue was ground with a mortar and pestle and resuspended with 2 ml of rpmi-1640 medium (sigma-aldrich inc, st. louis, mo). the homogenates were centrifuged at 10,000 g for 15 minutes at 4°c and the supernatants were stored at -70°c [40] . ifnγ and il-10 duoset canine kits (r&d systems inc., minneapolis) were used to measure cytokine levels in tissue supernantants and plasma samples according to the instructions of the manufacturer. dogs were infected with a dose of 7 × 10 7 l. (l.) mexicana promastigotes via intradermal route. all animals showed a local erythema nine days post-infection at the site of injection. at 3-4 weeks post-infection, we observed a papule in all animals, which ulcerated and expanded, to form a typical oval crater-like lesion ( figure 1 ). the majority of animals presented a lesion of about 1 cm in diameter, with some variation (range of 0.4 -1.8 cm in diameter). active skin ulcers were present for a median of 8 weeks until healing, with large individual variations of this duration, ranging from six to 11 weeks (figure 1 ). the lesion in one male dog never healed, as it was still active after 15 weeks of infection. some effect of the litter of origin was observed in size and duration of the lesions. beagles 1 and 6 belonging to one litter had larger lesions, while beagles 2 and 3 from another litter presented smaller but long-lasting lesions and beagles 4 and 5 from a third litter had intermediate size lesions. daily rectal temperature measurements showed weekly increases >38.6°c in all dogs. pyrexia was also observed in two dogs on day 25 post-infection, just after ulcer formation, reaching 39.3 and 40°c, respectively. infected animals were euthanized at week 15 postinfection, when lesions had healed except in one dog, and necropsy was carried out to detect any gross abnormality. all analyzed tissues appeared macroscopically normal. spleen was weighted and the data normalized to body weight. four of the six dogs (three males and one female) presented a splenomegaly outside the normal range ( figure 2) . biopsies from different tissues were also analyzed for histopathologic damage. skin tissue from the healed lesions showed focal inflammatory cell infiltration in the dermis around adipose tissue, and a loss of epidermis integrity ( figure 3c ). prescapular lymph nodes presented disseminated atrophy with interstitial edema and an enlarged cortical layer with necrotic fibrinoid zones. there was also an intense vasculitis in this tissue ( figure 3a ). the liver of beagle 4 and 6 showed hydropic and lipid severe degeneration around the central area and multiple focus of oncotic necrosis ( figure 3b ). in the kidneys, a severe case of inflammation and interstitial nephritis was found in beagle 4. there was an extensive histiocytic and lymphocytic infiltrate displacing renal tubules ( figure 3d ). some inflammation was observed in the kidneys of the other infected animals. however, no parasites or infected cells were observed in any of the tissue sections. parasite burden was measured at the site of the healed lesion and in the prescapular lymph node by qpcr targeting l. (l.) mexicana kdna. parasites were detected in all biopsies from all infected animals, with similar parasite burdens in the skin and in the lymph nodes ( figure 4 ). parasite burden varied from 36-3750 parasites equivalent/mg in the lymph nodes and between 57-301 parasites equivalent/mg in the healed lesions. a higher parasite burden in the healed lesion tended to be associated with increased splenomegaly, although this did not reach statistical significance (r 2 = 0.62, p = 0.06), and no association was found with the parasite burden in the lymph nodes. the immune response of the infected dogs was analyzed to determine whether potential immune responses correlated with disease progression. we measured serum antibody levels as well as ifnγ and il-10 cytokine levels in serum and tissues at different time points. anti-leishmania total igg rapidly increased following infection to reach a maximum level at 8 weeks post-infection. antibody levels were maintained up to 12 weeks post-infection ( figure 5a ). high igg levels were significantly associated with a large lesion size (r 2 = 0.87, p = 0.005). analysis of antibody isotypes showed a gradual igg isotype switching over the course of infection, from a predominant igg2 isotype at four weeks post-infection to a predominant igg1 isotype at 12 weeks post-infection, as indicated by the changes in igg1/igg2 ratio ( figure 5b) . a high igg1/igg2 ratio at 12 weeks post-infection was significantly associated with splenomegaly (r 2 = 0.68, p = 0.04), and tended to be associated with an increased parasite burden in the lesion (r 2 = 0.42, p = 0.15). we also evaluated nh36-specific antibodies. anti-nh36 igg levels reached their maximum at 8 weeks post-infection, but then presented some decrease at 12 weeks post infection ( figure 5c ). ifnγ and il-10 cytokines were measured in liver and spleen tissue homogenates, as well as in serum samples at 15 weeks post-infection, by elisa. il-10 could not be detected in spleen or plasma samples, and it was only detected in liver samples ( figure 6 ). on the other hand, ifnγ was found in all samples and appeared to be the predominant cytokine compared to il-10 ( figure 6 ). no significant associations were found between cytokine levels and the other immune or parasitological parameters (p > 0.05), although higher ifnγ levels in the spleen and in the liver tended to be associated with lower anti-leishmania igg, as well as a shorter duration and smaller size of the skin lesions. while visceral leishmaniasis has been extensively studied in dogs and several experimental models have been described [20, 26, 34, [41] [42] [43] , very little is known about canine cutaneous leishmaniasis. we present here a first [33] . lesion duration in the l. (v.) braziliensis model was also highly variable, ranging from 16 to 32 weeks [33] . this elevated variability and time length made this former model of limited practical use. a higher parasite dose (1 × 10 9 ) has also been used in a model of canine visceral leshmaniasis, but it led to limited clinical signs [44] . the clinical and pathological evolution of l. (l.) mexicana we described in this study was also very similar to what has been described for naturally-occurring cutaneous leishmaniasis in dogs. thus, dogs naturally infected with l. (v.) braziliensis or l. panamensis present skin lesion varying in size from 0.4-10 cm [27, 29] . similarly, the histopathologic damage that we observed in the skin and other tissues is very similar to that observed in naturally infected animals [32, 45] . remarkably, our observation of lymphoid infiltration in the kidney is a first report indicating possible renal damage in experimental cutaneous leishmaniasis. the induction of glomerulonephritis during infection with l. (l.) infantum has been recently attributed to the deposition of immune complex following a limited cellular immunity [46] . in addition, it is important to note that l. (l.) mexicana can cause a rather wide spectrum of clinical disease, including diffuse and visceral forms in some cases, particularly in patients with low immune status [47] [48] [49] . also similar to the natural infection is the apparent absence of parasites in the healed lesion as assessed microscopically in tissue sections [32] . however, using a technique with higher sensitivity such as qpcr, we clearly demonstrated an important parasite burden up to 15 weeks after infection, both in the skin and in the secondary lymphoid organs of all six infected animals. while this assay relies on the detection of parasite dna, the presence of live parasites is highly likely, as observed in many animal models of leishmania infection. these important results suggest that infected dogs have reached an asymptomatic stage, but parasite persistence indicated that, on one hand infection may be reactivated upon alteration of their immune status, and on the other hand, they remain a potential source of infection and may serve as a reservoir of the parasite. in brazil, it has been clearly established that domestic dogs are the main reservoir of l. chagasi [20, 21, 50] , but the role of dogs in the transmission of leishmania species causing cutaneous forms of the disease is not well understood [51] . observations of long lasting ulcer lesions and a high susceptibility to infection in dogs from argentina suggested that they could play an important epidemiological role in the transmission of leishmania parasites species causing cutaneous leishmaniasis [51] . this has important implications for the definition of epidemiological control measures. indeed, the massive euthanasia of dogs in brazil has had a questionable efficacy to reduce infection rate in humans [17] , and as a consequence, the world health organization has now prioritized the development of veterinary vaccines [34, 41] . therefore, our model of canine cutaneous leishmaniasis may be very helpful for the evaluation of vaccine candidates. nonetheless, further development of this animal model may include the evaluation of the addition of sand fly saliva and/or infection by sand fly bites, which have been shown to dramatically alter host immune response to leishmania infection in mice [52] , even though its effects in dogs have been less straightforward [41, 42] . analysis of the immune response of l. (l.) mexicana infected dogs indicated an increase in anti-parasite igg and anti-nh36 igg levels eight weeks post-infection, which seems much faster than that observed during infection with l. (l.) chagasi/infantum in which case antibody levels took more than a year to increase [42, 50] . however, igg levels appeared to rise faster when intradermal infection was used [53] . nonetheless, similar to the visceral form of the disease, for which higher igg levels are associated with symptomatic disease [21] , we found that a stronger humoral response was associated with larger lesion size. interestingly, the analysis of igg isotypes revealed a gradual shift from a predominant igg2 to a predominant igg1 isotype over the course of infection, suggesting an initial th1 type immune response at the beginning on the infection, followed by progression to a th2 type response at later stages. this change in immune profile would explain the initial control of the lesion leading to its healing, as well as the failure of the immune system to achieve parasite elimination, allowing the detection of leishmania parasite dna in the healed lesion and lymph nodes. it is also in agreement with the pathogenic role reported for igg1 with l. (l.) mexicana infection in murine models [54] . however, cytokine measurements showed a significant ifnγ production in plasma, liver and spleen, while il-10 was only detected in the liver. a rather mixed th1/th2 response is thus more likely to be occurring, as reported in canine visceral leishmaniasis, for which a clear immune polarization has been difficult to observe [43, [55] [56] [57] [58] [59] [60] [61] [62] . nonetheless, a th1 immune response characterized by predominant igg2 antibodies and higher ifnγ production seem rather associated with parasite control, as evidenced by a significant association with a reduced splenomegaly, and a tendency to lead to a decreased lesion size and duration. this is in agreement with established mouse models [63] , as well as observations on canine visceral leishmaniasis. the analysis of additional cytokines and tissues, such as draining lymph nodes, should help further characterize the immune response in this animal model. we present here the first model of canine cutaneous leishmaniasis caused by l. 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infection: analysis of its first steps: a review relationship of leishmania-specific igg levels and igg avidity with parasite density and clinical signs in canine leishmaniasis immunologic indicators of clinical progression during canine leishmania infantum infection cytokine and transcription factor profiles in the skin of dogs naturally infected by leishmania (leishmania) chagasi presenting distinct cutaneous parasite density and clinical status anti-leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs immunopathology of leishmaniasis: an update a canine model of experimental infection with leishmania (l.) mexicana this work was funded in part by project priori-2010-004 from the universidad autónoma de yucatán, yucatan, mexico. we thank jorge canto and angel ramos for logistical support and jesus valenzuela for critical revisions of the manuscript. http://www.parasitesandvectors.com/content/7/1/361 received: 29 january 2014 accepted: 3 august 2014 published: 9 august 2014 the authors declare that they have not competing interest.authors' contributions jvc and ed conceived and designed the study, contributed with data analysis, drafted and revised the manuscript. aca, lev and ppm collected the samples. jlg and ppm performed the necropsies. aca and lev performed molecular and serological assays. mer and mjr revised the manuscript. all authors read and approved the final version of the manuscript. key: cord-351357-8ahlir5y authors: colella, vito; nguyen, viet l.; tan, do y.; lu, na; fang, fang; zhijuan, yin; wang, jiangwei; liu, xin; chen, xinghui; dong, junyan; nurcahyo, wisnu; hadi, upik k.; venturina, virginia; tong, kenneth b.y.; tsai, yi-lun; taweethavonsawat, piyanan; tiwananthagorn, saruda; le, thong q.; bui, khanh l.; watanabe, malaika; rani, puteri a.m.a.; annoscia, giada; beugnet, frédéric; otranto, domenico; halos, lénaïg title: zoonotic vectorborne pathogens and ectoparasites of dogs and cats in eastern and southeast asia date: 2020-06-17 journal: emerg infect dis doi: 10.3201/eid2606.191832 sha: doc_id: 351357 cord_uid: 8ahlir5y to provide data that can be used to inform treatment and prevention strategies for zoonotic pathogens in animal and human populations, we assessed the occurrence of zoonotic pathogens and their vectors on 2,381 client-owned dogs and cats living in metropolitan areas of 8 countries in eastern and southeast asia during 2017–2018. overall exposure to ectoparasites was 42.4% in dogs and 31.3% in cats. our data cover a wide geographic distribution of several pathogens, including leishmania infantum and zoonotic species of filariae, and of animals infested with arthropods known to be vectors of zoonotic pathogens. because dogs and cats share a common environment with humans, they are likely to be key reservoirs of pathogens that infect persons in the same environment. these results will help epidemiologists and policy makers provide tailored recommendations for future surveillance and prevention strategies. a sia is the largest continent in the world, known for its thriving biocultural diversity. today, countries in asia are experiencing a rapid social, demographic, and economic transformation, thereby placing this region as an ever-growing economic powerhouse in the years to come. sustained economic growth in asia has resulted in increased demand for products and services and substantial urbanization (1) . these factors have triggered a series of human-mediated environmental alterations, such as deforestation and encroachment of humans into natural ecosystems, that now link previously isolated ecologic niches and give pathogens new opportunities to thrive (2) . during the past century, asia has been in the limelight for emergence and pathogenicity of a large number of infectious diseases that have taken a substantial toll on the health of millions of persons (1) . striking examples include the emergence of severe acute respiratory syndrome, infections with the highly pathogenic avian influenza a(h5n1) virus, and coronavirus disease . recently, human modification of natural habitats resulted in the emergence of a tick vector of kyasanur forest disease virus, a zoonotic vectorborne flavivirus that causes severe hemorrhagic fever with a fatality rate of 3%-10% (3) . also implicated in the changing epidemiology of pathogens of public health concern in eastern and southeast asia are dogs and cats (4) (5) (6) . in remote areas of eastern and southeast asia, three quarters of dogs are classified as stray or community dogs (7) . vito colella, viet l. nguyen, do y. tan to provide data that can be used to inform treatment and prevention strategies for zoonotic pathogens in animal and human populations, we assessed the occurrence of zoonotic pathogens and their vectors on 2,381 client-owned dogs and cats living in metropolitan areas of 8 countries in eastern and southeast asia during 2017-2018. overall exposure to ectoparasites was 42.4% in dogs and 31.3% in cats. our data cover a wide geographic distribution of several pathogens, including leishmania infantum and zoonotic species of filariae, and of animals infested with arthropods known to be vectors of zoonotic pathogens. because dogs and cats share a common environment with humans, they are likely to be key reservoirs of pathogens that infect persons in the same environment. these results will help epidemiologists and policy makers provide tailored recommendations for future surveillance and prevention strategies. surge in the number of pet dogs and cats living in metropolitan settings (8, 9) . in china, the population of pet dogs is estimated to grow by 5 million per year. along with this increase in companion animal ownership, the risk of acquiring parasitic zoonoses from companion dogs and cats represents an ongoing, yet neglected, threat (10, 11) . implementation of effective measures to control zoonotic diseases must rely on the elucidation of pathogens and reservoir hosts in a given area. for most countries in asia, limited knowledge about the agents parasitizing dogs and cats, including those transmissible to humans, hinders the establishment of proper strategies for treatment and prevention of zoonotic pathogens in animal and human populations. although previous investigations have explored the occurrence of zoonotic diseases in animals living in remote areas (4) (5) (6) (7) , our year-long multicenter study explored the occurrence of vectorborne pathogens and ectoparasites in pet dogs and cats from metropolitan areas in eastern and southeast asia. our study involved academic institutions and private facilities of eastern asia (china and taiwan) and southeast asia (indonesia, malaysia, the philippines, singapore, thailand, and vietnam). to provide capacity building and compliance with the study procedures, trainings were performed at local institutions as needed. the protocol of this study was approved by the ethics committee of the department of veterinary medicine, university of bari (protocol no. 13/17). at partner institutions, animal owners read, approved, and signed an owner informed consent, which contained information about study procedures. during 2017-2018, local investigators sampled 10 client-owned dogs and 10 client-owned cats each month for 12 months in each country, except china, where 40 dogs and 40 cats each month were sampled. inclusion criteria were a history of regular outdoor access and having not received recent antiparasitic treatments. data on the animals' location, age, breed, and sex were recorded. veterinarians performed a complete examination of the animals, reporting abnormalities in rectal temperature, overall physical condition, demeanor, nasal discharge, skin/haircoat, eyes, superficial lymph nodes, respiratory system (breathing), cardiovascular system (mucous membranes), and fecal consistency. the examinations included checking for the presence of ectoparasites (ticks, fleas, lice, and mites) by examining the whole-body surface for >5 minutes. the veterinarians inspected both eyes, including a thorough examination under the third eyelid to detect adult thelazia callipaeda eyeworms. they also performed testing for lesions evocative of sarcoptic mange or demodicosis (deep skin scraping), cheyletiellosis (tape test), or otoacariosis (earwax examination). sampled parasites were stored in vials containing 70% ethanol and sent for morphologic and molecular identification at the university of bari (bari, italy), where we examined adult and nymph ticks under a stereomicroscope. we clarified tick larvae, fleas, lice, and fur mites in 10% potassium hydroxide overnight, mounted in hoyer's medium and observed under an optical microscope (12) . we used morphologic keys to identify all ectoparasites to the species level (13) (14) (15) (16) (17) (18) (19) (20) . for mite identification, we minced crusty skin lesions by using disposable surgical blades, added drops of saline solution on a glass slide, observed the slides under an optical microscope, and identified the mites according to morphologic appearance (18, 21) . we mounted anterior and posterior extremities of adult t. callipaeda eyeworms in lactophenol and identified them (22) . to confirm morphologic identifications of ectoparasite species, we subjected a representative subpopulation (≈20%) of the ectoparasites to dna extraction and amplification of target genes (appendix table, https://wwwnc.cdc.gov/eid/article/26/6/19-1832-app1.pdf). for ticks, fleas and lice, we isolated genomic dna (gdna) by using the dneasy blood and tissue kit (qiagen, https://www.qiagen.com) according to the manufacturer's instructions. we isolated gdna from a small portion of the idiosoma of ticks (23) and from the anterior dorsal part of the abdomen of fleas (24) . we selected individual lice and mites under an optical microscope and extracted gdna by using a qiaamp dna micro kit (qiagen). from each study animal, we collected ≈2 ml of blood in a tube with anticoagulant and processed it as follows. kit (qiagen) according to manufacturer instructions. we tested all gdna isolated from dried blood samples by conventional pcr (cpcr) (appendix table) . we detected leishmania protozoa by quantitative pcr (qpcr) and further tested only samples scoring positive in duplicates by qpcr by cpcr on the internal transcribed spacer 2 region and kinetoplast dna for species identification (appendix table) . for all pcrs, we included positive controls (dna of pathogen-positive blood samples) and negative controls (dna of pathogen-negative blood samples). we visualized pcr amplicons from nematodes and apicomplexan protozoa by capillary electrophoresis by using a qiaxcel dna screening gel cartridge on a qiaxcel system (qiagen for each) and used a qx dna size marker (qiagen) to size pcr products. we injected a qx alignment marker (qiagen), which consisted of 15-bp and 3,000-bp fragments, onto the cartridge with each sample. we then determined the pcr product sizes by using qiaxcel screen gel 1.4.0 software (qia-gen). we subjected cpcr products from leishmania spp. protozoa, thelazia spp. eyeworms, ticks, fleas, lice, and mites to electrophoresis in a 2% agarose gel stained with gel red (vwr international pbi, https://it.vwr. com) and visualized them on a kodak gel logic 100 gel documentation system (https://www.kodak.com). we purified all cpcr amplicons obtained and sequenced them in both directions in an automated we calculated frequency values as the proportion of positive animals to the total number of examined animals and the relative frequency of occurrence of each species of parasite as the proportion of animals infested by a given parasite species/group within the total number of positive results within a given parasite species/group. we calculated 95% cis by using the wilson score interval. we categorized animals into 3 age groups (<1, >1 to <5, and >5 years). we used the χ 2 test to investigate associations between parasitic infection/exposure or infestation by ectoparasites and age group or clinical observations. we analyzed the cohen κ coefficient and dependent and independent variables by using graphpad prism 8 (http://www.graphpad.com). we considered p<0.01 to indicate significance. our study sample consisted of 2,381 animals (1,229 dogs and 1,152 cats). samples were collected from animals living in 23 main cities (and neighboring localities) in 8 countries in asia, specifically china (beijing, nanjing, shanghai, and guangxi province), taiwan (taipei, taoyuan, changhua, pingtung, and hualien), indonesia (jakarta, bogor, and yogyakarta), 22 .8% (95% ci 20.5%-25.2%) of dogs and 0.5% (95% ci 0.2%-1.1%) of cats were detected with or exposed to >1 vectorborne parasite. t. callipaeda eyeworms were detected in 1.7% (95% ci 0.8%-3.3%) of dogs from china, specifically in 6.7% (95% ci 3.4-12.7) of dogs and 0.6% (95% ci 0.2%-1.8%) of cats from beijing. we detected co-infections with hepatozoon canis and d. immitis heartworms in 4 dogs (2 each from thailand and the philippines). no tick infestations were found on dogs infected with b. gibsoni, but rhipicephalus sanguineus ticks were detected on 50% of animals with h. canis infection. h. canis infection was correlated with infestation by r. sanguineus ticks (p = 0.0125). a total of 4 dogs (0.3%, 95% ci 0.1%-0.8%) were positive for antibodies against leishmania infantum; 2 of these dogs were from china (0.4%, 95% ci 0.1%-1.5%) and 1 each from vietnam (0.8%, 95% ci 0.1%-4.6%) and the philippines (0.9%, 95% ci 0.2%-5.0%). in addition, the 2 seropositive dogs from china were positive for l. infantum by qpcr and cpcr sanger sequencing (table 6) . a total of 2 dogs (0.2%, 95% ci 0-0.6) were positive for antibodies against borrelia burgdorferi sensu lato. one was in the philippines (0.9%, 95% ci 0.2%-4.9%), and 1 was in indonesia (1.1%, 95% ci 0.2%-5.7%). overall, 5.2% (95% ci 4.1%-6.7%) of dogs were infected with filarial parasites according to antigen testing (3.5%, 95% ci 2.6%-4.6%), cpcr (2.7%, 95% ci 1.9%-3.7%), or both. cpcr-coupled sequencing identified brugia spp. in 0.4% of dogs (95% ci 0.2%-0.9%) and in 15.2% (95% ci 6.6%-30.9%) of the samples positive for filariae by cpcr. specifically, b. pahangi was found in dogs in thailand (1.7%, 95% ci 0.5%-5.9%) and malaysia (2.2%, 95% ci 0.4-11.6), and b. malayi was found in dogs in vietnam (0.8%, 95% ci 0.1-4.6) and thailand (0.8%, 95% ci 0.1%-4.6%). using the kappa statistic, we found slight to fair agreement between antigen testing and cpcr (κ = 0.271, 95% ci 0.079-0.463) for the diagnosis of d. immitis in dogs. one cat (1.3%, 95% ci 0.2%-6.9%) from indonesia was positive for d. immitis antigen. h. canis infection was diagnosed for 1 cat from the philippines (0.9%, 95% ci 0.2-5.1) and b. gibsoni for 3 cats in china (0.6%, 95% ci 0.2%-1.8%) and 1 cat in singapore (0.8%, 95% ci 0.1-4.3). fiv antibodies were detected in 5.2% (95% ci 4.0%-6.6%) of cats and felv antigens in 2.9% (95% ci 2.1%-4.0%). we compiled statistically significant associations for the detection of/exposure to >1 ectoparasite or vectorborne pathogen, to ectoparasites or vectorborne parasites only, and to filarial parasites in dogs in different age classes (appendix figure 1 ). the finding of clinical signs (e.g., respiratory, lymph nodes, ocular, and skin abnormalities and increased body temperature) was statistically associated with the overall detection of/exposure to >1 parasite (appendix figure 2 ) and with ectoparasite infestation or detection of/ exposure to vectorborne parasites in dogs (appendix figure 3 ). for cats, we found no association between age group and detection of parasites, whereas clinical signs (i.e., enlarged lymph nodes and skin abnormalities) were statistically associated with detection of ectoparasitic infestation (appendix figure 4) . we found no statistical association between seropositivity for fiv antibodies and felv antigens and detection of ectoparasites, vectorborne pathogens, or both. the detection of zoonotic pathogens in client-owned dogs and cats living in metropolitan areas indicates that these animals serve as hosts for several parasitic agents in asia. we provide data for an extended geographic distribution of zoonotic pathogens (e.g., l. infantum protozoa and zoonotic species of filariae) and of arthropods infesting animals (e.g., ticks of the haemaphysalis and rhipicephalus genera) where prior data unavailability made treatment and disease control strategies unachievable. nearly half of the dogs and one third of the cats in this study were infested with >1 ectoparasite or exposed to vectorborne pathogens; prevalence peaked in countries with a humid tropical climate (e.g., the philippines, where 67% of dogs were infested with ticks, and malaysia, where 89% of cats were infested with fleas). such findings raise concern that vectorborne pathogens are responsible for several zoonotic diseases in southeast asia (25) . the most prevalent tick on dogs and cats in this study was r. sanguineus. the taxonomic status of this tick group is a matter of debate with regard to r. sanguineus sensu lato including 2 lineages, so-called temperate and tropical (26) (27) (28) . the tropical lineage of the r. sanguineus s.l. tick is prevalent in most countries in asia and has been deemed accountable for the transmission of pathogens causing babesiosis, ehrlichiosis, and several rickettsial diseases in asia (25, 29, 30) . despite the high proportion of tick-infested animals, the paucity of data on the ecology of r. sanguineus s.l. ticks in asia makes their role as a vector difficult to ascertain. unexpectedly, we found tick species not classically associated with companion animals but with the potential to transmit zoonotic disease-causing pathogens in dogs. for example, haemaphysalis hystricis thorax is short and legs are small, ending with a single claw (arrow). b) trichodectes canis male louse with short thorax, flattened head with quadrangular shape, broader than long; each leg with only 1 claw on tarsus (arrow). c) heterodoxus spiniger female louse with subtriangular head, rounded anteriorly. the thorax is considerably longer than wide. each leg has 2 claws on the tarsus (arrow). scale bar indicates 1 mm. ticks have been implicated as vectors of a novel borrelia species closely related to the relapsing fever group (31) , and haemaphysalis wellingtoni ticks are vectors of kyasanur forest disease virus, which causes fatal epidemics among monkeys and leads to hospitalization of ≈500 persons/year in india (3). moreover, dogs seropositive to b. burgdorferi s.l. in this study were from indonesia and the philippines. this finding is unexpected, considering that these bacteria have been detected outside the known distribution area of ixodes tick species, the main vectors of b. burgdorferi s.l., and indicates a need for in-depth epidemiologic surveys of this group of pathogens in southeast asia. these results update the list of pathogens and ectoparasites affecting companion animals in asia, including ticks with multihost feeding behavior, which has the potential to extend the network of pathogen transmission further into urban areas. the same holds true for pet dogs, suggesting that these animals might have been overlooked as potential pathogen reservoirs in metropolitan settings in this geographic area. similarly, the ctenocephalides orientis flea was identified in one fifth of flea-positive dogs. the host spectrum of this flea is wider, but apparently its geographic distribution is more limited than that of the well-known cat flea ctenocephalides felis, and it is involved in the transmission of rickettsiae, including rickettsia sp. genotype rf2125 and rickettsia sp. th2014 (32, 33) . the morphologic ambiguity of the c. orientis flea (probably misidentified as ctenocephalides canis and previously reported as a subspecies of c. felis) has contributed to a substantial dearth of information on its global distribution and role as a vector. in contrast, the cosmopolitan c. felis flea has colonized different bioclimatic niches, mainly through humanmediated migration (34) . as human and animal global transportation increase in asia, constant vigilance regarding the introduction of c. orientis fleas outside their known range of distribution in developed and developing countries is essential, as supported by the recent report of detection of fleas of this species in iran (35) . in singapore, one of the countries with the highest human development index (36) and lowest proportion of animals affected by parasites, lynxacarus radovskyi, a mite for which little is known regarding its ecology, was detected on 35% of sampled cats. we have provided molecular data and updated morphologic information on this listophorid mite, which is an agent of papular dermatitis in humans (37) . furthermore, availability of appropriate diagnostics for this species or data on the efficacy of ectoparasiticides against it are limited. refined diagnostics are essential for assessing the distribution of filarial species in canine populations. for instance, the poor agreement (κ = 0.271) between cpcr and antigen-detection tests for d. immitis advocates for the use of integrated diagnostics to better appreciate the epidemiologic status of this species of filariae. furthermore, the use of both tests revealed b. pahangi and b. malayi to also (in addition to d. immitis) affect companion animals in the regions investigated. these 3 species of filariae cause clinical manifestations in humans: lymphatic filariosis for b. malayi and b. pahangi (38, 39) and pulmonary granulomas for d. immitis (40) . in particular, lymphatic filariosis is among the most debilitating neglected tropical diseases; an estimated 70 million persons are infected, among which >50% live in southeast asia (41, 42) , and d. immitis infection of humans poses significant diagnostic challenges (40) . hence, for development and enactment of global elimination programs (41) , surveillance of filarial species should be extended to animal populations in filariae-endemic countries (42) . similarly, leishmania spp. parasites currently cause ≈500,000 human infections/year in 62 countries (43), although their occurrence in eastern and southeast asia is poorly documented. we detected dogs positive for l. infantum by serology, qpcr, and sequencing in china and seropositive dogs in thailand and vietnam. in thailand, the recent emergence of l. martiniquensis and l. siamensis caused immunocompetent and immunocompromised persons to seek medical assistance (44) . a range of animals is involved in the zoonotic cycle of these 2 species (44, 45) , but dogs are the main reservoir for zoonotic leishmaniosis caused by l. infantum (46) . the role of leishmania spp. in human infections and as agents of disease in southeast asia requires urgent attention. further complicating knowledge of the transmission of zoonotic parasites in these regions of asia are the large populations of free-roaming animals; the increased number of pet dogs and cats; and the complex social, economic, and ecologic changes currently occurring in asia, (1, 2, 4, 25, 47, 48) . integrated strategies that address all of these factors are therefore fundamental for the control of such parasitic agents. we investigated the presence of pathogens and ectoparasites in pet dogs and cats living in metropolitan areas in close proximity to humans. these animals share a common environment with humans, which makes them likely key reservoirs for pathogens with the potential to infect persons living in such areas and settings. the epidemiologic data presented in this study can be pivotal for building knowledge bases about the occurrence of zoonotic parasites infecting companion dogs and cats in eastern and southeast asia. this information could help epidemiologists and 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tropics ticks and associated pathogens from dogs in northern vietnam we are grateful to isabelle richtofen, jianwei zhang, evonne lim, clair cheng, nadine duperray, and marielle servonnet for their expertise and contributions to managing this logistically challenging study. dr. colella is a postdoctoral research fellow at the university of melbourne, australia. his main research is focused on the development of epidemiologic studies and strategies for interventions against zoonotic pathogens. key: cord-262612-6mxzwm0h authors: townsend, wendy m. title: canine and feline uveitis date: 2008-02-23 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2007.12.004 sha: doc_id: 262612 cord_uid: 6mxzwm0h the clinical signs of uveitis occur as a result of inflammation within the vascular coat of the eye, which causes breakdown of the blood-aqueous barrier and blood-retinal barrier. many infectious and noninfectious causes can incite episodes of uveitis. although a complete diagnostic evaluation is highly recommended to identify any underlying etiologic agent, many cases remain idiopathic in nature. the goals of therapy are preserving vision, minimizing pain, and halting inflammation. the uvea is composed of the highly vascular and often pigmented iris, ciliary body, and choroid. the iris and ciliary body comprise the anterior uvea. the choroid comprises the posterior uvea. the uveal tract regulates the quantity of light allowed into the eye by varying the pupillary aperture; produces aqueous humor, which provides nourishment and removes waste from the cornea and lens; and provides nutrients to the outer layers of the retina [1] . uveitis is simply inflammation within the uveal tract. more precise terminology to describe the portion of the uveal tract involved includes iritis (inflammation of the iris), iridocyclitis or anterior uveitis (inflammation of the iris and ciliary body), choroiditis or posterior uveitis (inflammation of the choroid), and panuveitis (inflammation of the entire uveal tract) [2] . differentiating whether inflammation arises from the iris or the ciliary body can be difficult because of their close anatomic proximity and the similar clinical signs [3] . posterior uveitis can occur independent of anterior uveitis [3] . endophthalmitis occurs when ocular inflammation is confined to three or more tissues inside the eye [2] . panophthalmitis indicates that ocular inflammation involves all layers of the eye, including the sclera [2] . determining the extent of involvement is clinically important, because involvement of the posterior segment may decrease the likelihood of maintaining a visual globe. therapeutic agents must also be administered systemically to achieve effects within the posterior segment. uveitis is a significant cause of ocular disease in dogs and cats [4, 5] . uveitis occurs after damage to uveal tissue or vasculature disrupts the ocular bloodaqueous barrier (bab) or the blood-retinal barrier [6] . the bab is composed of an epithelial barrier at the level of the nonpigmented ciliary epithelium and an endothelial barrier at the level of the iridal blood vessels [7, 8] . the blood-retinal barrier is created by tight junctions within retinal capillaries and [9, 10] . the bab prevents the movement of molecules across the vascular endothelial surface [6] , which results in aqueous humor protein concentrations 200 times less than that of plasma [11] . disruption of the bab causes the aqueous humor protein concentration to increase to greater than normal levels, and the resultant light scattering (tyndall effect) makes the beam from the slit-lamp visible as it traverses the anterior chamber [12] . this phenomenon is known as flare and is a hallmark of uveitis. disruption of the blood-retinal barrier results in retinal edema, retinal hemorrhage, and detachment of the neurosensory retina [13] . the acute inflammatory phase of uveitis begins with brief arteriolar vasoconstriction, followed by prolonged vascular dilation [3] . prostaglandins and leukotrienes mediate the vasodilation and cause increased vascular permeability, which results in the breakdown of the bab. during episodes of anterior uveitis, the intraocular prostaglandin concentrations may increase 200-fold [14] . prostaglandins also induce hyperemia and reduction in intraocular pressure (iop) [3] . pgf 2a constricts the iris sphincter muscle, causing miosis and pain. in a recently reported study measuring inflammatory mediators in aqueous humor after anterior chamber paracentesis, an increase in pge 2 levels was noted along with elevations in inducible cyclooxygenase (cox)-2 and nitrites and nitrates [15] . breakdown of the bab allows proteins, cells, and additional inflammatory mediators entry into the iridal stroma and aqueous humor. cytokines and chemokines are important chemotactic factors that recruit inflammatory cells. one particularly important cytokine seems to be leukotriene b4, a classic chemoattractant that triggers adherence of leukocytes to the endothelium and recruits granulocytes and macrophages to the site of inflammation [16] . in an experimental model of uveitis in mice, blockade of the leukotriene b4 receptor greatly reduced the intensity of the ongoing disease [16] . blepharospasm, photophobia, excessive lacrimation, and enophthalmos are nonspecific signs of ocular discomfort noted with uveitis but also with ulcerative keratitis, scleritis, and glaucoma [17] . the globe often appears red because of hyperemia of the deep perilimbal anterior ciliary vessels [3, 17] . the engorgement of these radially oriented vessels is called ciliary flush. the vascular dilation occurs secondary to the elevation in prostaglandin levels. deep vessels may be distinguished from superficial conjunctival vessels by manipulation of the conjunctiva and application of 1:1000 epinephrine or 2.5% phenylephrine solution [17] . deep vessels do not move with the conjunctiva and do not readily blanch after the application of topical epinephrine. uveitis may result in corneal edema. corneal edema may occur in association with uveitis as a result of reduction of the endothelial sodium potassium (nak)-atpase or epithelial nachlorine (cl) pump activities or may be related to a rupture of the endothelial cell-cell junction barrier [18] . either mechanism allows hydration of the corneal stroma noted clinically as corneal edema. the presence of aqueous flare confirms a diagnosis of anterior uveitis. aqueous flare denotes breakdown of the bab and increased permeability of the ocular vasculature. because of this increase in vascular permeability, inflammatory cells may be visualized within the aqueous humor or vitreous body. an accumulation of purulent material within the anterior chamber is termed hypopyon (fig. 1) . blood within the anterior chamber is termed hyphema (fig. 2) . both may be noted during episodes of anterior uveitis. aggregates of inflammatory cells may adhere to the corneal endothelium and are then called keratic precipitates (fig. 3) . typically, the keratic precipitates are visualized on the ventral one half to one third of the cornea, where they are deposited by the aqueous humor thermal convention currents. blepharospasm or an elevated third eyelid may prevent visualization of the keratic precipitates by obscuring the ventral portion of the cornea. larger fatty-looking clusters of keratic precipitates have been termed mutton fat keratic precipitates (fig. 4) and often indicate granulomatous inflammation [19] . miosis, sometimes quite marked, occurs in response to prostaglandins, particularly pgf 2 [20, 21] , and other inflammatory mediators that act directly on the iris sphincter muscle [3] . the miosis and associated ciliary muscle spasm contribute greatly to the pain associated with anterior uveitis. failure to dilate completely in response to the topical application of tropicamide 1% ophthalmic solution can be a subtle sign of anterior uveitis [3] . iridal swelling may cause the iris to appear engorged or darker in color, possibly even yellow in animals with normally blue irides. the iop typically decreases during uveitis, because the inflammatory process leads to a reduction in active secretion of aqueous humor, possibly by means of interference with active transport mechanisms [22] . prostaglandin release may also contribute to the ocular hypotony by increasing aqueous humor outflow through the uveoscleral route [23] . subtle ocular hypotony, for example, an iop within the normal range but 5 mm hg less than the fellow eye is a significant finding that may be an early indication of inflammation [3, 24, 25] . examination of the posterior segment may reveal a cellular infiltrate within the vitreous body as inflammatory cells diffuse into the vitreous from the pars plana and pars plicata of the ciliary body. a complete fundic examination is necessary to evaluate alteration within the retina and choroid. because of the close anatomic proximity of the retina and choroid, the choroid is infrequently inflamed as a sole process. the retina is typically involved primarily or secondarily. areas of grayish discoloration over the tapetal fundus and grayish to white areas within the nontapetal fundus may occur as a result of retinal edema or cellular infiltration [13] . more extensive inflammation may result in areas of retinal detachment (fig. 5 ). the detachments may be bullous with a fluid exudate in the subretinal space, which allows visualization of the underlying tapetum (fig. 6 ). retinal detachments may also be characterized by a cellular infiltrate in the subretinal space, which appears as grayish to pink-white accumulations of material beneath the retinal detachment. hemorrhage may be present within the vitreous, retina, or subretinal space. close inspection of the retinal vasculature may reveal changes in vascular caliber and tortuosity. sheathing of the retinal vessels by inflammatory cells, called perivascular cuffing, occurs with some forms of uveitis. chronic inflammation of previous bouts of inflammation may incite multiple changes within the globe. chronic inflammation stimulates ingrowth of peripheral corneal vascularization. the vessels bud from the limbal vasculature [26] . matrix metalloproteinase 2 within the anterior chamber may be one of the stimuli that incite corneal angiogenesis [27] . the combination of inflammatory cells, fibrin, fibroblasts, iridal swelling, and miosis may create adhesions of the iris to the lens capsule or cornea [3] . posterior synechiae occur if the iris is adherent to the anterior lens capsule. anterior synechiae occur if the iris is adherent to the corneal endothelium. if extensive, posterior synechiae may occlude the flow of aqueous from the posterior chamber through the pupil into the anterior chamber, causing iris bombé, which is an anterior ballooning of the iris, and secondary glaucoma [19] . preiridal fibrovascular membranes may form on the anterior surface of the iris. angiogenic factors released by ischemic retina, neoplasms, or leukocytes involved in ocular inflammation can incite endothelial budding from vessels in the anterior iridal stroma [28] . the membranes may extend onto the anterior lens capsule or into the iridocorneal angle. clinically, the term rubeosis iridis is applied, because the neovascular membrane on the anterior iridal surface causes a reddish cast to the iris (fig. 7 ) [19] . chronic inflammation often induces the formation of cataracts, presumably by the diffusion of inflammatory mediators across the lens capsule causing lens epithelial metaplasia, necrosis, or posterior migration and lens fiber degeneration, liquefaction, and necrosis [29, 30] . chronic uveitis may also result in lens luxation. the inflammatory products within the aqueous humor cause degradation of the zonular fibers, which then allows the lens to move from its normal position within the patellar fossa [31] . although this process seems to be relatively rare in dogs [32] , chronic uveitis seems to be a frequent cause of lens luxation in the cat [33] . secondary glaucoma may occur from pupillary block as a result of iris bombé or lens luxation or may be attributable to occlusion of the iridocorneal angle by peripheral anterior synechiae [5] . resolution of chorioretinitis may leave areas of retinal degeneration demarcated as areas of tapetal hyperreflectivity in the tapetal fundus as the overlying retina has thinned and mottled pigmentation in the nontapetal fundus. hypertrophy of the retinal pigment epithelium may be noted as areas of dense pigmentation in areas of previous retinal detachment. if marked choroidal inflammation was present, there may be changes in tapetal coloration, pigment clumping, or loss of choroidal pigment, which exposes the choroidal vessels or sclera. retinal vascular attenuation may be generalized or occur overlying the areas of retinal degeneration. finally, phthisis bulbi may occur as chronic cyclitis, and the resultant tissue atrophy and fibrosis destroy the ability of the ciliary body to produce aqueous humor. because the normal iop can no longer be maintained, the globe begins to shrink [3] . the histologic hallmarks are an atrophic and disorganized globe typically characterized by a cyclitic membrane and variable degrees of chronic inflammation [34] . fibrous or osseous metaplasia may occur as well. fluorescein staining should be performed routinely to rule out the presence of ulcerative keratitis and the possibility of a reflex uveitis [17] . the presence of ulcerative keratitis precludes the use of topical corticosteroids. because many significant systemic diseases can induce uveitis, a thorough physical examination, complete blood cell count, serum biochemistry profile, and urinalysis should be performed. during the physical examination, particular attention should be paid to the integument, lymphatic system, thoracic auscultation, and abdominal palpation. thoracic radiographs, abdominal ultrasonographic examination, and select serologic titers are often valuable tools in the diagnostic investigation. if a marked cellular infiltrate is present, cytologic evaluation of aqueous humor may be beneficial in identifying etiologic agents or neoplastic cells, particularly in cases of lymphoma [35] . the risks of aqueous centesis are low in the hands of an experienced ophthalmologist but do include cataract formation if one contacts the anterior lens capsule, hyphema [36] , and exacerbation of the uveitis. if marked vitreal infiltrates or cellular retinal detachments are present, cytologic examination of those infiltrates is often more rewarding [37] . in a report by brightman and colleagues [37] , an etiologic agent was identified in 13 (65%) of 20 of cases using vitreous centesis and cytologic examination. aqueocentesis can also be used to determine the level of intraocular antibody production. the ratio of aqueous antibody titer to serum titer is known as the goldman-witmer coefficient, or c value. if greater than 1, this shows that an intraocular infectious agent is causing iridal plasma cells to produce antibody, thus demonstrating that an infectious agent is causing the uveitis rather than merely being a bystander [38, 39] . primary treatment goals for uveitis are halting inflammation, stabilizing the bab, minimizing sequelae, decreasing pain, and preserving vision. the agents used to attain those goals include topical mydriatics, topical (and, in select cases, systemic) corticosteroids, and nonsteroidal anti-inflammatory drugs (nsaids). if an underlying etiology can be detected, therapy should be directed toward removal of the inciting agent or alleviating the associated systemic disease. the mydriatic agent most often selected is 1% atropine ointment or solution. atropine is a selective, reversible, direct-acting anticholinergic agent [40] . topical administration results in pharmacologic blockage of the sphincter muscles of the iris and ciliary body, leading to pupillary dilation and cycloplegia [40] . the resultant pupillary dilation decreases the possibility of posterior synechia formation. the pupillary dilation may exacerbate congestion of the iridocorneal angle, however, and thereby decrease aqueous outflow. therefore, atropine must be used with caution in patients that have or are at risk for secondary glaucoma. the cycloplegia greatly lessens the pain associated with ciliary spasm. administration of atropine also decreases the permeability of ocular blood vessels to proteins and intravenously administered fluorescein, thereby stabilizing the bab [41, 42] . in a normal canine or feline globe, the onset of action is within 30 to 60 minutes [43, 44] and mydriasis may persist for up to 10 days after administration of the last dose [45] . in uveitic globes, atropine is initially applied up to four times daily to achieve mydriasis and is then administered once to twice daily to maintain mydriasis. because the agent is bitter tasting, hypersalivation or, infrequently, vomiting may be noted after the instilled topical solution travels through the lacrimal puncta and nasolacrimal ducts and is tasted [46] . cats may have particularly marked reactions [44] . therefore, use of the ointment preparation is indicated in feline or potentially sensitive patients. anti-inflammatory therapy is a key element of therapy for uveitis. corticosteroids are frequently used to suppress inflammation because they reduce production of metabolites of the arachidonic acid (inflammatory) cascade [47] . glucocorticoids upregulate lipocortin expression [48] . lipocortin is a key inhibitor of proinflammatory substances, such as phospholipase a 2 , the enzyme responsible for initiating the arachidonic acid cascade [48] . glucocorticoids can also directly reduce pge synthesis and increase vascular stability [49] . to control anterior uveitis, topical application of the corticosteroid is often the preferred route because it allows for high local drug concentrations and minimal systemic side effects [47] . prednisolone acetate 1% ophthalmic suspension and dexamethasone 0.1% are able to penetrate an intact corneal epithelium [50] . therefore, they are the only topical corticosteroids that can achieve therapeutic concentrations in the aqueous humor [50] . each of these agents may inhibit 40% to 50% of protein exudation from the iris and ciliary body [51] . the initial frequency of application may be every 2 to 4 hours depending on the severity of the inflammation. once clinical improvement is noted, the frequency may be decreased but the therapy should be gradually tapered to diminish the likelihood of recurrence [47] . topical corticosteroids are contraindicated in the presence of corneal ulceration because they delay normal wound healing. use of glucocorticoids, such as prednisone, prednisolone, or dexamethasone, to suppress inflammation within the posterior segment, for example, chorioretinitis, requires systemic administration of anti-inflammatory or, occasionally, immunosuppressive doses [47] . the dose is then incrementally decreased based on the response to therapy [50] . when using corticosteroids, one risks exacerbation of clinical signs if an infectious cause is present but has not yet been identified [47] . systemic side effects, including endocrinopathies, may result with long-term use. the nsaids can be particularly useful in cases of mild anterior uveitis, as adjunctive therapy when combined with topical corticosteroids, and to control posterior segment inflammation when an infectious etiology has not been completely ruled out. the nsaids block the conversion of arachidonic acid to prostaglandins by cox [52] . prostaglandins are key mediators of ocular inflammation, causing breakdown of the bab, exacerbating photophobia, and lowering the ocular pain threshold [53] . the nsaids are also beneficial because they have been shown to suppress polymorphonuclear cell locomotion and chemotaxis [54] , decrease expression of inflammatory cytokines [55] , and function as free radical scavengers [56] . although many systemic nsaids are currently available, only a few have been evaluated to assess their efficacy in controlling ocular inflammation. flunixin meglumine and aspirin have been shown to stabilize the bab in experimental models of uveitis [57, 58] . flunixin meglumine had good effects, whereas the effects of aspirin were moderate when compared with the placebo. both drugs induced some gastrointestinal bleeding, however. in a pilocarpine-induced model of uveitis in dogs, carprofen resulted in a 68% inhibition of aqueous flare [59] . in a clinical study, tolfenamic acid was shown to control postoperative intraocular inflammation [60] . when selecting a systemic nsaid, one must be cognizant of the potential systemic side effects, particularly the adverse gastrointestinal effects in all species [61] , and the potential for bone marrow suppression and hemorrhage in cats [62] . topical nsaids may be used to control mild inflammation or may be combined with topical corticosteroids to improve control of more severe ocular inflammation [52] . the application frequency typically varies from two to four times per day [62, 63] . the relative efficacies have been studied in an anterior chamber paracentesis model and the order of bab stabilizing efficacy was as follows: diclofenac greater than flurbiprofen, flurbiprofen greater than suprofen, and suprofen greater than tolmetin, which was equal to the control solution [64] . one must use caution in canine eyes with the potential for secondary glaucoma because the topical nsaids have been found to elevate the iop [65] . one must also exercise caution when using topical nsaids in the presence of corneal ulceration. in people, topical nsaids have been associated with marked corneal collagenolysis [3] . cases of immune-mediated uveitis that require long-term maintenance with systemic glucocorticoids or that fail to respond to conventional therapy may require use of immunosuppressive drugs, such as azathioprine. azathioprine is a purine analogue with relatively select cytotoxicity for t helper lymphocytes [66] . conditions like uveodermatologic syndrome and pigmentary uveitis are the conditions in which azathioprine is most frequently used [67, 68] . the initial dose in dogs is 2 mg/kg every 24 hours [69] . the dose for long-term therapy is typically decreased to 0.5 to 1 mg/kg every other day. the lag period before successful treatment is recognized ranges from 3 to 5 weeks [69] . complete blood cell counts should be monitored, because bone marrow suppression is a concern. gastrointestinal side effects and hepatotoxicity may be noted as well [69] . a plethora of etiologies may incite uveitis. infectious diseases, neoplasia, and immune-mediated conditions may all present with clinical signs of uveitis. for the purposes of this discussion, the etiologies are grouped into noninfectious and infectious causes of uveitis. unfortunately, most cases of uveitis remain idiopathic despite intensive systemic evaluations. in a study by massa and colleagues [4] , 60% of cases of dogs that had uveitis were classified as idiopathic or immune mediated, because an underlying systemic cause could not be identified. the dogs with idiopathic uveitis were typically middle aged, did not exhibit any signs of systemic illness, and more often were presented with unilateral uveitis. in the study by massa and colleagues [4] , the degree of inflammation and ocular lesions did not vary between those dogs with infectious, neoplastic, or idiopathic uveitis. in studies of feline uveitis, approximately 30% to 62% of affected cats had no identifiable concurrent systemic disease [70, 71] . although the underlying etiology may often remain obscure, a complete diagnostic investigation remains essential because of the severity of the systemic diseases associated with uveitis. an excellent review article on this topic has been written by van der woerdt [72] , and the reader is referred to that text for additional information. two different forms of uveitis may be initiated by lenticular pathologic findings. a lymphoplasmacytic inflammatory process, termed phacolytic uveitis, occurs in association with hypermature or rapidly forming cataracts [31] . phacolytic uveitis is typically mild. the prevalence has been reported to be as high as 71% in dogs screened for cataract surgery [73] . the high prevalence is not surprising, because fluorophotometric studies have demonstrated breakdown of the bab in association with all stages of cataracts [74] . the lens-associated inflammation is proposed to occur after deviation of the normal low level of t-cell-mediated tolerance to lens proteins [72, 75] . a more dramatic form of uveitis, termed phacoclastic uveitis, occurs in association with rupture of the lens capsule and release of lens proteins and membrane-associated antigens [31] . histologic examination of affected globes revealed intralenticular neutrophils and a surrounding inflammatory response that ranges from suppurative to lymphocytic in nature [76] . in a study by davidson and colleagues [77] , prompt surgical removal of the lens material resulted in a visual eye in most cases. in contrast, attempts at medical management resulted in the loss of vision. in cats, because of the risk for traumatic ocular sarcoma, surgical removal of the lens material or globe is recommended if the eye cannot be salvaged. rupture of the lens capsule is believed to induce traumatic ocular sarcoma, although the time from trauma to detection of the tumor averages 5 years [5, 78] . blunt trauma and penetrating trauma can incite uveitis and are common causes of uveitis in domestic animals [17] . hyphema and varying amounts of fibrin are often present in cases of traumatic uveitis [5] . in cases of penetrating trauma, one must assess the extent of damage within the globe, including whether the lens capsule has been ruptured. ultrasonographic examination may be required to evaluate the extent of the damage fully. the administration of broad-spectrum systemic antibiotics is strongly suggested, because bacterial or fungal contamination may occur at the time of globe penetration [5] . endophthalmitis can progress rapidly and cause loss of the globe. if marked hyphema is present, administration of tropicamide can mobilize the pupil and assist in preventing the development of synechia [5] . administration of tropicamide is preferable to atropine, because tropicamide produces greater iridoplegia than cycloplegia, and thus less often produces increases in iop [40] . primarily reported in the golden retriever, pigmentary uveitis is characterized by anterior segment inflammation and the deposition of pigment on the anterior lens capsule, often in a radial fashion [67] . no systemic signs are associated with this condition. the mean age at presentation is 8.6 years [67] . in a report by sapienza and colleagues [67] , common sequelae were cataract formation (37%) and secondary glaucoma (46%). in a report by deehr and dubielzig [79] , uveal cysts were noted in 15 of 18 eyes and were thought to be an important factor in the development of glaucoma. in the report by sapienza and colleagues [67] , uveal cysts were a common finding on histologic examination of the enucleated glaucomatous blind eyes, whereas they were only noted clinically in 13% of cases. interestingly in those globes, microscopically, little inflammation was noted. therapy often consists of combinations of topical and systemic corticosteroids, topical nsaids, medications to control secondary glaucoma, and azathioprine [67] . administration of topical nsaids seems to exacerbate ocular hypertension frequently [65, 67] . uveodermatologic syndrome, or vogt-koyanagi-harada-like syndrome, is an autoimmune condition of dogs in which melanocytes become the target of the cellular response [80] . an immunohistochemical examination of affected tissues from two dogs revealed that the skin lesions were mediated by t cells and macrophages (t helper [th] 1 immunity), whereas the ocular lesions were more consistent with a b-cell and macrophage response (th2 immunity). the breeds primarily affected are the akita, samoyed, siberian husky, and shetland sheepdog [3] . the condition does occur sporadically in other breeds, however. affected patients are usually presented with anterior uveitis or panuveitis characterized by iridal or choroidal depigmentation, bullous retinal detachment, or blindness [3] . the ocular lesions may precede the cutaneous lesions, which include poliosis and vitiligo of the facial mucocutaneous junctions, nasal planum, scrotum, and footpads [81, 82] . generalized vitiligo may also occur [81] . because of the chronic nature of the disease, affected patients typically develop extensive posterior synechia, iris bombé, cataract, and secondary glaucoma. immunosuppressive drugs are the mainstay of therapy [3] . azathioprine is often combined with or substituted for corticosteroids to avoid the side effects associated with chronic systemic corticosteroid administration. the presence of any neoplastic process, whether primary or metastatic, within the globe may induce clinical signs of uveitis, such as iris hyperpigmentation, intraocular fibrin exudation, and hemorrhage [83] . therefore, the possibility of associated neoplasia must always be considered in patients that have uveitis, particularly if the inflammatory response or secondary glaucoma precludes complete visualization of the intraocular compartments [83] . the more common primary intraocular tumors include melanomas (dogs and cats) [5, 84] and iridociliary epithelial tumors (dogs) [85] . lymphosarcoma is the most frequent metastatic intraocular tumor in cats and dogs [5, 86, 87] . ocular involvement in canine lymphoma may include anterior uveitis, posterior uveitis, panuveitis, retinal hemorrhage, and superficial disease [87] . metastasis of angioinvasive pulmonary carcinoma has been described in four cats [88] . ophthalmic examination revealed wedge-shaped tan discoloration of the tapetal fundus, variable but mild serous exudation under the retina, and profoundly attenuated retinal vasculature [88] . the more common infectious causes of uveitis in cats are feline infectious peritonitis (fip), feline leukemia virus (felv), feline immunodeficiency virus (fiv), toxoplasmosis, and the systemic mycoses. bartonella henselae has also been proposed as a frequent cause of feline uveitis [89] . according to previously published reports, between 38% and 70% of cats with uveitis have an associated systemic disease [70, 71] . feline infectious peritonitis fip is caused when the immune response to feline coronavirus induces granulomatous necrotizing phlebitis and periphlebitis, protein-rich effusions into body cavities, and granulomatous inflammatory lesions in multiple organs [90, 91] . the associated clinical signs include febrile episodes, weight loss, anorexia, depression, debility, and variable thoracic and abdominal involvement [5] . the uveitis associated with fip is typically a panuveitis or panophthalmitis with diffuse and severe corneal edema, marked anterior uveitis, marked cellular infiltration of the vitreous (ie, vitritis), chorioretinitis, inflammatory retinal detachments, or optic neuritis [5] . mutton fat keratic precipitates with occasional admixed hemorrhage occur most often with fip because of the granulomatous nature of the infection. the granulomatous periphlebitis may be noted as perivascular cuffing surrounding the retinal vasculature. the diagnosis of fip can be challenging, because enteric coronaviruses crossreact and cause positive results on serologic tests and reverse transcriptase (rt)-polymerase chain reaction (pcr) assays. according to recommendations from the fip workshop symposium [92] , the first step in establishing a diagnosis of fip is to compare the signalment, history, and clinical findings with those of the typical individual infected with fip. most cats with fip are from 6 months to 3 years of age, come from shelters or catteries, and show signs of cyclic antibiotic-resistant fevers and specific physical manifestations depending on the form of the disease and location of lesions [92] . diagnostic test findings supportive of a diagnosis of fip include characteristic analysis of peritoneal or pleural effusions, leukocytosis with neutrophilia and lymphopenia, hyperglobulinemia, hypoalbuminemia, increased fibrinogen, and nonregenerative anemia of chronic disease [92] . finally, in a cat with suspect fip, one may also submit effusions or surgical biopsies for immunohistochemistry or rt-pcr [92] . serology or rt-pcr performed on serum samples can confirm exposure to feline coronavirus but must be paired with appropriate clinical signs to ensure an accurate diagnosis [92] . no therapy has been proved effective in the management of fip [92] . therapy remains symptomatic. bartonella spp b henselae was first suggested as a cause of feline anterior uveitis by lappin and black [89] in 1999 after a feline patient had a c value for igg antibodies to bartonella spp of 4.42, which indicated antibody production by ocular tissues [38, 39] , and no clinical response to therapy was noted until doxycycline was administered [89] . since the initial report, ketring and colleagues [93] have demonstrated elevated serum antibody production against bartonella in cats with uveitis. in a more recent study by fontanelle and colleagues [94] , however, healthy cats were more likely to have elevated bartonella titers than cats with uveitis. therefore, serum antibody tests alone do not seem to be sufficient to confirm a diagnosis of bartonella-induced uveitis. a definitive therapeutic protocol to resolve bartonella infection does not currently exist [95, 96] . the authors of one article [93] suggest azithromycin, doxycycline, or rifampin. illness in felv-infected cats results from the direct effects of the virus on bone marrow or lymphoid tissue [97] . the uveitis in association with felv infection is primarily a manifestation of lymphosarcoma. in the study by peiffer and wilcock [98] , felv-associated lymphosarcoma was the third most frequent cause of uveitis in cats after idiopathic lymphoplasmacytic uveitis and fip-associated uveitis. felv-induced lesions range from inflammatory cells and fibrin within the anterior chamber to small iris nodules or extensive neoplastic infiltration [99] . funduscopically, one may note the characteristic lesions of retinopathy of anemia, which may occur secondary to felv-related anemia [100] . cytologic examination of aqueous humor usually reveals variable amounts of lymphocytes and occasional plasmacytes and neutrophils [5] . the presence of lymphosarcoma may be confirmed based on bone marrow aspiration, lymph node biopsy, and direct biopsy of intraocular masses [101] . an elisa antigen test of peripheral blood can demonstrate the presence of the felv antigen [99] . the diagnosis of a persistent infection should be confirmed by performing an immunofluorescent antibody (ifa) test or repeating the elisa antigen test in 3 to 4 months [99] . direct viral damage of ocular tissues, initiation of secondary immune phenomena, or opportunistic infection secondary to immunosuppression may all contribute to the uveitis associated with fiv infection [102] . clinically, pars planitis is often a significant feature of the disease, creating a ''snow banking'' phenomenon as cells are deposited throughout the anterior vitreous with a greater density toward the periphery [102] . keratic precipitates are uncommon findings [102] . coinfection with toxoplasma gondii seems to increase the severity of clinical signs in cats that are infected with fiv [71, 103] , perhaps because the defense mechanisms for t gondii depend on cd4þ cells [103] . elisa tests may be used to detect antibodies to fiv [104] . according to a recent study [105] , the snap combo plus (idexx laboratories, atlanta, ga) is the best performing in-hospital test kit, and the mapic fiv test (sinovus biotech, inc., lund, sweden) should not be used because of the large number of invalid results or results that are difficult to interpret. unfortunately, vaccination of cats for fiv produces antibodies that are indistinguishable from those used to diagnose fiv infection [104] . currently, there is no method by which to differentiate vaccinal antibodies from those produced by natural infection. therefore, attaining a definitive diagnosis in a cat whose vaccination status is unknown becomes nearly impossible. symptomatic therapy is used to control the uveitis. t gondii is a well-recognized cause of retinitis, choroiditis, and anterior uveitis [106] . tachyzoites target the eye and multiply intracellularly within ocular tissues [107] . the classic funduscopic appearance is multifocal dark gray lesions in the tapetal fundus and fluffy white infiltrates in the nontapetal fundus [106] . t gondii has frequently been implicated as a main cause of acute and chronic idiopathic feline anterior uveitis [106] . supportive evidence for this hypothesis includes the higher seroprevalence rate to t gondii in cats with uveitis [71] . the only definitive diagnosis is histologic identification of the organism [108] . because a histologic diagnosis is frequently not available, however, serologic testing is the primary diagnostic modality. exposure to the organism is widespread within the feline population. therefore, paired serologic titers are recommended, and most cases remain suspect rather than confirmed [106] . an elisa test for igm antibodies with a single titer greater than 1:256 or rising igg titers is considered consistent with an active infection [108, 109] . more recently, pcr (b1 gene) tests for t gondii-specific igm and igg have been evaluated in serum and aqueous humor samples [110, 111] . t gondii-specific igm or igg was detected in the serum but not in the aqueous humor in 34.8% of healthy cats. in cats that had uveitis, t gondii-specific igm or igg was detected in the serum of 72% and in the aqueous humor of 39.5%, suggesting that the combination of serum and aqueous humor t gondii titers may be the most informative and useful method of testing. anti-inflammatory therapy should be used to control the uveitis in conjunction with clindamycin hydrochloride at a dose of 12.5 mg/kg administered orally twice daily for 14 to 21 days [112] . a granulomatous anterior uveitis, often with concurrent chorioretinitis, may be associated with cryptococcosis [113] , histoplasmosis [114] , blastomycosis [115] , or coccidioidomycosis [116] . from the literature, the incidence of blastomycosis in cats seems to be rare, except for sporadic clusters [117, 118] . in most cases, hematogenous spread is the likely route of ocular involvement. ocular cryptococcosis seems to be an exception, however, because extension from the central nervous system occurs more frequently. the diagnostic protocol for mycotic uveitides includes a complete physical examination, hematology, and clinical chemistries [5] . diagnosis is often achieved by identification of the organism during cytologic examination of aspirates or impression smears from lymph nodes, bone marrow aspirates, cerebrospinal fluid, or cutaneous lesions [5] . aqueous or vitreous paracentesis may be useful depending on the degree of ocular involvement (ie, anterior or posterior segment) [5] . histoplasmosis may be effectively treated using itraconazole [119] . blastomycosis may be treated with itraconazole or fluconazole, although the response is often poor [117, 118] . an excellent review article on this disease has been published by wanke [120] . endophthalmitis, chorioretinitis, and hyphema have all been reported in association with brucella canis infection [121] . vinayak and colleagues [121] reported that 14.2% of patients that had b canis infection demonstrated ocular signs. other clinical signs include reproductive tract lesions (eg, abortions, epididymitis, failure to conceive) [120] , diskospondylitis, osteomyelitis, splenomegaly, glomerulopathy, and meningoencephalitis [122, 123] . isolation of the organism is considered the ''gold standard'' diagnostic test [120] . this can be difficult, however. the rapid slide agglutination test is sensitive and can be performed early in the stage of infection [120] . positive results are confirmed with other tests, including the tube agglutination test, agar-gel immunodiffusion, indirect fluorescent antibody test, and elisa [122] . achieving complete eradication of the organism is difficult [120] . various suggested therapeutic regimens include minocycline and streptomycin, tetracycline and streptomycin, long-acting oxytetracycline [120] , enrofloxacin [124] , and gentamicin [121] . complete resolution of ocular clinical signs and clearance of the organism have only been reported in one case in the literature [121] . borrelia burgdorferi (lyme disease), ehrlichia spp, including canis, platys, and risticii, and rickettsia rickettsii (rocky mountain spotted fever [rmsf] ) have all been implicated as causative agents in cases of uveitis [122, 125] . the ocular lesions are similar and include anterior uveitis, hyphema, retinal hemorrhage, and retinal detachment (see fig. 7 ). the diagnosis is typically based on the combination of clinical signs and serologic testing. serum elisa and ifa serologic tests can be used to document exposure to lyme disease [126] . in patients previously vaccinated for lyme disease, western blot immunoassays may be used to differentiate natural exposure from vaccinal response [126] . the serum fluorescent antibody test is most reliable for the diagnosis of ehrlichial agents [125] . ifa and elisa serum antibody titers are available for the diagnosis of rmsf [125] . the current therapeutic recommendations are also similar. doxycycline is administered at a dosage of 10 mg/kg every 24 hours for 1 month as the primary therapy for lyme disease in patients with positive serology and clinical signs of disease [126] . two case reports document favorable responses to administration of doxycycline, sometimes paired with anti-inflammatory doses of corticosteroids in cases of canine ehrlichiosis [127, 128] . doxycycline is also used in the therapy of rmsf, and combination with systemic prednisolone has not been shown to decrease efficacy [129] . uveitis is a relatively infrequent presentation of this re-emerging disease [122, [130] [131] [132] . reported cases have had anterior uveitis [122] and, in one case, bilateral serous retinal detachment [132] . the diagnosis is most commonly achieved using a serum microscopic agglutination test. a single high titer or rising titers are considered indicative of infection [133] [134] [135] [136] [137] therapy is directed toward elimination of the organism. high doses of penicillin, ampicillin, and amoxicillin can clear the leptospiremia phase, usually within hours of administration [138] . these drugs do not eliminate the carrier state, however [133, 134] . current recommendations are to use a 2-week course of doxycycline to clear the carrier state in dogs [138] . blastomycosis, cryptococcosis, coccidiomycosis, and histoplasmosis are the systemic mycoses most commonly involved with uveitis [139, 140] . patients may be presented with anterior uveitis, chorioretinitis, panuveitis, endophthalmitis, or optic neuritis. a complete physical examination with particular attention to the cutaneous examination, thoracic auscultation, and abdominal palpation can aid in establishing a diagnosis. as discussed previously, cytologic identification of the organism within aspirates or impression smears is the gold standard for establishing a diagnosis. serologic tests are available. the latex cryptococcal agglutination test detects cryptococcal antigen and can be a useful test for establishing a diagnosis and monitoring the response to therapy. the serologic tests for histoplasmosis, blastomycosis, and cocciciomycosis detect antibody production. false-negative responses, at least for blastomycosis, are not uncommon [141] . the preferred systemic therapy for each type of mycosis varies [3] . the current preferred therapy for blastomycosis is the administration of itraconazole [142] . although many clinicians do not advocate systemic corticosteroid treatment in dogs with systemic mycoses [143] , a recent retrospective study of dogs infected with blastomycosis by finn and colleagues [144] did not note any change in survival times and suggested that combination therapy of systemic prednisone and itraconazole may have assisted in the maintenance of vision. the clinical signs of uveitis occur as a result of inflammation within the vascular coat of the eye, which causes breakdown of the bab and blood-retinal barrier. clinical signs include blepharospasm, photophobia, conjunctival hyperemia, circumlimbal corneal vascularization, corneal edema, aqueous flare, inflammatory cells within the anterior chamber, keratic precipitates, iridal congestion, ocular hypotony, retinal hemorrhage, and retinal detachment. sequelae to uveitis include cataracts, posterior synechiae, secondary glaucoma, and retinal degeneration. many infectious and noninfectious causes can incite episodes of uveitis. therefore, complete ocular and physical examinations are recommended for all patients that have uveitis. a complete blood cell count, serum biochemistry profile, urinalysis, thoracic radiographs, and select serologic tests may be performed in an effort to identify any underlying etiologic agents. despite exhaustive workups, however, the underlying cause is not determined in many cases. the goals of therapy are preserving vision if possible, minimizing pain, and halting inflammation. additional therapeutic agents may be used if the underlying etiologic agent can be identified. immunopathology of uveitis diseases and surgery of the canine anterior uvea causes of uveitis in dogs: 102 cases (1989-2000) feline ophthalmology laser flaremetric evaluation of experimentally induced blood-aqueous barrier disruption in cats the structural basis of the blood-ocular barriers the blood-ocular barriers studies on the permeability of the blood-retinal barrier. i. on the existence, development, and site of a blood-retinal barrier the intercellular junctional complexes of retinal pigment epithelia adler's physiology of the eye adler's physiology of the eye: clinical application diseases of the canine ocular fundus mechanism of steroid action in ocular inflammation: inhibition of prostaglandin production measurement of inflammatory mediators in aqueous humor following paracentesis of the anterior chamber in dogs. presented at 38th annual conference of the american college of veterinary ophthalmology blockade of the interaction of leukotriene b4 with its receptor prevents development of autoimmune uveitis uveitis in the dog and cat corneal epithelial cl-dependent pump quantified dictionary of eye terminology effects of indomethacin and prostaglandins on the dog iris sphincter and dilator muscles effects of prostaglandin f2 alpha and leukotriene d4 on pupil size, intraocular pressure, and blood-aqueous barrier in dogs aqueous humor dynamics in experimental iridocyclitis comparison of the hypotensive and other ocular effects of prostaglandins e2 and f2a on cat and rhesus monkey eyes textbook of veterinary ophthalmology. philadelphia: lea & febiger fundamentals of veterinary ophthalmology corneal vascularization in uveitis and graft rejection matrix metalloproteinases in disease and repair processes in the anterior segment the pathogenesis and significance of pre-iridal fibrovascular membrane in domestic animals principles and practice of ophthalmology. philadelphia: wb saunders philadelphia: wb saunders disease of the canine lens and cataract formation clinical and pathological observations concerning the aetiology of primary lens luxation in the dog feline lens displacement: a retrospective analysis of 345 cases. progress in veterinary and comparative fundamentals of veterinary ophthalmic pathology examination of the aqueous humor as a diagnostic aid in anterior uveitis diagnostic anterior chamber paracentesis in uveitis: a safe procedure vitreous centesis as a diagnostic procedure enzyme linked immunosorbent assays for the detection of toxoplasma gondii specific antibodies and antigens in the aqueous humor of cats toxoplasma gondii-specific antibodies in the aqueous humor of cats with toxoplasmosis nonhypotensive autonomic agents in veterinary ophthalmology experimental studies on the blood-aqueous barrier a comparative study of the effects of mecholyl, doryl, eserine, pilocarpine, atropine, and epinephrine on the blood aqueous barrier mydriatics for canine ophthalmoscopy evaluation of mydriatics in the cat applications of adrenergic drugs in ophthalmology salivation induced in dogs by conjunctival instillation of atropine the use of corticosteroids to treat ocular inflammation lipocortin and the mechanism of action of the glucocorticoids glucocorticoids inhibit prostaglandin synthesis not only at the level of phospholipase a2 but also at the level of cyclo-oxygenase/pge isomerase control of ocular inflammation effects of flunixin meglumine and dexamethasone on aqueous protein values after intraocular surgery in the dog nonsteroidal anti-inflammatory drugs in veterinary ophthalmology topical nonsteroidal anti-inflammatory agents in ophthalmology in vivo effects of indomethacin and flurbiprofen on the locomotion of neutrophils elicited by immune and non-immune inflammation in the rat anti-inflammatory and antiallergic effects of ketorolac tromethamine in the conjunctival provocation model cyclo-oxygenase inhibitors in ophthalmology acute effects of anti-inflammatory drugs on neodymium:yttrium aluminum garnet laser-induced uveitis in dogs comparison of the blood-aqueous barrier stabilizing effects of steroidal and nonsteroidal anti-inflammatory agents in the dog. progress in veterinary and inhibition of pilocarpine-induced aqueous humor flare, hypotony, and miosis by topical administration of anti-inflammatory and anesthetic drugs to dogs tolfenamic acid in the control of ocular inflammation in the dog: pharmacokinetics and clinical results obtained in an experimental model the gastroduodenal effects of buffered aspirin, carprofen, and etodolac in healthy dogs diagnosis and treatment of feline uveitis topical nonsteroidal anti-inflammatory drugs for ophthalmic use: a safety review comparative efficacy of topically applied flurbiprofen, diclofenac, tolmetin, and suprofen for the treatment of experimentally induced blood-aqueous barrier disruption in dogs effect of flurbiprofen on facility of aqueous outflow in the eyes of dogs immunosuppressive drugs in immune and inflammatory ocular disease prades-sapienza a. golden retriever uveitis: 75 cases (1994-1999) vogt-koyanagi-harada syndrome in humans and dogs. compendium of continuing education for the practicing veterinarian drugs used to treat immune-mediated disorders in animals. presented at wsava congress. granada feline uveitis: a retrospective study of 45 cases seroepidemiologic and clinical observations of 93 cases of uveitis in cats. progress in veterinary and lens-induced uveitis the effect of lens-induced uveitis on the success of extracapsular cataract extraction: a retrospective study of 65 lens removals in the dog fluorescein concentrations in the aqueous of dogs with cataracts human t-cell mediated response to homologous lens antigen the pathology of lens-induced uveitis in dogs traumatic anterior lens capsule disruption feline ocular sarcomas a histopathological study of iridociliary cysts and glaucoma in golden retrievers experimentally induced vogt-koyanagi-harada disease in two akita dogs uveodermatologic syndrome (vogt-koyanagi-harada-like syndrome) with generalized depigmentation in a dachshund an immunohistochemical study of uveodermatologic syndrome in two japanese akita dogs ciliary body epithelial tumors in the dog and cat: a report of 13 cases primary intraocular tumors in the dog iridociliary epithelial tumors in 100 dogs and 17 cats: a morphological study ocular changes in malignant lymphoma of dogs prevalence of ocular involvement in dogs with multicentric lymphoma: prospective evaluation of 94 cases. veterinary and comparative angioinvasive pulmonary carcinoma with posterior segment metastasis in four cats bartonella spp infection as a possible cause of uveitis in a cat cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis morphologic features and development of granulomatous vasculitis in feline infectious peritonitis recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium bartonella: a new etiological agent of feline ocular disease prevalence of serum antibodies against bartonella species in the serum of cats with or without uveitis use of enrofloxacin for treatment of large-form haemobartonella felis in experimentally infected cats efficacy of enrofloxacin or doxycycline for treatment of bartonella henselae or bartonella clarridgeiae infection in cats hematopoietic tumors of cats histopathologic study of uveitis in cats: 139 cases (1978-1988) feline leukemia virus and feline immunodeficiency virus ocular disease in felv-positive cats: 11 cases (1981-1986) laboratory testing for infectious diseases of dogs and cats intraocular disease associated with feline immunodeficiency virus infection in cats feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis effect of vaccination against feline immunodeficiency virus on results of serologic testing in cats quality of different in-clinic test systems for feline immunodeficiency virus and feline leukaemia virus infection toxoplasmosis of animals and man histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952-1990) clinical feline toxoplasmosis. serologic diagnosis and therapeutic management of 15 cases polymerase chain reaction for the detection of toxoplasma gondii within aqueous humor of experimentally-inoculated cats detection of toxoplasma gondii parasitemia in experimentally inoculated cats feline uveitis: diagnosis and treatment feline cryptococcosis: a review and seven case reports multifocal ocular histoplasmosis in a dog and cat ocular changes in a cat with disseminated blastomycosis coccidioidomycosis in 48 cats: a retrospective study (1984-1993) blastomycosis in indoor cats: suburban chicago clinical aspects of natural infection with blastomyces dermatitidis in cats: 8 cases (1991-2005) management of bilateral uveitis in a toxoplasma gondii-seropositive cat with histopathologic evidence of fungal panuveitis canine brucellosis clinical resolution of brucella canis induced ocular inflammation in a dog canine systemic bacterial infections canine brucellosis: a diagnostician's dilemma use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial) canine rickettsial infections acvim small animal consensus statement on lyme disease in dogs: diagnosis, treatment, and prevention ocular signs of canine monocytic ehrlichiosis: a retrospective study in dogs from ocular manifestations of natural canine monocytic ehrlichiosis (ehrlichia canis): a retrospective study of 90 cases prednisolone at anti-inflammatory or immunosuppressive dosages in conjunction with doxycycline does not potentiate the severity of rickettsia rickettsii infection in dogs diagnosis of leptospirosis: a reemerging disease of companion animals leptospirosis: a re-emergent zoonosis leptospirosis panuveitis in a dog canine leptospirosis in new jersey and michigan: 17 cases (1990-1995) infectious diseases of the dog and cat treatment and outcome of dogs with leptospirosis: 36 cases (1990-1998) naturally acquired leptospirosis in 36 dogs: serological and clinicopathological features leptospirosis in dogs. compendium of continuing education for the practicing veterinarian leptospirosis a re-emerging zoonotic disease comparative ophthalmic pathology systemic mycoses infectious disease of the dog and cat treatment of blastomycosis with itraconazole in 112 dogs diseases and surgery of the canine anterior uvea visual outcome in a group of dogs with ocular blastomycosis treated with systemic antifungals and systemic corticosteroids key: cord-022300-9w0lehal authors: hoskins, johnny d. title: the liver and pancreas date: 2009-05-15 journal: veterinary pediatrics doi: 10.1016/b978-0-7216-7665-4.50015-2 sha: doc_id: 22300 cord_uid: 9w0lehal nan liver enzyme activities. the serum activities of alanine aminotransferase (alt) and aspartate aminotransferase (ast) in newborn and growing puppies and kittens are usually within the normal range or slightly higher than those of healthy adult dogs and cats (keller, 1981) . the serum alkaline phosphatase (alp) and gamma-glutamyltransferase (ggt) activities of 1-to l o-day-old puppies are 20-to 25fold greater than those of healthy adults. selected laboratory values used as indicators of hepatobiliary dysfunction in newborn and growing puppies and kittens are listed in table 11 -1. the source of the profound alp and ggt activity is probably of placental, colostral, and! or intestinal origin. these profound serum alp and ggt activities after 10 to 14 days postpartum will decrease to moderately increased activities. although the bone alp isoenzyme derived from active osteoblast activity may increase the serum alp activity in growing animals, the magnitude of increase usually is only two-to threefold the normal level (center et ai, 1995) . colostrum is rich in both alp and ggt activity, and it is possible that these enzymes may be absorbed from the colostrum or intestinal tract during the first days of life. alternatively, the ingestion of colostrum may stimulate intestinal growth and enzyme production. therefore, colostrum-deprived puppies can be identified by their serum alp and ggt activity level, particularly during the first week of 200 life, as an indicator of successful ingestion of colostrum. thus, increased serum alp and ggt activities cannot be used in the diagnosis of acute liver dysfunction during the first 10 to 14 days of life. such increases in serum alp and ggt activities cannot be used to detect colostrum-deprived kittens. serum bile acids. serum bile acids are used to identify hepatic and hepatoportal circulatory dysfunction. the use of the serum bile acid measurement as a test of hepatic dysfunction is of value for the young dog and cat using 12-to 24-hour fasted and 2-hour postprandial serum samples (center et al, 1985a,b) . the normal fasting and 2-hour postprandial values established for adults may be used for puppies and kittens as young as 4 weeks. this test is also reliable for detecting portal circulatory abnormalities (center et al, 1986a; meyer, 1986) . extramedullary hematopoiesis. extramedullary hematopoiesis is commonly observed in the livers of the puppy and kitten through 4 months of age. in older puppies and kittens, disorders associated with blood loss and a subsequent need for erythron mass replenishment may also be associated with hepatic extramedullary hematopoiesis. congenital gallbladder disorders. congenital division of the gallbladder is the most common anomaly and has been referred to as an accessory, cleft, or diverticular gallbladder (bartlett, 1951; boyden, 1926) . these anomalies may develop as an initial subdivision of the primary cystic diverticulum of the embryo or as a bud from the neck of the embryonic gallbladder. the gallbladder may become trilobed or bilobed ( fig. 11-1 ). other anomalies include accessory gallbladders; the development of two separated gallbladders with cystic ducts uniting in a common duct; ductular bladders developing as supernumerary vesicles derived from either hepatic, cystic, or common bile ducts; and trabecular bladders derived from vesicular outgrowths of liver trabeculae. these malformations are infrequently associated with signs of hepatobiliary dysfunction in puppies and kittens. congenital hepatic cysts. congenital hepatic cysts are infrequently found in the young dog and cat (black, 1983; crowell et ai, 1979; mckenna and carpenter, 1980) . cystic lesions may be parenchymal or ductal in origin and may be either solitary or multiple (polycystic). cystic lesions vary in size from a few millimeters to several centimeters. congenital hepatic cysts are typically asymptomatic. rarely, fluid may accumulate in the abdominal cavity as a consequence of cyst rupture or portal hypertension resulting from impingement of major vessels. cyst contents are usually a clear or modified transudate, although cysts may contain bile or blood. acquired hepatic cysts are usually solitary, whereas congenital or developmental hepatic cysts are commonly multiple. polycystic hepatic cysts may be associated with cystic lesions in other organs, most notably the kidneys. polycystic kidneys and liver have been identified in cairn terrier dogs and persian cats during the first few months of life (crowell et ai, 1979; mckenna and carpenter, 1980) . abdominal radiographs may reveal an irregular hepatic margin or focal densities if a few large hepatic or biliary cysts are present. ultrasonography readily reveals the cystic nature of these lesions and the extent of parenchymal or biliary tract involvement ( fig. 11-2 ). treatment is usually not needed for a congenital hepatic cyst unless a large cyst is causing abdominal discomfort or fluid accumulation. if cystic lesions are symptomatic, surgical resection of solitary lesions, partial cyst wall, or a liver lobe may be required. congenital biliary tract malformations. congenital anomalies of the biliary tract are rare in the young dog and cat. one case of suspected biliary atresia in the cat is reported (blood, 1947) . shunts. the hepatic portal system develops from the umbilical and omphalomesenteric systems. the mesenteric portions of the omphalomesenteric veins become the tributaries of the portal vein. small anastomoses develop between the portal and systemic circulation routes that become the normal portosystemic venous communications (heath and house, 1970; khan and vitums, 1971; sleight and thomford, 1969; viturns, 1959 ). in the fetus, blood from the umbilical vein flows directly to the caudal vena cava through the ductus venosus, thus bypassing the liver. by passively responding to changes in the systemic or hepatic circulation, the ductus venosus stabilizes the venous return to the fetal heart as the umbilical venous return fluctuates (edelstone, 1980) . functional and morphologic closure of the ductus venosus does not occur at the same time after birth (lohse and suter, 1977) . functional closure develops gradually during the second and third days after birth in the puppy. morphologic closure occurs as the ductus atrophies, resulting in the formation of a thin fibrous band, the ligamentum venosum, within the liver. the ductus closure depends on changes in pressure and resistance across the hepatic vasculature that follows the postnatal obliteration of the umbilical circulation. morphologic closure of the ductus occurs by 1 to 3 months after birth. congenital portosystemic venous shunts (pss) are abnormal vascular connections between the portal and systemic venous circulations boothe et ai, 1996; bostwick and twedt, 1995; carr and thornburg, 1984; gandolfi, 1984; scaveui et al, 1986) . several different types of congenital pss occur in young dogs and cats, including but not limited to (1) persistent patent fetal ductus venosus, (2) direct portal vein to caudal vena cava, (3) direct portal vein to azygos vein, (4) combination of portal vein with caudal vena cava into the azygos vein, (5) left gastric vein to vena caval shunt, (6) portal vein hypoplasia or atresia with secondary anomalous vessel, and (7) anomalous malformation of the caudal vena cava (center et ai, 1995) . the most cornman types of congenital pss are illustrated in figure 11 -3. in the cat, the most cornman congenital pss involves the left gastric vein . a congenital pss in puppies and kittens is usually a single anomalous vessel in extrahepatic or intrahepatic locations, whereas an acquired pss most commonly occurs as multiple extrahepatic smaller vessels that become patent during sustained portal hypertension. the consequences of the anomalous portal circulation are that the portal blood contains toxins absorbed from the intestines that is delivered directly to the systemic circulation without benefit of hepatic detoxification, contributing to signs of hepatic encephalopathy, and that hepatotrophic factors in the visceral circulation draining the gastrointestinal tract and pancreas do not circulate directly to the liver, causing inadequate liver development and reduced functional liver tissue. signs of hepatic dysfunction associated with congenital pss are usually exhibited at a young age in puppies and kittens. puppies may exhibit the signs as early as 6 to 8 weeks of age. the signs in puppies are variable but may include vomiting, diarrhea, anorexia, small body stature, weight loss, intermittent fever, polyphagia, polydipsia, hematuria, hypersalivation, intolerance to anesthetic agents or tranquilizers that require hepatic metabolism or excretion, atypical behavior, and, rarely, ascites or icterus. intermittent neurologic abnormalities associated with ingestion of protein-laden food or resolving hemorrhage are common and may include episodic aggression, amaurosis, ataxia, incessant pacing, circling, head pressing, and seizures. some puppies present with ammonium biurate uroliths located in the urinary tract (marretta et al, 1981) . most cases of congenital pss in kittens (by 6 months of age in 75% of affected kittens) occur in himalayan, persian, and mixed breed cats, although any breed may have a congenital pss (levy, 1997) . kittens may exhibit signs as early as ages 6 to 8 weeks. the signs of congenital pss are usually hypersalivation, seizures, ataxia, tremors, and depression. intermittent or permanent blindness and mydriasis are also ob-the liver end pencrces i 203 served. other less often noted signs may include vomiting, diarrhea, anorexia, tachypnea, dyspnea, and nasal discharge. polyuria and polydipsia are observed infrequently. dysuria may be observed in kittens with ammonium biurate calculi. about two thirds of affected kittens will have small body stature and be thin and unkempt. a definitive diagnosis of congenital pss in puppies and kittens is often not possible by routine laboratory evaluations. complete blood counts (cbcs), serum chemistry profiles, and urinalysis may help rule out other causes of presenting signs such as acute renal failure, electrolyte derangements, hypoglycemia, and urinary tract disorders. the cbc findings may include microcytic, normochromic erythrocytes and/or a mild nonregenerative anemia (griffiths et al, 1981) . poikilocytosis has been observed in peripheral blood films of some kittens with congenital pss. urinalysis may reveal ammonium biurate crystals when viewed under magnification (x 400) to determine their typical color and shape. serum chemistry profiles may reveal mild increases in the serum activities of alt, as'f, and alp. because of the young age of the animals at initial diagnosis, the serum alp activity is usually two-to threefold higher than normal. the serum activity levels of the alt and ast are less frequently increased. in some cases, active liver disease coexists with a congenital pss, and the animals thus affected have mildly to moderately increased liver enzyme activity and notable hepatic inflammation and/or fibrosis on histopathologic examination. in most animals with congenital pss, the total bilirubin value is normal. albumin values may be mildly decreased. coagulation profiles including prothrombin time, activated partial thromboplastin time, and fibrinogen are usually normal. serum glucose values may be normal, mildly reduced, or markedly hypoglycemic. in some cases, the hypoglycemia-produced neuroglycopenia may complicate the recognition of the congenital pss. animals with congenital pss may become hypoglycemic due to insufficient glycogen stores, abnormal responsiveness to glucagon, hyperglucagonemia, or abnormal insulin metabolism (lickley et al, 1975; magne and macy, 1984) . these abnormalities, coupled with the metabolic immaturity of the young animal's liver, may cause profound hypoglycemia during the first weeks of life. toy-breed puppies appear to be at increased risk for profound hypoglycemia. the blood urea nitrogen concentration may be low or in the low normal range in any young animal with hepatic dysfunction. the most reliable and consistent blood test for the detection of liver dysfunction in puppies and kittens with congenital pss is the 12-to 24-hour fasted and 2-hour postprandial serum bile acid concentrations (center et ai, 1986b; meyer, 1986) . diagnostic imaging of a puppy or kitten with abnormal serum bile acid values helps determine if a suspected congenital pss is present (table 11 -2) (lamb, 1996; lamb et ai, 1996) . animals with congenital pss frequently have reduced hepatic size (i.e., rounded contour of the caudal edge of the liver and cranial displacement of the stomach radiographically) (lamb, 1997) . in addition, these animals may have opaque ammonium biurate calculi. ultrasonographic findings in puppies with congenital pss include small liver, reduced visibility of intrahepatic portal vasculature, and an anomalous blood vessel draining into the caudal vena cava or sometimes into the azygos vein (lamb, 1996) . two-dimensional, gray-scale ultrasonography is used to image through a ventral abdominal wall; however, in most large puppies the optimal approach to the portal vein is through a lateral abdominal wall using the right intercostal spaces (lamb, 1997) . the portal vein is normally visible by ultrasound imaging as ultrasound waves enter the liver at the porta hepatis, ventral to the caudal vena cava. lobar branches of the portal vein have echogenic walls. congenital intrahepatic portocaval shunts are identified on the basis of their ultrasonographic appearance as left-divisional, central-divisional, or right-divisional intrahepatic shunts (lamb, 1997) . left-divisional intrahepatic shunts have a relatively consistent bent tubular shape and drain into the left hepatic vein. central-divisional intrahepatic shunts take the form of a foramen between dilated portions of the intrahepatic portal vein and caudal vena cava. rightdivisional intrahepatic shunts appear as large, tortuous vessels that extend far to the right of midline. the morphology of the left-divisional shunts is compatible with patent ductus venosus. the irish wolfhound and deerhound are predisposed to left-divisional intrahepatic shunts; the old english sheepdog and australian cattle dog are predisposed to central-divisional intrahepatic shunts; labrador and golden retrievers are affected by both left-divisional and central-divisional intrahepatic shunts (lamb, 1997) . animals with extrahepatic congenital pss typically have an anomalous vessel that drains into the caudal vena cava between the right renal vein and the hepatic veins; because of the dorsal location, this anomalous vessel may be visible only through the right dorsal intercostal spaces (lamb, 1997) . congenital portoazygos shunts may also be visualized using the right dorsal intercostal approach; looking for the shunting vessel at the point where it drains into the caudal vena cava is more accurate than trying to examine the various tributaries of the portal vein (lamb, 1997) . extrahepatic shunts may be difficult to identify ultrasonographically if access to the relevant structures is hindered by the skill of the person performing the ultrasana graphic study, the animal's large body size, a lack of acoustic windows as a result of reduced hepatic size, or the presence of excessive intestinal gas. duplex doppler ultrasonography may be used to measure portal blood flow velocity in puppies with suspected congenital pss (lamb and mahoney, 1994) . normal portal blood flow is relatively uniform and nonpulsatile, average portal blood flow velocity being approximately 15 cm/s in healthy, unsedated puppies. the caudal vena cava normally contains variable blood flow because of the influence of changing right atrial and pleural pressures. in most cases, congenital pss represents a low resistance pathway for blood to bypass the liver and enter the caudal vena cava. in affected puppies with congenital pss, the portal vein is exposed to right atrial and pleural pressure changes, and the pattern of portal blood flow may become similar to that noted in the caudal vena cava. puppies with portal hypertension have reduced mean portal blood flow velocity, which correlates with the presence of multiple extrahepatic anomalous vessels. puppies with portal hypertension as a result of hepatic arteriovenous fistula have pulsatile hepatofugal flow in the portal vein. most extrahepatic and intrahepatic congenital psss are detectable using two-dimensional, gray-scale ultrasonography; however, use of color-flow doppler ultrasonography aids in the detection of small extrahepatic shunting vessels. a congenital extrahepatic pss usually drains into the caudal vena cava close to the cranial pole of the right kidney, and on color-flow images a localized area of turbulent flow in the caudal vena cava indicates the shunt's location. when two-dimensional gray-scale, duplex doppler, and color-flow doppler modalities are used in combination, the accuracy for ultrasonographic diagnosis of congenital pss in puppies and kittens can be at least 94% (lamb, 1997) . various techniques for opacification of the portal vein and its hepatic branches have been used, including operative mesenteric portography, cranial mesenteric angiography, and percutaneous splenoportography (suter, 1975) . operative mesenteric portography is the most frequently performed opacification procedure for suspected congenital pss, where abdominal radiographs are made immediately after injection of contrast medium into a indwelling catheter placed surgically in a mesenteric vein . obtaining lateral and ventrodorsal portograms usually provides an excellent view of the intrahepatic or extrahepatic shunt. this technique can also be used in combination with surgery, in which case the mesenteric vein catheter is also used to measure portal blood pressure during the congenital shunt ligation or attenuation procedure. repeating the portogram after shunt ligation or attenuation enables the surgeon to assess the patency of the intrahepatic portal vessel and to check if there is any other congenital shunt(s) present. portal scintigraphy with tc 99m-pertechnetate that is absorbed into the portal circulation after administration per rectum is currently being used in puppies and kittens to detect congenital pss (forstervan hijfte et ai, 1996; koblik et al, 1990 ). by acquiring a dynamic series of gamma camera images of the thorax and cranial abdomen immediately after administration of tc 99m _ pertechnetate and comparing the rate of accumulation of tc 99m-pertechnetate activity in the liver and heart, congenital pss may be detected with a high degree of accuracy. tc 99m-pertechnetate activity in the portal vein normally accumulates first in the liver, but in animals with congenital pss the distribution of tc 99m_per_ technetate activity is altered as it bypasses the liver, reaching the heart before the liver. the severity of the congenital pss can be quantified as a shunt index that provides an estimate of the proportion of portal blood that bypasses the liver. normal puppies have a shunt index of less than 15%; most puppies with congenital pss have a shunt index greater than 60%. an alternative technique for portal scintigraphy involves ultrasound-guided injection of a radiochemical directly into a splenic vein (meyer et al, 1994) . this method of injection combined with the use of tc 99m-iabeled macroaggregates that are normally trapped in the cap-illaries or sinusoids of the target organ enables accurate quantification of the degree of congenital shunting. typical values of a shunt index using this technique are less than 5% for normal puppies and greater than 90% for puppies with congenital pss. whether the administration per rectum or splenic vein injection technique is used, portal scintigraphy can provide a comparison between the shunt index before and after surgical treatment (van vechten et al, 1994) . the shunt index is usually markedly decreased after surgery to attenuate or ligate an anomalous vessel, although it may not be in the normal range. a continued high shunt index is a poor prognostic sign. surgical ligation or shunt vessel attenuation is the definitive treatment for congenital pss and is the preferred method of long-term management (birchard, 1984; breznock et ai, 1983; vogt et al, 1996; wrigley et ai, 1983) . the extrahepatic congenital pss is more amenable to surgical ligation or shunt vessel attenuation than are intrahepatic congenital psss. medical management should be given to the affected animal before and after surgical correction until improvement in hepatic function is shown. mfected animals undergoing surgery should have their body temperature stabilized and should receive intravenous fluids supplemented with 2.5% or 5.0% dextrose solution and with potassium chloride. in addition, owners should be cautioned that any animal with a congenital pss probably would have a shortened life expectancy despite satisfactory surgical correction and their conscientious care. manometric determination of baseline portal blood pressure should be completed before shunt vessel ligation (hardie, 1997) . after visual observation of the anomalous vessel, a ligature is temporarily placed while manometric determination of the portal blood pressure is made. equilibration of the manometer pressure usually takes several minutes. if the relative change in portal pressure exceeds 10 cm h 20 or the postligation pressure exceeds 20 em h 20, the ligature should be loosened. assessment of visceral perfusion by color change (cyanosis and/ or injection) or of arterial vasospasm causing a throbbing of the mesenteric circulation is not a reliable method of determining the safe tautness of the shunt vessel ligature. in many cases, only a partial surgical ligation, or shunt vessel attenuation, can be completed. further ligation may be possible in several weeks or months. incomplete ligation of a congenital pss can result in marked clinical improvement of the animal and owner satisfaction. partial shunt vessel ligation may result in eventual complete shunt closure within 6 months in some animals. complications after shunt vessel ligation or attenuation are frequent and may lead to the animal's death (hardie, 1997) . it is important that the owner understand the potential complications before surgery is attempted because many times the financial and emotional costs of complications are great. the immediate complication rate for performing a laparotomy on puppies and kittens with congenital pss is between 14% and 25%. because many of the complications are life threatening, it is reasonable to tell the owner that this surgery carries a 15% risk of death due to unexpected complications. intrahepatic shunt ligation requires longer surgery times than does extrahepatic shunt ligation, but the risk of death is no higher than with extrahepatic shunt ligation when performed by a experienced surgeon. in the immediate postoperative period, the animal is closely monitored for signs of portal hypertension, as indicated by acute abdominal swelling, abdominal pain, shock, vomiting, or bloody diarrhea (holt, 1994) . if portal hypertension is suspected, shock therapy is initiated and the animal is returned to surgery for immediate ligature removal. feeding may precipitate portal hypertension, and animals should be given small amounts of food and monitored closely after each feeding. if thrombosis of the shunt vessel or the portal vein occurs, signs of portal hypertension may occur up to several days after surgery, and treatment is usually futile. bleeding after surgery can also result in abdominal distention and signs of shock. at reoperation these animals are usually found to have diffuse hemorrhage. conservative treatment with packed red blood cells, fresh frozen plasma, or whole blood may be a more appropriate treatment than reoperation, if bleeding is suspected (hardie, 1997) . serious complications encountered in animals undergoing congenital pss surgery include intraoperative cardiac arrest, life-threatening hemorrhage, portal hypertension, seizures that usually start 2 to 3 days after surgery and may progress to status epilepticus, hyperthermia, gastric dilation-volvulus, acute pulmonary edema, and biliary pseudocyst formation (hardie, 1997) . the more manageable complications include abdominal distention, hypotension, hypothermia, hypoglycemia, mild gastrointestinal disturbances, and postoperative pain. predictor signs of immediate postoperative complications include low packed cell volume before surgery, absence of arborizing intrahepatic vasculature the liver lind plincrells i 207 during the mesenteric portogram, partial shunt vessel ligation, and hypothermia in the postoperative period. kittens are especially prone to developing seizures after shunt vessel ligation and should be administered phenobarbital at therapeutic serum concentrations for the entire perioperative period (hardie, 1997) . seizures usually occur 12 hours to 4 days after surgery. kittens that have no evidence of seizures in the peri operative period are weaned off the phenobarbital 1 month after surgery. long-term complications are encountered in animals whose congenital pss is not completely ligated and in animals that are older than 2 years of age at the time of surgery (hottinger et ai, 1995) . approximately 50% of the single shunts cannot be completely ligated at the first surgery because complete occlusion results in portal pressures greater than 20 ern h 2 0 or a rise in portal pressure greater than 10 em h 2 0 . within this group of partial vessel ligation, recurrence of pss signs may occur in as many as 40% of animals if a further vessel ligation is not performed. multiple extrahepatic shunts may form even with partial vessel ligation and are often associated with recurrence of pss signs. for animals with multiple extrahepatic shunts due to portal hypertension, the 2-year survival rate is 50% regardless of whether they are treated medically or surgically (hardie, 1997) . postoperative improvement is apparent from observation of the animal's activity at home but should always be followed by assessment of serum chemistry profile and serum bile acid concentrations for hepatic dysfunction (hardie, 1997) . medical management should be maintained until postoperative improvement has been unequivocally demonstrated. if serum bile acid values remain increased and the shunt index is greater than 15% at 60 to 90 days after surgery, reoperation is indicated. reoperation, however, has its own complications, mostly associated with the risk of inadvertently cutting a structure surrounded by scar tissue. reoperation of congenital intrahepatic shunts can be extremely difficult. if a mattress suture was placed across the anomalous vessel, the long ends of the suture material can be identified and the mattress suture tightened further. in some instances, it is necessary, however, to place additional sutures. to avoid the risks associated with reoperation of congenital intrahepatic shunts, a new technique has been developed in which an extrahepatic shunt is created between the portal vein and the vena cava using a jugular vein graft . this shunt is created at the time of the first surgery, and the intrahepatic shunt is completely closed. the venous graft prevents portal hypertension from developing at the time of the first surgery. the venous graft may slowly occlude, resulting in a normal shunt ndex .60~o 9~days after surgery. if the shunt mde~is std.l hi~h, reoperation is simply a matter of .either ligating or placing an ameroid constrictor band on the vein graft. in some cases, serum bile acid values remain abnormal despite a remarkable improvement in the animal's signs (hardie, 1997) . if serum chemistry profile and serum bile acid concentrations. indicate ongoing hepatic dysfunction, then medical management should be continued. medi~al manageme~t is directed at minimizing the signs of hepatic encephalopathy and includes manipulation of dietary proteins and intestinal flora and avoidance of medications or s?bstances capable of inducing encephalopathic signs. a restricted protein diet (2.0 to 2.5 mg/ kg) composed of proteins rich in branchedchain amino acids with comparatively smaller amounts of aromatic amino acids is recom-m~nded. foods containing milk protein (dried milk or cottage cheese) are best. the bulk of the caloric intake should consist of simple carbohydrates such as boiled white rice. meals should be frequent and in small amounts to maximize digestion and absorption so that minimal residue is passed into the colon where intestinal anaerobic bacteria degrade~itrogenous compounds to ammonia. commercial diets formulated for liver or renal dysfunction and a diet formulated for intestinal disease are used with success in most puppies and kittens with congenital or acquired pss. manipulation of intestinal flora with antimicrobial a~e.nts.and lactulose also produces mar~ed :limcallmprovement. for animals presennng m encephalopathic crisis intravenous isotonic electrolyte solutions suppl~mented with 2.5 %. or 5.0%~extrose solution and potassium chloride, cleansing enemas with warmed 0.9% saline solution, or enemas with added neomycin (15 to 20 ml of 1% solution three to four times daily), lactulose (5 to 10 ml diluted 1:3 with water three to four times daily), or betadine solution (10% solution, rinse after 10 minutes with wa~water) are recommended. for long-t~rm medical m~nagement of encephalopathic signs, lactulose is given orally at a dosage of 0.~5 to 1.0 ml per 4.5 kg body weight, the dose adjusted to the frequency and consistency of the stools passed each day. two to three soft or pudding-consistency stools indicate an optimal dose. too great a dose may result in flatulence, severe diarrhea, dehydration, and acidemia. to f\1rther manipulate the intestinal flora, neomyem (22 mglkg orally two to three times daily) etronidazole (7.5 mg/kg orally two to threå¨ rnes daily), aijolpicillin (5 mglkg orally two to three urnes. dally), or amoxicillin (2.5 mg/kg orally two ttmes a day) may be used intermittently for several weeks. congenital hepatic arteriovenous fistulas. congenital hepatic arteriovenous fistulas etween the~epatic artery and portal vein occur in both puppies and kittens (easley and carpenter, 1975; legendre et al, 1976â· moore and rogers et al, 1977) . these congenital malformations are the result of failure of e com~on embryologic anlage to differentiate l1~to artenes and veins. congenital hepatic artenovenous fistulas are associated with portal hyertension and shunting of blood through multtple portosystemic venous collaterals (table 11 -3): in~reased pressure in the portal vein, hepa~c. vel?, and hepatic sinusoids is caused by arterialization of the portal circulation. arteriovenous fistulas located in other areas of the body increase cardiac output and produce signs of heart failure (gomes and bernatz 1970â· moore and whiting, 1986) . the interpositioãµ f the hepatic sinusoids between the heart and fistula. cushions the hemodynamic effects, influencing heart function in animals with congenital hepatic arteriovenous fistulas. animals with congenital hepatic arteriovenous fistulas have multiple portosystemic shunts and ascites. portal venography by splenic pulp injection or mesenteric vein catheterization demonstrates multiple anomalous shunts but does not show e fistulas. diagnosis is made by nonselective jugular venography , selective celiac angiography, technetium scintigraphy, ultrasonography, or observation of an abnormal liver lobe during laparotomy. affected liver lobes are large and may have numerous pulsating surface vessels. unaffected liver lobes are small. a continuous murmur accentuated during systole may be auscultated near the lesion. palpation of the area may reveal a thrill. ultrasonographically, congenital hepatic arteriovenous fistulas are identified on the basis of finding multiple large, tortuous, and pulsatile hepatic vessels and en-lar~ement of the celiac and common hepatic artenes, congenital hepatoportal microvascular~p lasia. hepatoportal microvascular dyspla-sl~is char~ct~nzed by the presence of multiple tnlcroscopic intrahepatic shunts (phillips et al, 1993; schennhorn et ai, 1996) . the microvascular dysplasia occurs in the same dog breeds that have congenital pss, possibly being an inherited disorder in cairn terriers. most dogs with microvascular dysplasia are asymptomatic, probably because only a small amount of blood is being shunted away from the liver. when signs are present, they are similar to those seen in dogs with congenital extrahepatic and intrahepatic pss, with the exception that most dogs with microvascular dysplasia usually present at an older age. the most prominent laboratory abnormality is increased serum bile acid concentrations. there is no ultrasonographic, sur-gical, or portographic evidence of a congenital pss, and the rectal portal scintigraphy is normal. medical treatment is the same as for any suspected congenital pss. asymptomatic dogs with increased serum bile acids as their only detectable abnormality do not require treatment. congenital storage disorders. congenital storage disorders affecting the function or availability of lysosomal enzymes or effector proteins essential for catabolism of glycoproteins, glycolipids, glycosaminoglycans (mucopolysaccharides), gangliosides, and glycogen have been identified in puppies and kittens. these disorders are characterized by tissue accumulation of undegraded storage products. signs are usually progressive in association with tissue accumulation of storage material. hepatomegaly may develop from the undegraded storage product accumulating in hepatocytes and kupffer cells. mannosidosis resulting from a deficiency in acid mannosidase activity that causes the intralysosomal accumulation of a mannoside oligosaccharide occurs in kittens (jezyk et al, 1986; vendevelde et ai, 1982) . clinical findings include hepatomegaly, neurologic dysfunction (including tremors, ataxia, hypermetria, and/or weakness), stunted growth, facial dysmorphia, and early death. histopathologic examination reveals extensive cytoplasmic vacuolation in hepatocytes and neurons and the presence of unusual axonal spheroids. the mucopolysaccharide storage disorders are caused by a defect in lysosomal enzymes responsible for the degradation of dermatan sulfate, heparan sulfate, or keratan sulfate-normal constituents of the connective tissue matrix. these disorders are clinically progressive and are associated with tissue accumulation of glycosaminoglycans. clinical features vary with the specific enzyme deficiency. hepatosplenomegaly may develop from the accumulation of incompletely degraded mucopolysaccharides in parenchymal and reticuloendothelial cells. clinical findings may include facial dysmorphia (rounded broad forehead, small ears, and dished face), corneal opacity, bone and joint lesions (including odontoid hypoplasia, intervertebral disk degeneration, spinal canal and vertebral exostoses, osteoporosis, coxofemoral luxation, lytic areas in long bones and vertebrae, joint effusions, and degenerative joint disease), cardiac murmurs, stunted growth, metachromatic granules in leukocytes, neurologic abnormalities (mental slowness, cervical or thoracolumbar myelopathy), and early death. mucopolysaccharidosis i (a-l-iuronidase deficiency) has been described in a kindred of plott hounds and in domestic short-haired cats (haskins et ai, 1983; shull et ai, 1984) . mucopolysaccharidosis vi (arylsulfatase b deficiency) has been described in siamese and domestic short-haired cats (breton et al, 1983; haskins et ai, 1980 haskins et ai, , 1981 . mucopolysaccharidosis vii has been described in a dog (haskins et al, 1984) . a presumptive diagnosis of mucopolysaccharidosis can be made by a positive urine toluidine blue spot test. definitive diagnosis is made by measurement of the activity of specific enzymes in fresh serum, cultured dermal fibroblasts, or leukocytes. treatment with bone marrow transplantation has been reported to result in clinical improvement (dial et al, 1985; . gangliosidosis occurs from incomplete catabolism of certain gangliosides and glycolipids and retention of these substrates within lysosomes. gangliosidosis has been reported in the puppy and kitten (alroy et al, 1985; baker and lindsey, 1974; barnes et al, 1981; cork et al, 1977 cork et al, , 1978 n euwelt et al, 1985; read et ai, 1976; wenger et al, 1980) . affected animals develop neurologic signs as early as 2 or 3 months of age. progressive, fine generalized muscle tremors, ataxia, and paresis are the usual clinical findings. gangliosides accumulate in the central nervous system and in visceral organs, including the liver. membrane-bound cytoplasmic bodies are observed in cells from affected individuals. glycogen storage disorder associated with hepatomegaly has been diagnosed in a kindred of lapland dogs and in a german shepherd dog (rafizuzzaman et ai, 1976; walvoort et al, 1982 walvoort et al, , 1984 . affected animals showed signs as early as 2 months of age that were slowly progressive over many months. signs included weakness, weight loss, and gradual abdominal distention associated with profound hepatomegaly. glycogen is freely dispersed in the hepatocellular cytoplasm. a deficiency of amylo-l,6-g1ucosidase was demonstrated in a german shepherd dog (ceh et ai, 1976) , and a deficiency of a-glucosidase was demonstrated in a lapland dog (walvoort et al, 1982) . copper storage disorders of the bedlington terrier. a chronic active liver disease associated with an age-related accumulation of hepatic copper occurs in bedlington terrier dogs (hultgren et al, 1986 ). an autosomal recessive the liver .nd p.ncre.s i 2ii mode of inheritance is involved; only individuals homozygous for the recessive gene develop the excess copper accumulation in hepatic lysosomes. the adverse effects of retained copper are not noted during the first few years of life in affected bedlington terrier dogs by the protective lysosomal sequestration of copper. once lysosomal storage is overwhelmed, a progressive hepatopathy and clinical evidence of chronic active liver disease ensue. in affected dogs, copper accumulation begins before 1 year of age and continues for at least 5 or 6 years. hepatic copper concentrations exceeding 2000 j.lg/g dry tissue are consistently associated with morphologic and functional evidence of the progressive hepatopathy that over time progresses to chronic active hepatitis and cirrhosis (fig. 11 -6a) (hultgren et al, 1986; twedt et al, 1979) . affected dogs can be identified at 6 months of age on the basis of hepatic biopsy results (lohnson et ai, 1984) . liver tissue can be qualitatively and quantitatively evaluated for copper accumulation. routine staining with hematoxylin and eosin reveals dark cytoplasmic granules in hepatocytes of affected dogs early in the disease. tissue-bound copper can be stained with rubeanic acid, rhodanine, or timm's stain for qualitative and semiquantitative estimation of the degree of copper retention (fig. 11-6b) (johnson et al, 1984; . tissues should be stored embedded in paraffin blocks rather than formalin solution if examination is delayed for several months, because copper staining is reduced after prolonged storage in formalin solution . quantitative assessment of hepatic copper is accomplished by atomic absorption spectroscopy of tissue previously preserved in formalin or paraffin block or frozen. normal dog liver has less than 400 j.lg copper per gram of dry tissue twedt et al, 1979) .affected bedlington dogs may develop hepatic copper content up to 2000 j.lg/g during the first year of life before developing histopathologic evidence of hepatocellular injury. dogs showing evidence of abnormal copper storage in hepatic biopsy material by 1 year of age should not be used for breeding. affected bedlington dogs may have evidence of increased hepatic copper as early as 8 to 12 weeks of age. diagnosis of copper-associated hepatopathy in bedlington terriers can be made by examination of hepatic tissue for excessive copper storage or by performing genetic tests on dna samples collected from suspected dogs. an autosomal recessive mode of inheritance is invalved in copper-associated hepatopathy in bedlington terriers. the frequency of the recessive gene in bedlington terriers is estimated to be as high as 50% in the united states, with a similar frequency in england. this means that more than 25% of bedlington terriers are "affected," and another 50% are "carriers." the dna samples can be collected with a soft cheek brush that is provided by a commercial genetic laboratory (vetgen, 3728 plaza drive, suite 1, ann arbor, mi 48108; 1-734-669-8440, toll free: 1-800-4-vetgen, fax: 1-734-669-8441; or see their web site: www.vetgen.com). by gently brushing the inside of the dog's cheek, cells containing dna are removed. the collected dna samples then are analyzed to determine the genetic status of the suspect dog. useful for dogs of any age, the dna sample collection and analysis activities can be completed before puppies are purchased at 6 to 10 weeks. the results of the dna testing also can be formally registered with the orthopedic foundation for animals. (for further information about the orthopedic foundation for animal's registry for copper toxicosis in bedlington terriers, contact orthopedic foundation for animals, 2300 e. nifong boulevard, columbia, mo 65201-3856 or telephone 1-573-442-0418.) n-penicillamine, a copper chelator, is recommended for the treatment of the copper-associated hepatopathy in bedlington terriers as soon as the disorder is confirmed. the recommended dose of n-penicillamine is 125 to 250 mg/day (adult dogs), given 30 minutes before feeding (hardy, 1983) . the most common adverse effects from o-penicillamine administration are vomiting and anorexia. vomiting may be manageable by dividing the daily dose into two or three doses. if o-penicillamine is not tolerated, another copper chelator, 2,3,2-trientine, administered at 10 to 15 mg/kg orally one to two times daily or zinc acetate administered at 50 to 200 mg orally once a day, decreases intestinal absorption of copper and may be used. in addition to the decopper drugs, vitamin c and prednisolone have been recommended. vitamin c is known to facilitate the excretion of copper in urine, and large doses may reduce the intestinal absorption of copper. dosages of 500 to 1000 mg/day have been suggested (hardy, 1983) . prednisolone at 0.5 to 1.0 mg/kg per day is recommended only for those dogs showing evidence of active hepatic necrosis. limitation of the dietary intake of copper is usually not possible. most dog foods contain 5 to 10 mg/ kg of copper, which may result in a higher copper intake per kilogram than is appropriate. hepatopathies associated with increased concentrations of hepatic copper have been recognized in young dogs and cats with chronic active hepatitis, cirrhosis, and chronic bile duct obstruction (rolfe and twedt, 1995) . copper may aggravate the underlying pathologic process in these disorders by direct injury to cellular organelles or by promotion of fibrogenesis (hultgren et al, 1986) . the decreased ability to excrete biliary copper probably underlies abnormal hepatic copper retention when a primary cholestatic disease exists. primary hepatobiliary disease associated with an increased accumulation of hepatic copper, albeit smaller amounts of tissue copper than in bedlington terriers, has been described in doberman pinscher, skye terrier, west highland white terrier, and american and english cocker spaniel dogs (crawford et al, 1985; thornburg and rottinghaus, 1985; thornburg et al, 1986) . the chronic active hepatitis associated with an increased liver copper content in doberman pinschers occurs primarily in middle-aged females. although the youngest dog reported with this disorder was 1.5 years old, it is unknown whether younger dogs are symptomatic. it is suspected that affected dogs could be identified at a younger age on the basis of routine screening serum chemistry profiles revealing increased liver enzyme activity. a familial copper-associated liver disease occurs in west highland white terrier dogs (thornburg et ai, 1986) . increased hepatic copper concentrations are detected in asymptomatic dogs as young as 7 months of age. in three affected dogs younger than 9 months of age, the hepatic copper concentration ranged between 1500 and 1750 f.lg/g dry weight. hepatic copper concentrations in affected dogs have ranged as high as 3500 ppm, considerably lower than the maximal values recorded for bedlington terriers. in an attempt to decrease the perpetuation of this disorder, it has been recommended that relatives of west highland white terrier dogs dying of liver disease be evaluated by hepatic biopsy before 1 year of age. those animals with increased hepatic copper content should not be used for breeding purposes. liver disease has also been observed with unexpected frequency in american and english cocker spaniel dogs (thornburg and rottinghaus, 1985) . dogs as young as 9 months have been diagnosed as having chronic active hepatitis. the liver disease appears to be progressive, and dogs dying of cirrhosis have had hepatic copper concentrations three to five times normal. a genetic defect in the dalmatian dog's liver results in an inability to convert uric acid into allantoin, the soluble excretory product of purine metabolism in non-dalmatian dogs (briggs, 1985; briggs and harley, 1986; giesecke and tiemeyer, 1984) . this genetic defect is transmitted by homozygosity for a recessive trait. serum uric acid concentrations in dalmatian dogs are consistently increased, and urinary excretion of uric acid is markedly greater than in non-dalmatian dogs. typical serum uric acid concentrations in dalmatian dogs range between 2 and 4 mg/dl versus less than 1 mg/dl the liver .nd p.ncre.s i 213 in other breeds of dogs (kruger and osborne, 1986; schaible, 1986) . urine excretion of uric acid in dalmatians ranges between 400 and 600 mg in 24 hours versus 10 to 60 mg in 24 hours in non-dalmatians (kruger and osborne, 1986) . urine uric acid-to-creatinine values have ranged between oj and 0.6 for normal puppies and 1.3 and 4.6 for pedigree dalmatian puppies at 3 to 7 weeks of age and between 0.2 and 0.4 for normal dogs and 0.6 and 1.5 for purebred adult dalmatians (schaible, 1986) . the increased urinary excretion of uric acid puts the dalmatian at increased risk for the formation of urate uroliths, although not all affected dogs develop uroliths. hepatic lipidosis. most puppies and kittens that present with hepatic lipidosis have primary disease in other organ systems or an infectious disease, and therefore it is possible that the hepatic lipidosis was the consequence of acquired nutritional inadequacies. a variety of metabolic disorders can disturb the mobilization of triglycerides from the liver. whenever intrahepatic lipid synthesis or the hepatocellular uptake of fat exceeds the dispersal of triglycerides from the liver, hepatic lipidosis develops (fig. 11-7) (miettinen, 1981; pulito et ai, 1976) . severe hepatic lipidosis occurs most commonly in toy-breed puppies, which become hypoglycemic and die after prolonged anorexia or fasting (van toor et ai, 1991) . clinically, kittens appear to be more susceptible to hepatic triglyceride accumulation than puppies. any serious medical problem in the kitten can be associated with excessive hepatic lipid accumulation characterized by cytoplasmic vacuole formation that adversely influences hepatic function. nutritional management that ensures adequate intake of calories, essential amino acids, and essential fatty acids is the best recommended symptomatic therapy. in addition, nutritional management for the mother during pregnancy can be important in possibly preventing hepatic lipidosis in the newborn. neonatal icterus. neonatal icterus often occurs in puppies and kittens as a result of immunohemolytic anemia (cain and suzuki, 1985; giger et al, 1991; young et al, 1951) . icterus often occurs in kittens within 3 days of birth with hemolysis from neonatal isoerythrolysis (see chapter 3). noncirrhotic portal hypertension in young dogs. most young dogs with noncir-rhotic portal hypertension are younger than 19 months old, pedigree dogs, and female (see table 11 -3) (bunch, 1997; demarco et ai, 1998; rand et al, 1988; rutgers et al, 1993; van den ingh and rothuizen, 1994; van den ingh et al, 1995) . typical signs are apathy, ascites, vague gastrointestinal upset (anorexia, vomiting, diarrhea), neurologic derangements, and polydipsia! polyuria. the affected dogs typically have smallsized livers, acquired pss, and splenomegaly. common trends in serum chemistry profiles are increased liver enzyme activities and evidence of hepatic dysfunction (e.g., hypoalbuminemia, increased serum bile acid content, and hyperammonemia). microcytosis is a consistent finding. liver biopsy is required for an accurate diagnosis in the affected dog. histopathologic findings include preserved to altered liver architecture, portal hypoperfusion, and variable degrees of fibrosis; there are usually no cytopathic indications of destructive processes such as necrosis or inflammation. responses to symptomatic and specific hepatic treatment of affected dogs vary with the degree of portal hypertension present and the length of time hypertension has existed. symptomatic measures to decrease ascites and signs of hepatic encephalopathy are indicated. colchicine (0.025 mglkg orally once daily) and/or prednisone (0.5 to 1.0 mglkg orally daily initially, then every other day) have been the medications reported to be useful in a small number of cases (rutgers et ai, 1990) . it seems that affected dogs have the potential to have a good quality of life for an indefinite period of time. feline inflammatory liver disease. inflammatory liver diseases of young cats is probably best referred to as feline cholangitis/cholangiohepatitis syndrome (cchs) (center, 1997) . this syndrome can then be described as being either a suppurative cchs or a nonsuppurative cchs. affected cats with suppurative cchs usually are 3 months and older and usually are males. a sudden-onset history of vomiting and diarrhea is common. older cats are icteric, febrile, lethargic, and dehydrated on initial presentation. less than 50% of cats have hepatomegaly. the most common organisms associated with suppurative cchs are escherichia coli, staphylococcus, a-hemolytic streptococcus, bacillus, actinomyces, bacteroides, enterococcus, enterobacter, and clostridium species. most cats with suppurative cchs show a moderate increase in serum alt, as'f, alp, and ggt activities. some cats have left-shifted leukograms with an accompanying leukocytosis. on ultrasonography, severe ascending cholangitis associated with thickening of the extrahepatic biliary system and inflammation within the lumen of the intrahepatic bile ducts may be observed. ultrasonography also may show coexisting extrahepatic bile duct obstruction (enlarged gallbladder, distended and tortuous common bile duct, and obvious intrahepatic bile ducts), cholecystitis (thickened, laminar appearance of the gallbladder wall, adjacent fluid accumulation), and pancreatitis (prominent, easily visualized enlarged pancreas with adjacent hyperechoic fat). cytologic evaluation of liver aspirates or imprints may reveal suppurative inflammation. most cats with nonsuppurative cchs are 1 year of age or older and have been ill for several months (center, 1997) . clinical signs are subtle and may include only episodic vomiting, diarrhea, and anorexia. most cats have hepatomegaly, are icteric, and may have ascites. concurrent disorders frequently include inflammatory bowel disease, low-grade lymphocytic pancreatitis, and cholecystitis. cats with lymphoplasmacytic inflammation tend to have greater magnitudes of increased serum alt, ast, alp, and ggt activities than cats with just lymphocytic inflammation. cats with lymphocytic inflammation may develop a lymphocytosis (total lymphocyte counts greater than 14,000/f..d) without other evidence of malignant lymphoproliferative disease. similar to cats with suppurative cchs, abdominal radiographs rarely show important diagnostic information. in most cats with nonsuppurative cchs, a multifocal hyperechoic pattern is recognized ultrasonographically, which represents peribiliary inflammation and fibrosis. in some cats, ultrasonography may fail to show any abnormalities. cytologic preparations from liver aspirates may lack evidence of inflammation or may disclose only a few inflammatory cells. a wedge biopsy of the liver for histopathology is preferable for a definitive diagnosis because it more reliably demonstrates whole acinar units and portal triads (center, 1997) . treatment of suppurative cchs incorporates appropriate antimicrobial therapy based on identification of infectious organisms. if bacteria are cytologically observed, a gram's stain facilitates selection of antimicrobial agents. cats with extrahepatic bile duct obstruction should have their biliary occlusion decompressed, if possible. if biliary tract decompression cannot be accomplished, the biliary pathway may be rerouted by a cholecystoenterostomy. biliary diversion is a vital early therapeutic intervention in the prevention or control of sepsis in obstructive suppurative cholangitis. aerobic and anaerobic bacterial cultures should be collected from bile, tissue adjacent to any focal lesion, gallbladder wall, and liver tissue. any icteric cat suspected of having suppurative or nonsuppurative cchs should be evaluated for coexistent extrahepatic bile duct obstruction, pancreatitis, and inflammatory bowel disease as well as coexistent hepatic lipidosis. if lipid vacuolation is detected, nutritional support with a commercially prepared feline diet should be included in the treatment plan. immunosuppressive therapy for cats with nonsuppurative cchs includes a combination of prednisolone (initial dose of 2 to 4 mglkg orally once a day or divided twice daily), with titration to the lowest effective dose over the the liver ind plncfels i 215 next several months, and metronidazole (7.5 mglkg orally two to three times daily) (center, 1997) . supplementation with l-carnitine 250 mg/cat per day, water-soluble vitamins (two times the normal maintenance dose), and vitamin k 1 (0.5 to 1.5 mglkg) subcutaneously or intramuscularly for three doses at 12-hour intervals and then once a week for 1 or 2 additional weeks may be provided. oral vitamin e can also be added as a supplement to ensure its adequacy as a free radical scavenger; a dose of 100 to 200 iu per day is used. ursodeoxycholic acid (10 to 15 mglkg orally per day) is given to all cats with cchs once extrahepatic bile duct obstruction is corrected. monthly serum liver enzyme activities and total bilirubin concentrations may be used to monitor treatment response as well as how well the cat is doing at home. hepatic abscessation. hematogenous, omphalogenic, biliary, and peritoneal extension are sources of infecting organisms that cause hepatic abscesses to appear in puppies and kittens (hargis and thomassen, 1980; valentine and porter, 1983) . postpartum umbilical infection appears to be the most common cause of hepatic abscessation. once clinical signs develop, animals deteriorate and die within 2 to 4 weeks. occasionally, seemingly healthy puppies die unexpectedly, the cause being discovered on histopathologic examination. most affected puppies are between 3 and 70 days of age and are from large litters (hargis and thomassen, 1980) . organisms frequently isolated from hepatic abscesses in puppies and kittens include escherichia coli and staphylococcus, streptococcus, and salmonella species. puppies and kittens with hepatic abscesses are usually stunted, emaciated, and dehydrated and may have enlarged abdomens due to hepatomegaly and peritonitis. unaffected liver lobes usually show multifocal necrosis on histopathologic examination. suspected hepatic abscesses in puppies and kittens should be managed with antimicrobial drugs and other supportive care (see chapter 5). hepatic parasitism. hepatic trematode infection may be diagnosed in kittens as young as 4 months of age. the most common liver fluke in cats in north america is platynosomum amcinnum. other species of flukes that may infect cats include amphimerus pseudofelineus, opisthorchis tenuicollis, metorchis albidus, and metorchis conjuctus. cats acquire platynosomum con-cinnum infection by the ingestion of the second intermediate hosts: a land snail (subulina octona) and a lizard or marine toad. once ingested, the infective stage of the parasite migrates up the common bile duct into the gallbladder and bile ducts, where in 8 to 12 weeks it matures into the adult fluke. embryonated eggs are passed in the feces and are the basis for diagnosis. clinical signs are noted by 7 to 16 weeks after infection and may include inappetence, lethargy, weight loss, hepatomegaly, emaciation, mucoid diarrhea, depression, vomiting, and abdominal tenderness. many naturally infected cats show no clinical signs. in heavy infections, clinical signs may develop before the fecal shedding of ova, which occurs as early as 8 weeks after infection. concentration of eggs in feces by sedimentation is the most reliable diagnostic test. transient increases in the serum ast and alt activities develop during fluke migration through the liver. the serum alp activity may remain normal or may increase. cats with heavy fluke infection may become jaundiced. persistent fluke infections and bile duct obstruction may result in biliary cirrhosis. treatment with praziquantel (20 to 40 mglkg daily for at least 3 days) is clinically effective. hepatobiliary lesions produced by ascarid larval migration are commonly observed during necropsy of young dogs and cats. these lesions are usually not associated with clinical signs or laboratory abnormalities. severe hepatic and peritoneal migration, gallbladder rupture, and bile peritonitis may, however, occur in a few puppies. in young dogs and cats, after ingesting eggs, the larval forms of 'toxocara canis and 'toxocara cati penetrate the wall of the alimentary canal and pass by way of lymphatics or the portal circulation to the liver. ascarids may also migrate from the gastrointestinal tract directly through the peritoneal cavity to the liver. diseases. canine herpesvirus infection is an acute, rapidly fatal disease that is associated with hepatic necrosis. puppies acquire canine herpesvirus in utero, during passage through the birth canal, by exposure to infected littermates, or from orona sal secretions of the dam. abortions and stillbirths may occur if infection is acquired in utero (poste and king, 1971) . generalized, fatal infections develop in puppies during the first 3 weeks of life. puppies infected when older than 3 weeks are comparatively resistant and develop mild or inapparent infection. an incubation period of 4 to 6 days follows initial exposure. a diffuse necrotizing vasculitis and spread of virus into parenchymal organs, including the adrenals, kidneys, lungs, spleen, and liver, results in multifocal organ necrosis. meningoencephalitis that causes seizure activity is common in canine herpesvirus infections. in survivors, permanent neurologic deficits may persist, most common of which are cerebellar vestibular defects. ocular involvement causing panuveitis, cataracts, keratitis, retinitis, and subsequent blindness may occur. clinical signs of canine herpesvirus infection in puppies may include depression, diminished suckling response, persistent crying, yellowgreen diarrhea, abdominal pain, and incoordination. petechial hemorrhages may be notable on mucous membranes. cutaneous lesions may include an erythematous rash with red papules progressing to vesicles. papular or vesicular lesions may develop in the vulvovaginal orifice, prepuce, and/or oral cavity. neurologic signs may occur during the terminal stages of the disease. death frequently occurs within 24 to 48 hours after onset of clinical signs in infected puppies. definitive diagnosis of canine herpesvirus infection in puppies is made on the basis of history, clinical signs, histopathologic changes, and virus isolation. hematologic and biochemical abnormalities are nonspecific and variable. thrombocytopenia may be present in ill puppies. widespread hepatic necrosis causes increased serum activities of alt and ast. icterus does not occur. gross pathologic findings include disseminated multi focal petechial hemorrhages ( fig. 11-8 ) and areas of necrosis that are distinctly circumscribed in the liver, kidney, and lungs (greene and kakuk, 1984) . hepatomegaly, splenomegaly, and lymphadenopathy are common. histopathologic lesions are characterized by perivascular necrosis associated with a mild neutrophil and lymphocyte infiltration, hemorrhages, and occasional intranuclear inclusions (fig. 11-9 ). treatment for canine herpesvirus infection is usually unrewarding owing to its rapidly fatal progression. rectal temperature elevation to about 37.7 0 c (100 0 f) and adequate nutritional support may improve puppy survival during an outbreak. focal hepatitis and hepatic cord disorganization may develop in puppies and kittens infected with canine or feline parvovirus. two-to fivefold increases in serum activities of alt and ast may develop. in some cases, hepatic involvement is progressive, resulting in icterus. seemingly, a poor prognosis is warranted when hepatic involvement becomes clinically apparent. coronavirus infection causing feline infectious peritonitis (fip) most often affects cats between 6 months and 2 years of age. coronavirus infection has been diagnosed as a cause of stillborn kittens and fading kittens and as an effusive disease in kittens younger than 4 weeks of age. clinical signs of fip usually develop in several siblings in a litter, and death losses may span a 6-to 12-month interval. cats with liver involvement may demonstrate cranial abdominal pain and hepatomegaly. serum alt and ast activities are usually increased from 2-to 10-fold in cats with liver involvement. icterus may develop in those cats with severe, diffuse hepatic lesions. a coagulopathy and thrombocytopenia develop in cats with diffuse vascular the liver and pancreas i 217 injury, in those with severe inflammation and subsequent activation of clotting factors, or in severe hepatic involvement (weiss et ai, 1980) . immunosuppression may help prolong the survival of some cats. unfortunately, kittens showing signs of hepatic involvement are usually poor candidates for immunosuppressive therapy. infection of kittens with feline leukemia virus or feline immunodeficiency virus may occur by horizontal or vertical transmission. by virtue of their oncogenic potential and ability to immunologically compromise the host, these viruses may be associated with neoplastic conditions and infectious diseases involving the liver. lymphosarcoma and myeloproliferative disease can develop in infected cats within weeks or months of exposure. affected kittens demonstrate hepatomegaly when they have liver involvement. icterus develops with diffuse hepatic involvement, periportal infiltration, or major bile duct occlusion. serum chemistry profile abnormalities are variable, depending on the extent of hepatic involvement. bacterial-induced hepatic diseases. enteric organisms such as salmonella species and escherichia coli can be a source of hepatic parenchymal and biliary tract infections in young dogs and cats (greene, 1984) . salmonella species may exist in young dogs and cats as a part of the normal enteric flora. transmission of salmonella species from carrier animals to susceptible hosts may result in gastroenteritis, bacteremia, parenchymal organ or lymph node colonization or abscessation, endotoxemia, stillbirths, or a fading puppy or kitten syndrome. signs of gastrointestinal infection may develop after 3 to 5 days of exposure or after some unusual environmental or physical stress. initial signs may include fever (104 0 to 106 0 f), malaise, anorexia, vomiting, abdominal pain, and diarrhea. diarrhea can be voluminous and usually contains mucus and fresh blood. further signs may develop, including weight loss, severe dehydration, weakness, hypotension, pale mucous membranes, and, in some cases, evidence of neurologic involvement. icterus may develop as a result of endotoxemic effects on the liver, hepatic infarction, or bacterial colonization of hepatic tissue. serum chemistry profile evidence of liver involvement includes increases in the serum activities of al'f, as'f, and alp. hyperbilirubinemia is an inconsistent finding. multifocal necrosis is the most common histopathologic lesion of salmonellosis. a necrotizing pneumonia may also occur in puppies with hepatic involvement. definitive diagnosis of salmonellosis relies on culture of the organism from involved tissues or body fluids that are normally free of this organism. positive culture of fecal specimens does not confirm the causal relationship of the organism to the animals' clinical disease. successful treatment requires attention to supportive nursing care, plasma transfusion for severe hypoproteinemia, and selection of an appropriate antimicrobial agent. the prognosis for puppies and kittens with salmonellosis is generally poor. efforts to improve kennel or cattery sanitation, to improve nutrition, and to reduce stress on puppies and kittens may curtail further infection. bacillus piliformis, the causative agent of tyzzer's disease, is a gram-negative, spore-forming, obligate intracellular bacterium that can cause enteric and hepatic infections in young dogs and cats, most commonly seen at the time of weaning. animals subject to infection develop necrotizing enteritis and multifocal hepatitis. infection in most young dogs and cats is thought to occur by their ingestion of bacterial spores passed in rodent feces. signs of natural disease in young dogs and cats include a sudden onset of lethargy, depression, anorexia, diarrhea, and abdominal tenderness. icterus may occur in some affected kittens. within 24 to 48 hours after the onset of illness, affected animals become hypothermic and severely depressed. death rapidly follows. identification of the bacterium is aided by the use of special stains such as giemsa's stain or gomori's methenamine silver stain (greene, 1984) . isolation of the causative agent cannot be accomplished on routinely used bacterial culture media. toxoplasma gondii infections vary depending on the chronicity of infection, immune status of the host, mode of infection, and target organs affected. young dogs and cats are particularly at risk when immunocompromised and debilitated. in utero infection can occur and lead to stillbirths or neonatal disease and death. mfected puppies and kittens may appear normal at birth but become depressed, inappetent, and dyspneic and develop a mucopurulent oculonasal discharge and progressive neurologic disease; they eventually die. dissemination to multiple organs usually occurs. hepatic lesions are typified as a multifocal necrotizing hepatitis. hepatic inflammation may be associated with cranial abdominal pain and peritoneal effusion and is usually associated with vomiting, diarrhea, and inappetence. animals may become icteric o.wing to the diffuse nature of the hepatic necro-sis. laboratory abnormalities associated with toxoplasmosis are variable, depending on the target organs affected and the chronicity of infection. early hematologic features may include a panleukopenia with a degenerative left shift. a leukocytosis may follow during the recovery period (greene and prestwood, 1984) . serum chemistry profile abnormalities indicating hepatic involvement include marked increases in serum al'f, ast, and alp activities and hyperbilirubinemia. definitive diagnosis of toxoplasmosis is made on the basis of tissue examination for toxoplasma gondii organisms or demonstration of a rising serologic antigen and/or antibody titer. recommended treatments for toxoplasmosis include the use of pyrimethamine, trimethoprim-sulfonamide, and clindamycin (greene and prestwood, 1984) . the pancreas is a unique organ possessing both exocrine (digestive) and endocrine (hormonal) functions. inflammatory pancreatic disease affecting only the exocrine portion is extremely uncommon in young dogs and cats (strombeck and guilford, 1990) . consequently, inflammatory pancreatic disease, that is, acute pancreatitis or relapsing pancreatitis that more commonly affects older dogs and cats, has been rarely identified in dogs and cats younger than 6 months of age. the likely causes of inflammatory pancreatic disease in the young dog and cat are abdominal trauma and infectious agents. abdominal trauma may induce pancreatitis in dogs that are traumatized by motor vehicles and in cats that have fallen or jumped from high places (high-rise syndrome) (drazner, 1986 ). in addition, abdominal surgery may result in acute pancreatitis due to traumatic injury to the pancreas (spearing the pancreas with a surgical instrument) or excessive manipulation of the pancreas. infectious agents can occasionally contribute to inflammatory pancreatic disease. pancreatic necrosis can be found on postmortem examination of an occasional dog afflicted with canine parvovirus infection (drazner, 1986) . it is not known whether the canine parvovirus is directly cytotoxic to the pancreatic tissue or pancreatitis occurs secondary to the invasion of enzymes and bacteria of the intestinal tract into the pancreas. in cats, pancreatitis may be associated with the effusive form of fip (barlough and weiss, 1983) . other infectious agents directly associated with inflammatory pancreatic disease in the young dog and cat would be extremely unusual and most likely a one-time occurrence. although seldom required, laboratory confirmation of inflammatory pancreatic disease includes a complete blood count, serum chemistry profile, serum amylase and lipase determinations, serum trypsin-like immunoreactivity (tli) assay, and survey radiographs and/or ultrasonography of the abdomen. normal values for serum amylase and lipase activities in dogs and cats younger than 6 months of age are generally indicative of normal adult values. hyperamylasemia and hyperlipasemia combined with typical clinical features of inflammatory pancreatic disease, as seen in adult animals, establish the diagnosis of inflammatory pancrefigure 11 -10. photograph of the pancreas from a young dog with congenital pancreatic hypoplasia. note the generalized reduction in amount of pancreatic tissue present and the absence of any inflammatory pancreatic disease. the liver end pencreas i 219 atic disease until proved otherwise. the serum tli assay may be increased-tli values of more than 35 ij.gll in young dogs and more than 50 ij.gll in young cats are consistent with pancreatitis. its treatment is entirely supportive and is managed in a manner similar to that for the afflicted older dog or cat. by far the most common cause of noninflammatory pancreatic disease in the young dog is congenital pancreatic hypoplasia (harris, 1985; jubb, 1983; sherding, 1979) . this disorder of young dogs is characterized by generalized reduction in pancreatic exocrine (acinar) cells, but the islets of langerhans remain intact (fig. il-10) . the disorder is more common in large breeds of dogs, that is, german shepherd (alsatian), doberman pinscher, irish setter, labrador retriever, and saint bernard, but has also been seen in the beagle (hill et ai, 1971; prentice et al, 1980) . there may be a sex predilection favoring females (anderson and low, 1965) , and young dogs that are symptomatic generally present before 1 year of age. congenital pancreatic hypoplasia has not been recognized in the young cat. most dogs affected with congenital pancreatic hypoplasia present with signs of weight loss or failure to gain adequate weight and poor physical appearance (i.e., dull, dry haircoat and excessive shedding) despite exhibiting a good to voracious appetite (sherding, 1979) . varying degrees of frequent (6 to 10 stools per day), foul-smelling, bulky, greasy, loose stools are de-scribed by the dog owner. often, coprophagia is noted. affected dogs commonly eat their stools because of their high fat content and because of a dietary energy deficit. the diarrheic stools contain undigested sugars and fats that are being altered by intestinal bacteria to become osmotically active particles (drazner, 1983) . the marked increase in osmotically active particles and the subsequent efflux of water into the lumen of the intestinal tract result in the colon's inability to resorb the increased volume, and diarrhea ensues. the volume of unabsorbed intraluminal water produces marked intestinal distention and altered motility, which may be severe enough to cause intestinal and colonic bacterial overgrowth (drazner, 1983) . unabsorbed fatty acids may also impair the absorptive capacity of the small intestine by damaging the brush border, blunting the villi, and inhibiting colonic water absorption. the diagnostic evaluation of dogs suspected to have congenital pancreatic hypoplasia differentiates this disorder from intestinal mucosal malabsorption. diagnosis of congenital pancreatic hypoplasia is usually not difficult because the presenting signs are rather characteristic and the laboratory test results are helpful in its diagnosis. the serum tli assay values are classically decreased-tli values are consistently less than 2.5 f.lgll in affected dogs. "when the diagnosis is still in question or serum tli assay results are not available, an exploratory laparotomy can be used for confirmation. treatment of dogs with congenital pancreatic hypoplasia depends mainly on dietary management and supplementation with pancreatic digestive enzymes (lewis et al, 1987) . efforts to treat these dogs are usually rewarded with a favorable response. the expense of treatment and an unconscientious owner, rather than the ineffectiveness of the treatment regimen itself, are most often the reasons that successful treatment is not accomplished. the most effective dietary management for dogs with congenital pancreatic hypoplasia is a highly digestible, lowfiber, moderate-fat diet supplemented with pancreatic enzymes (lewis et ai, 1987) . commercial diets formulated for gastrointestinal disease may be fed. the dog's daily food intake is divided into two or three feedings or is fed free choice. the dietary replacement of pancreatic digestive enzymes is given orally with each meal. various pancreatic enzyme products are commercially available for this purpose (sherding, 1979) . reliable commercial products are available in both powder and tablet form. the usual effective dosage of the powder preparation is 1 to 2 tsp per meal for each 20 kg body weight. the pancreatic enzyme product is mixed with the commercially prepared canned or well-moistened dry dog food and fed without necessarily any preincubation time. "when diarrhea is in remission and the animal is gaining weight, the pancreatic enzyme product should be titrated to the minimum effective maintenance dose per feeding. "when pancreatic digestive enzymes are given orally, a high percentage of them are inactivated by gastric acid. even though only a fraction of the pancreatic enzymes administered reach the small intestine in an active state, they are still effective because only a slight increase in duodenal digestive enzyme activity is needed to achieve marked improvement in nutrient assimilation (drazner, 1986) . in some dogs, antimicrobial agents may be a helpful adjunctive therapy for the bacterial overgrowth of the small intestine that often accompanies malassimilation in congenital pancreatic hypoplasia (drazner, 1986) . medium-chain triglycerides may be added to the dog's diet if additional dietary energy is needed to increase weight gain or maintain condition in the dog that fails to respond otherwise. the medium-chain triglycerides can be used to provide up to 25% of the dog's caloric need and, when fully utilized, provide 8 kcal/ml (lewis et ai, 1987) . the dog's body weight, general condition, and stool character should be monitored weekly during the treatment of congenital pancreatic hypoplasia. stool volume should decrease precipitously, and gains in body weight should begin soon after initiation of dietary management and the supplementation of pancreatic digestive enzymes (sherding, 1979) . the dietary replacement of pancreatic digestive enzymes is generally required for the rest of the dog's life. neurovisceral and skeletal gml-gangliosidosis in dogs with betagalactosidase deficiency hepatic beta galactosidase and feline gm 1 gangliosidosis a divided intrahepatic gallbladder in a cat congenital feline portosystemic shunts ea: the accessory gall-bladder: an embryological and comparative study of aberrant biliary vesicles occurring in man and the domestic mammals a case of mucopolysaccharidosis vi in a cat briggs om: serum urate concentrations in the dalmatian coach hound presumptive neonatal isoerythrolysis in cats congenital portacaval shunt in two kittens 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significance of hepatic copper values in dogs with cirrhosis hereditary copper toxicosis in west highland white terriers clinical, morphologic, and chemical studies on copper toxicosis of bedlington terriers van den ingh tsgam, rothuizen j: lobular dissecting hepatitis in juvenile and young adult dogs experimental induction of fasting hypoglycaemia and fatty liver syndrome in three yorkshire terrier pups use of transcolonic portal scintigraphy to monitor blood flow and progressive postoperative attenuation of partially ligated single extrahepatic portosystemic shunts in dogs hereditary neurovisceral mannosidosis associated with alpha-mannosidase deficiency in a family of persian cats portosystemic communications in the dog gradual occlusion of extrahepatic portosystemic shunts in dogs and cats using the ameroid constrictor canine glycogen storage disease type ii: a biochemical study of an acid alpha-glucosidase deficient lapland dog canine glycogen storage disease type ii: a clinical study of four affected lapland dogs disseminated intravascular coagulation in experimentally induced feline infectious peritonitis niemann-pick disease: a genetic model in siamese cats a method for controlling portal pressure after attenuation of intrahepatic portocaval shunts ligation of ductus venosus in a dog using ultrasonographic guidance the pancreas anderson nv, low dg: juvenile atrophy of the canine pancreas feline infectious peritonitis mechanisms of diarrheal disease diseases of the pancreas juvenile pancreatic atrophy in a dog pancreatic degenerative atrophy in dogs juvenile pancreatic atrophy (pancreatic hypoplasia) in dogs pancreatic atrophy in young beagle dogs canine exocrine pancreatic insufficiency small animal gastroenterology key: cord-334894-v5mu9ey2 authors: gaykwad, c.; garkhal, j.; chethan, g. e.; nandi, s.; de, u. k. title: amelioration of oxidative stress using n‐acetylcysteine in canine parvoviral enteritis date: 2017-07-12 journal: j vet pharmacol ther doi: 10.1111/jvp.12434 sha: doc_id: 334894 cord_uid: v5mu9ey2 previously, antioxidants have not been evaluated for treatment of parvoviral diarrhea in dogs. in this study, antioxidant potential of n‐acetylcysteine (nac) in dogs infected with canine parvovirus with a nonblinded randomized clinical trial has been carried out. a total 18 parvo‐infected dogs were randomly divided into two groups: nine parvo‐infected dogs were treated with supportive treatment and nine parvo‐infected dogs were treated with nac along with supportive treatment. simultaneously, nine healthy dogs were kept as healthy control. in parvo‐infected dogs, marked hemoconcentration, leucopenia, neutropenia and oxidative stress were noticed compared to healthy dogs. the nac treatment progressively improved the leukocyte, neutrophil, monocyte, and eosinophil counts over the time in parvovirus‐infected dogs compared to dogs that received only supportive treatment. in addition, nac treatment significantly improved glutathione s‐transferase (gst) activity and decreased nitrite plus nitrate (nox) and malondialdehyde (mda) concentrations on day 3 and 5 compared to supportive treatment in parvo‐infected dogs. however, supportive treatment alone failed to ameliorate oxidative stress in the infected dogs till day 5. the results of this study suggest that nac represents a potential additional treatment option that could be considered to improve the health condition and minimize the duration of hospitalization in case of canine parvoviral diarrhea. canine parvovirus infection occurs worldwide in domestic dogs and wild canids. incidence is higher in animal shelters, pet stores, and breeding kennels. although cpv-2 can affect all breeds of dogs at any age, severe infection is most common in puppies between 6 weeks and 4 months old (hueffer et al., 2003) . the disease condition has been complicated further due to emergence of a number of variants, namely cpv-2a, cpv-2b and cpv-2c over the years and involvement of domestic and wild canines (buonavoglia et al., 2001; martella et al., 2004) . in absence of suitable antiviral therapy to protect the dogs against cpv infection, supportive treatment is the only option to reduce the mortality due to this disease. therapeutic efficacy of recombinant feline interferonω and recombinant canine granulocyte colony-stimulating factor has been evaluated in cpv, but there are limitations like commercially nonavailability in all the places and exorbitant price make these therapeutics for regular use (de mari, maynard, eun, & lebreux, 2003; duffy, dow, ogilvie, rao, & hackett, 2010; mylonakis, kalli, & rallis, 2016) . oseltamivir, a neuraminidase inhibitor, has also been tried for the treatment of cpv without any substantial benefit in terms of survival or duration of hospitalization (savigny & macintire, 2010) . in recent years, oxidative stress has been paid much attention due to its pivotal role in the pathogenesis of viral diseases (beck, handy, & levander, 2000; schwarz, 1996; valyi-nagy & dermody, 2005) . the oxidative stress occurs due to imbalance of production of reactive oxygen/nitrogen species or neutralizing antioxidant enzymes (kim, kim, & hahm, 2012; lykkesfeldt & svendsen, 2007) . free radical-induced oxidative stress causes oxidation of the polyunsaturated fatty acids of erythrocyte cell membrane, which results in lipid peroxidation. malondialdehyde (mda), the principal and most studied product of polyunsaturated fatty acid peroxidation, is a classical marker of free radical-induced cell damage (del rio, stewart, & pellegrini, 2005; kuhn & borchert, 2002) . determination of mda in plasma indicates the degree of lipid peroxidation and the concentration of free oxygen radicals indirectly (deger, deger, bicek, ozdal, & gul, 2009) . nitric oxide (no), another essential messenger molecule under physiological concentrations, becomes a free radical to generate peroxynitrite anion (onoo) under excess and is considered as a marker of oxidative stress (pacher, beckman, & liaudet, 2007; pierini & bryan, 2015) . glutathione s-transferase (gst), a metabolic isozyme, plays a crucial role in defense mechanisms against oxidative injury (röth et al., 2011) . gst protects cells from oxidative stress by detoxifying the secondary reactive oxygen species (ros) produced when ros react with cellular constituents (veal, toonem, jones, & morgan, 2002) . the oxidant/antioxidant imbalance has been reported in pathogenesis of enteric viral diseases like feline coronavirus, bovine herpesvirus-1, porcine reproductive and respiratory syndrome and rotavirus (de et al., 2014; durgut, ataseven, & öztürk, 2013; kayar et al., 2015; stukelj, toplak, & nemec svete, 2013) . recently, it has been observed that parvovirus infection is linked with oxidative stress, and marked enhancement of reactive oxygen/nitrogen species, lipid peroxidation, dna damage and poor antioxidant reserve (luo & qiu, 2013; nykky, vuento, & gilbert, 2014; panda, patra, nandi, & swarup, 2009) . in recent years, emphasis has been given on the antioxidants as the potential drugs of interest for management of viral diseases (beck, 1998; chandrasena et al., 2014; crump, langston, rajkarnikar, & grayson, 2013) . a strong association of cpv with oxidative stress suggests incorporation of antioxidants in therapeutic regimen in canine parvoviral diarrhea may help in ameliorating the clinical signs. n-acetylcysteine (nac), the body's primary cellular antioxidant, is a precursor to glutathione and its role on glutathione maintenance and metabolism is well established (kelly, 1998) . glutathione counteracts the harmful effect of reactive oxygen and nitrogen species through both direct and indirect scavenging (dean, giorlando, & berk, 2011) . cholangiohepatitis of dogs and other noninfectious systemic disease conditions of rat (dean et al., 2011; ribeiro et al., 2011; sadowska, manuel-y-keenoy, & de backer, 2007; seguro, poli de figueiredo, & shimizu, 2012; shahripour, harrigan, & alexandrov, 2014; stanislaus, gilg, singh, & singh, 2005; webster & cooper, 2009) . recently, antioxidant effect of nac has been reported in viral diseases including hiv and influenza (geiler et al., 2010; sgarbanti et al., 2014; staal, 2000; uchide & toyoda, 2011) . however, the antioxidant effect of nac in dogs naturally affected with parvoviral diarrhea has not yet been explored. therefore, this study was aimed to evaluate the antioxidant potential of nac in diarrheic dogs infected with canine parvovirus. the study was conducted at referral veterinary polyclinic and teaching veterinary clinical complex (rvp-tvcc) of the institute during february 2016 to may 2016. a total 26 dogs (age group 1.5-6 months) with complains of pyrexia, weakness, reduced appetite, severe dehydration, hemorrhagic diarrhea and vomition were taken for this study. the fecal samples were collected from the diarrheic dogs during the episodes of hemorrhagic gastroenteritis in virus transport media in separate tube and 20 μl of 3m sodium acetate and 1 ml of absolute ethanol were added. after mixing, the tubes were kept at −20°c overnight. next day the tubes were again centrifuged at 14,000 g for 10 min. the supernatant was discarded and 200 μl of 70% ethanol was added and repeated the centrifugation process. the supernatant was discarded and pellet was dried at 90°c and finally dissolved in 20 μl of nuclease-free water. the vp2 gene of cpv was used for diagnosis in this study (pereira, monezi, mehnert, d'angelo, & durigon, 2000) . the forward (5′ gaa gag tgg ttg taa ata ata 3′) and reverse (5′ cct ata tca cca aag tta gta g 3′) primer sets (imperial biomedics) were used to amplify part of vp2 gene of cpv to yield a product of 681 bp (pereira et al., 2000) . in pcr, 25 μl of pcr mastermix (2×), 1 μl each of forward and reverse primers (10 pmol), 5 μl of extracted dna as template and rest nuclease-free water (nfw) to make final volume of 50 μl. suitable no template control having nfw instead of template dna was included as negative control. whereas the cpv 2 isolated in the laboratory, amplified by pcr, cloned in cloning vector and sequenced was taken as positive control. the pcr was performed in a thermal cycler (applied biosystems, usa). the cyclic condition was denaturation at 95°c for 45 s, primer annealing at 55°c for 45 s and extension at 72°c for 45 s. the cyclic condition was repeated for 35 times and a final extension at 72°c was given for 10 min. after pcr, the amplified products were analyzed on 1.0% agarose gel containing ethidium bromide to a final concentration of 0.5 μg/ml. ten microliter of amplified product was mixed with 2 μl of bromophenol (6×) dye and loaded into the well and run along with 100 bp to 1 kbp dna ladder in 1× tae electrophoresis buffer at 5 volts/cm 2 and the progress of mobility was monitored by migration of dye. at the end of the electrophoresis, the gel was visualized under the uv transillumiator (nandi, pandey, sharma, & chauhan, 2008) . all the pcr products were gel eluted, cloned using clone jet pcr cloning kit and sequenced to substantiate the authenticity of result. the blood samples (approximately, 3.0 ml) were collected by venipuncture of either cephalic or recurrent tarsal vein in k 2 edtacontaining vial from each participated dog in association with routine clinical sampling before initiation of any treatment. of 3.0 ml, 0.5 ml blood was use for hematology and 2.5 ml blood was immediately centrifuged at 200 g for 10 min to separate plasma and stored at −20°c until analysis. the hematological analysis of blood was performed manually (jain, 1986) . the concentration hemoglobin (hb) in the whole blood was measured by modified sahil's acid hematin method. red blood cell (rbc) and white blood cell (wbc) count was measured by neubauer's hemocytometer. methanol fixed blood smear was stained with diluted giemsa stain (1:10) for 45 min and the differential leukocyte count (dlc) was done. the hematology was performed before treatment (day 0) and thereafter on day 3 and 5 of initiation of treatment. the oxidative stress was evaluated on the basis of measurement of the activity of glutathione s-transferase (gst) and concentration of nitric oxide (nox) and malondialdehyde (mda) in plasma before treatment (day 0) and thereafter on day 3 and 5 of initiation of treatment. lipid peroxidation was measured by determining the plasma malondialdehyde (mda) concentration by double heating method (draper & hadley, 1990 ). in short: 2.5 ml of 10% triochloroacetic acid (tca) was mixed with 0.5 ml plasma in a test tube. the mixture was kept in a boiling water bath for 15 min. the reaction mixture was cooled and centrifuged at 400 g for 10 min. two milliliters of the supernatant was taken out and mixed with 1.0 ml 0.67% tba in a separate tube and incubated in a boiling water bath for 15 min. the solution was cooled to room temperature and the absorbance was measured at 532 nm using a spectrophotometer. the concentration of plasma mda was calculated from the absorbance coefficient (1.56 × 105 cm −1 m −1 ) of the thiobarbituric acid-malondialdehyde (tba-mda) complex, and value was expressed in nmol/ml. the nox in the plasma was measured by reduction in nitrate with acid-activated copper-cadmium alloy followed by color development with griess reagent (sastry, maudgal, mohan, tyagi, & rao, 2002) . briefly, a reaction mixture consisting of 100μl plasma sample, 400 μl of carbonate buffer and 150-mg powder of copper-cadmium alloy was incubated for 1 hr at room temperature with frequent shaking. the reaction was stopped by the addition of 0.35-m naoh and 120-mm znso4. after vortexing, the reaction mixture was centrifuged at 400 g for 15 min, and 75μl supernatant was separated and added with 75μl griess reagent (0.1% naphthalene diamine dihydrochloride in 3 n hydrochloric acid and 1% sulfanilamide, 1:1 ratio) in 96-well microplate. the optical density was read at 545 nm in microplate reader after incubation for 10 min at room temperature. the value of nox production was calculated from a standard curve using different concentrations of potassium nitrate. the plasma gst activity was assayed by using commercially available kit (ezassaytm gst activity estimation kit, product code: cck028, himedia, mumbai, india) and value was expressed in μm ml −1 min −1 . the data were analyzed using statistical software package sas v 9. the one-way anova was used to compare the treatments to hematology and oxidative stress indices tested and, when an interaction was found, tukey's post hoc test was used to determine statistical significance between the different treatment groups. an individual dog was considered the experimental unit. a probability level (p) of .05 was selected as the statistical selection limit for all tests. when an interaction was present between the treatment and hematology or oxidative stress indices as determined by one-way anova, the pvalue obtained by tukey's post hoc test for the relevant treatment groups was presented; when an interaction was not present, the p-value obtained from the one-way anova was presented. results are expressed as means ± sem. on clinical examination, the canine parvovirus affected dogs exhibited the clinical signs of high rise of temperature, weakness, reduced appetite, severe dehydration, bloody diarrhea and vomition. of 26 affected dogs, 18 dogs were found positive for cpv infection by pcr using vp2 gene-specific primers in processed fecal sample as evident by presence of expected 681 bp dna product in agarose gel under uv transilluminator. the rest eight samples were found negative for cpv infection by pcr as there was no specific band visualized (figure 1 ). the mean hb concentration and rbc count were significantly higher (p < .05) in parvo-infected dogs (group b and group c) compared to control dogs (group a) before treatment. the supportive treatment and supportive treatment plus nac (st-nac) significantly (p < .05) improved the hb concentration and rbc count in parvo-infected dogs on day 3 and 5 as compared to pretreatment value. a statistically significant interaction was found between treatment and hb (p = .000) and rbc count (p = .020; figure 2 ). . the data were analyzed by tukey's post hoc test using repeated measure analysis. a statistically significant interaction was found between treatment and hb (p = .000) and rbc count (p = .020). superscripts a, b between the groups within a day and superscripts a, b between the days within the group differ significantly (p < .05) showed better result (p < .05) in terms of monocyte and eosinophil counts on day 3 and 5. when the mean monocyte and eosinophil counts were compared between parvo-infected dogs and control dogs, no significant difference was noticed on day 5. the mean lymphocyte and basophil counts remain unaffected throughout the study period in both the treated groups. a statistically significant interaction was found between the treatment and monocyte count (p = .049), but such interaction was not present for lymphocyte (p = .886), eosinophil (p = .771) and basophil count (p = .824; table 1 ). in parvo-infected dogs, significantly lower concentration of gst (p = .004) but higher concentration of nox (p = .000) and mda (p = .000) were noticed compared to healthy dogs. although the mean values of gst, nox and mda concentrations did not differ significantly throughout the study period in parvo-infected dogs that received only st (group b), there was significant elevation of the mean gst (p = .035) concentration and appreciable reduction in nox (p = .039) and mda (p = .035) concentrations on day 3 and 5 in st-nac-treated dogs (group c). when the values were compared between st-and st-nac-treated groups, it was found that mean gst (p = .007, .031) activity significantly increased and nox (p = .001, .033) and mda (p = .007, .037) concentrations significantly reduced on day 3 and 5 in st-nac-treated dogs compared to st-treated dogs. there was a statistically significant interaction between the treatment and nox concentration (p = .036), but not with gst activity (p = .063) and mda concentration (p = .100; figure 3 ). in this study, pronounced elevation of hb% and rbc count in infected dogs indicates hemoconcentration during parvoviral diarrhea. the parvoviral enteritis causes anemia that is attributed to cytotoxic effect the data were analyzed by tukey's post hoc test using repeated measure analysis. a statistically significant interaction was found between treatment and monocyte count (p = .049), but such interaction was not present for tlc (p = .281), neutrophil count (p = .054), lymphocyte count (p = .886), eosinophil count (p = .771) and basophil count (p = .824). the values were expressed as mean ± sem. superscripts a, b, c between the groups within a day and superscripts a, b, c between the days within a group differ significantly (p < .05). of virus on hematopoietic cells, bone marrow failure and subsequently erythroid hypoplasia during acute stages of the disease (grimes & fry, 2015; nandi & kumar, 2010; stann, digiacomo, giddens, & evermann, 1984) . in contrast, hemoconcentration in this study might be due to excessive fluid loss during the episodes of diarrhea. furthermore, leucopenia and neutropenia are the major hemogram alterations in the parvo-infected dogs (castro et al., 2013) . the leukopenia and neutropenia in parvo-infected dogs could be due to destruction of hematopoietic progenitor cells of the various leukocyte types in the bone marrow, inadequate supply for the massive demand for leukocytes in the inflamed gastrointestinal tract and loss of neutrophils through the damaged gastrointestinal tract (macartney, mccandlish, thompson, & cornwell, 1984; fulton et al., 199; goddard, leisewitz, christopher, duncan, & becker, 2008 much has been learned about the beneficial effect of antioxidants against viral diseases in recent years (beck, 2001; peterhans, 1997; zhang, wang, chen, chen, & tian, 2014) . studies derived from influ. the mean gst activity significantly elevated on day 5 (p = .035) from pretreatment value (day 0) in st-nac treated dogs, whereas no significant changes was observed in parvo-infected dogs received st only. the average mda and nox concentrations significantly reduced on day 5 (p = .035, .039) from pretreatment value (day 0) in st-nac treated dogs, whereas no significant changes was observed in parvo-infected dogs received st only. a statistically significant interaction was found between treatment and nox concentration (p = .036) but, such interaction was not present for gst activity (p = .063) and mda concentrations (p = .100). superscripts a, b between the groups within a day and superscripts a, b between the days within the group differ significantly (p < .05) (kurutas, cetinkaya, bulbuloglu, & kantarceken,2005; viviano & wielen, 2013 (dekhuijzen, 2004; garigliany & desmecht, 2011; guerrero, torres, garcía, guerrero, & acosta, 2014) . from the findings of this study, it can be concluded that antioxidants like nac represent a potential additional treatment option that could be considered in the case of parvoviral diarrhea. however, the therapeutic efficacy of nac involving large number of dogs with parvovirus infection needs further study to extract the full potential of the compounds with comprehensive results. we acknowledge director ivri for providing facilities to carry out the work. cg, cge and jg thank icar-ivri for granting research fellowship for their research program. the effect of n-acetylcysteine on pulmonary lipid peroxidation and tissue damage dna damage responses to oxidative stress the influence of antioxidant nutrients on viral infection antioxidants and viral infections: host immune response and viral pathogenicity the role of oxidative stress in viral infections n-acetylcysteine protects induced pluripotent stem cells from in vitro stress: 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parvoviral diarrhoea in dogs antioxidant enzymes (sod, gpx), biochemical and haematological parameters in pigs naturally infected with porcine reproductive and respiratory syndrome virus antioxidant therapy as a potential approach to severe influenza-associated complications role of oxidative damage in the pathogenesis of viral infections of the nervous system distinct roles for glutathione s-transferases in the oxidative stress response in schizosaccharomyces pombe effect of n-acetylcysteine supplementation on intracellular glutathione, urine isoprostanes, clinical score, and survival in hospitalized ill dogs therapeutic use of cytoprotective agents in canine and feline hepatobiliary disease antioxidants: potential antiviral agents for japanese encephalitis virus infection amelioration of oxidative stress using n-acetylcysteine in canine parvoviral enteritis key: cord-255011-7oqfod62 authors: erles, kerstin; brownlie, joe title: canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex date: 2008-05-22 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2008.02.008 sha: doc_id: 255011 cord_uid: 7oqfod62 infectious respiratory disease in dogs is a constant challenge because of the involvement of several pathogens and environmental factors. canine respiratory coronavirus (crcov) is a new coronavirus of dogs, which is widespread in north america, japan, and several european countries. crcov has been associated with respiratory disease, particularly in kenneled dog populations. the virus is genetically and antigenically distinct from enteric canine coronavirus; therefore, specific tests are required for diagnosis. r espiratory disease in dogs is generally of greatest importance in establishments in which dogs are housed in groups, such as shelters, boarding kennels, and veterinary hospitals. disease outbreaks involving only one species of infectious agent are possible, as seen in distemper outbreaks in susceptible populations [1] . most commonly, however, infectious respiratory disease in dogs has a multifactorial etiology and is best described as canine infectious respiratory disease (cird) complex (also known as ''kennel cough''). viruses detected in dogs with cird include canine parainfluenza virus (cpiv) [2] , canine adenovirus (cav) type 2 [3] and canine herpesvirus [4] . canine influenza virus, which recently has been detected in some parts of the united states, is likely to become part of the disease complex because it often causes mild respiratory disease characterized by nasal discharge and persistent cough [5] . bacteria are important in cird as primary pathogens and as a cause of secondary infections. bordetella bronchiseptica is the bacterium most frequently associated with cird [6] , but mycoplasmas, particularly mycoplasma cynos, have also been linked to the disease [6, 7] . streptococcus equi subsp. zooepidemicus has been isolated from severe cases of respiratory disease that were frequently fatal [8] . vaccines have been developed for protection against several canine respiratory pathogens. combination vaccines routinely contain canine distemper virus and cav-1 or cav-2. cav vaccines confer cross-protection against type 1 (the cause of canine infectious hepatitis) and type 2 (associated with respiratory disease). cpiv is also included in several multivalent vaccines, or it is available in combination with b bronchiseptica as a ''kennel cough vaccine.'' this work was supported by battersea dogs and cats home and the guide dogs for the blind association. canine respiratory coronavirus (crcov) was first detected in 2003 in dogs housed at a uk rehoming center [9] . the center had a high turnover of dogs and was reporting problems with enzootic respiratory disease despite regular vaccination. an investigation into pathogens associated with cird in this population led to the detection of a coronavirus in tracheal and lung samples by reverse transcriptase polymerase chain reaction (rt-pcr). coronaviruses are large enveloped viruses containing a positive-sense singlestranded rna genome. the structural proteins located in the viral envelope include the spike protein (s), the membrane protein (m), and the small membrane protein (e). initial sequence analysis of crcov showed a high similarity to bovine coronavirus (bcov) and human coronavirus oc43 (96% amino acid identity with bcov in the variable spike protein). coronaviruses had been described before in dogs with gastroenteritis [10] ; however, it was shown that crcov was distinct from the previously known canine coronavirus (ccov). the virus showed only 69% nucleotide identity in the highly conserved polymerase region and only 21% amino acid sequence identity in the spike protein, indicating that crcov was a novel coronavirus of dogs. members of the family coronaviridae are separated into groups according to their genetic similarities [11] . most members of group 2 of coronaviruses contain an additional gene coding for a surface hemagglutinin-esterase protein. this gene was found to be present in crcov, confirming its place in group 2 together with its closest relative, bcov (fig. 1) . ccov, in contrast is a member of group 1, which includes feline coronavirus and porcine transmissible gastroenteritis among others. currently, the oldest samples that tested positive for crcov are canine lung samples collected in canada in 1996 [12] . one of the reasons precluding earlier discovery of crcov may be its poor growth in cell culture and the requirement for specific host cells. the close genetic relation to bcov throughout the crcov genome indicates that the virus was probably transmitted to dogs from cattle [13] . interestingly, it has recently been shown that human coronavirus oc43 also may have emerged after viral transmission from cattle to people [14] . coronaviruses are a cause of respiratory disease in many species, including human beings, poultry, and cattle [15] [16] [17] [18] [19] [20] . the presence of crcov in dogs was first described in a large study of dogs with cird [9] . in this investigation, performed at a shelter, clinical signs were graded by veterinary clinicians into (1) no signs of respiratory disease; (2) mild cough; (3) mild cough and nasal discharge; (4) cough, nasal discharge, and inappetence; and (5) severe respiratory disease with evidence of bronchopneumonia. because of a small number of samples in grade 4, grades 3 and 4 were merged and referred to as ''moderate respiratory disease.'' crcov was most frequently detected in the trachea of dogs with mild clinical signs (grade 2). it was less frequently recovered from dogs with moderate or severe clinical signs or from dogs without clinical signs at the time of sampling. crcov was also detected in the lung, albeit less frequently. table 1 summarizes the detection of crcov in clinical samples from dogs. after 3 weeks of stay at a shelter, almost 100% of dogs tested positive for antibodies to crcov compared with 30% on the day of entry, indicating that the virus was highly prevalent in the population and was easily transmitted. it was also found that the presence of antibodies to crcov on the day of entry led to a significantly reduced risk for contracting cird, supporting the hypothesis that crcov played a role in the etiology of the disease [9] . after this initial investigation, crcov was also detected in two uk training kennels for working dogs [21] . serum samples had been collected during two outbreaks of respiratory disease at one of the kennels and 4 weeks after. almost all dogs housed at the kennel showed seroconversion to crcov after the outbreaks. moreover, crcov was detected by pcr in two oropharyngeal swabs taken from dogs with clinical respiratory disease. not all dogs that developed antibodies to crcov also showed signs of cird; nevertheless, this was the second study associating crcov with respiratory disease in dogs. two further studies identified crcov in nasal and oral swabs from dogs in japan that had respiratory disease [22, 23] . an analysis of 126 archival tissue blocks of cases of respiratory disease identified two crcov-positive samples by immunohistochemistry. both were from dogs with bronchitis and bronchiolitis, and crcov antigen was found to be present in respiratory columnar epithelial cells. one of those dogs was also positive for canine distemper virus [12] . the detection rate of crcov in the archival study may seem quite low; however, the tissue samples were mostly derived from dogs with severe respiratory disease with a fatal outcome, whereas crcov has mostly been associated with mild respiratory disease so far. serologic studies to determine the prevalence of antibodies to crcov have been performed to date for the united kingdom, republic of ireland, italy, united states, canada, and japan. the highest seroprevalence was detected in canada (59.1% of sera tested were found to be positive) and the united states (54.7% of sera tested were found to be positive). samples from the united states had been collected from 33 states, and positive samples were identified in 29 [24] . in those states that allowed a meaningful interpretation of seroprevalence (more than 10 samples), the prevalence ranged from 31.3% (maine) to 87.5% (kentucky). the prevalence in the united kingdom and the republic of ireland was lower, with 36% and 30.3%, respectively [24] . two studies performed in italy showed seroprevalence ranging from 20% to 32.5% [25, 26] . the lowest seroprevalence was detected in japan, with 17.8% [23] . further data are not yet available, but it is likely that crcov is present throughout the united states and in other european countries. although crcov was detected throughout the year in a kennel with enzootic cird, another study reported a seasonal occurrence of the virus in the winter months. no seroconversions to crcov and few cases of respiratory disease were recorded in the summer months [21] . a similar seasonality has been reported for human coronaviruses involved in the common cold [27] . crcov infections can occur in dogs of all ages. dogs younger than 1 year of age were significantly more likely to be seronegative than older dogs, however [24, 25] . this is in contrast to the prevalence of enteric ccov, which is frequently found in dogs younger than 1 year of age [10] . this may reflect different patterns of transmission of the two viruses. the seroprevalence of crcov was increasing after the age of 1 year for all studies and then reached a plateau between the ages of 2 and approximately 8 years. this is probably a consequence of the greater probability of exposure to the virus with increasing contact with other dogs. it is not certain how long crcov antibody levels in dogs remain stable after infection. one study showed a twofold decrease in antibody titers in 6 of 14 dogs tested and a fourfold decrease in 4 dogs in less than 1 year [21] . because these were naturally occurring infections, it is not known if the antibody response measured reflected primary or repeated infections. the viral dose encountered by dogs would also influence the level and duration of the antibody response. the rapid spread of crcov through kenneled populations indicates that the virus is highly contagious. this, in conjunction with the predominant detection of crcov in respiratory samples, suggests that it is mostly spread by means of respiratory secretions. crcov probably enters the respiratory tract by inhalation of droplets or contact with secretions and contaminated surfaces. it is not possible to discuss the pathogenesis and clinical signs associated with crcov without considering the cird complex as a whole. crcov has been detected in several studies in dogs with respiratory disease. in most of these cases, however, other respiratory pathogens were also present. in two detailed studies into the causes of cird in which evidence of crcov was reported, the dogs presented with the typical signs of a dry cough and nasal discharge [21, 28] . concurrent infections were most frequently caused by cpiv and b bronchiseptica. crcov has also been detected in dogs that have nonrespiratory disease. it was detected in the lung, spleen, mesenteric lymph nodes, and intestines of a dog that had died from hemorrhagic gastroenteritis [26] . the dog also tested positive for canine parvovirus type 2 and ccov. similarly, crcov was detected in a rectal swab from a dog with vomiting and diarrhea, which was also positive for ccov and cpiv [22] . in both cases, the concurrent infections with canine parvovirus or ccov are likely to have been the cause of the clinical signs. studies of the tissue distribution of crcov in 10 naturally infected dogs showed that crcov was most frequently detected in the nasal cavity, nasal tonsil, and trachea and less frequently in the lung, bronchial lymph nodes, and palatine tonsil. it was also detected in samples from the spleen, mesenteric lymph nodes, and colon but not in the enteric content (k. erles, unpublished data, 2004) . the tissue tropism of crcov therefore seems not to be exclusively respiratory, and fecal-oral transmission of crcov may be possible. crcov may show a dual tropism, similar to bcov, but the ability of the virus to replicate in the epithelium of the gastrointestinal tract and the clinical consequences need further investigation. experimental studies using crcov have not been reported to date. a study using bcov showed that dogs became infected and transmitted the virus to contact dogs. bcov was detected in rectal and oral swabs, and the dogs developed neutralizing antibodies to the virus [29] . the dogs did not develop fever or any clinical signs of respiratory or gastrointestinal disease. despite their high similarity, bcov may be less pathogenic in dogs compared with crcov. furthermore, the etiology of cird has been shown to involve multiple pathogens. viral infections can aid the entry of other pathogens by facilitating their attachment or by inhibition of the mucociliary clearance. many pathogens known to be involved in the cird complex have also been found in dogs without clinical signs, including cpiv and b bronchiseptica [28, 30] . when assessing the pathogenesis of complex diseases, it is important to consider the possible interaction of pathogens during coinfections and contributing factors, such as stress. because of the involvement of multiple pathogens in the etiology of cird, it is not possible to diagnose crcov solely by clinical signs. the most suitable test to diagnose crcov in respiratory samples is nested rt-pcr based on the spike glycoprotein gene [9] or the hemagglutinin-esterase gene [22] . this test has a high sensitivity, which is particularly useful when analyzing samples with a potentially low number of cells, such as oropharyngeal or nasal swabs. both pcr methods are specific for crcov and do not detect enteric ccov. because the virus was found most frequently in the nasal cavity, nasal swabs are suitable diagnostic samples. crcov has also been detected in oral swabs, and, furthermore, nasal or tracheal washes are likely to yield crcov during an active infection. if postmortem samples are available, nasal cavity, nasal tonsil, trachea, and lung samples should be collected for analysis. isolation of crcov in cell culture has been achieved; however, it is not recommended to use virus isolation alone to diagnose crcov. to date, isolation of crcov has only succeeded on the human rectal tumor cell line hrt-18 and its clone hrt-18g [13] . even on hrt-18 cells, the isolation of crcov from rt-pcr-positive samples is often unsuccessful [22, 23, 26] . the only isolate of crcov that has so far been studied in detail did not produce a cytopathic effect on hrt-18 cells, and infection had to be confirmed by using immunofluorescence or pcr. supernatants from infected cell cultures were found to agglutinate chicken erythrocytes at 4 c [13] . hemagglutination assays may aid in detection of crcov-infected cell cultures if isolation is attempted. crcov has also been detected by immunohistochemistry on formalin-fixed tissues using an antibody directed against bcov [12] . the sensitivity of immunohistochemistry in comparison to pcr has not been evaluated; however, this method is useful for testing archival respiratory samples. serology is a valuable tool for the detection of crcov infections if paired serum samples are collected during an outbreak of respiratory disease and at least 2 to 3 weeks afterward. the high similarity of crcov and bcov allows the use of bcov antigens to test canine sera by elisa [9, 26] . similarly, bcov has been used instead of crcov in serum neutralization tests [23] . a hemagglutination inhibition test based on bcov has also been evaluated but was assessed as having poor sensitivity and specificity compared with an elisa based on bcov [26] . an elisa assay using crcov antigen was found to have slightly higher sensitivity and specificity compared with an assay based on bcov; however, overall, the agreement between the two elisa tests was high [24] . antibodies to crcov have also been detected by using an immunofluorescence assay on crcov-infected hrt-18 cells [24] . specific tests for crcov are becoming increasingly available; however, most assays offered for the detection of coronaviruses in dogs are specific for enteric ccov. antibodies to crcov do not cross-react with enteric ccov. it is important to use an assay capable of detecting antibodies to crcov or related group 2 coronaviruses, such as bcov. consequently, the requirement for crcov detection should be discussed with the diagnostic laboratory before submitting samples for rt-pcr, virus isolation, or serology. there is no specific treatment for infections caused by crcov. as for other causes of cird, patient care should focus on the prevention and treatment of bacterial infections. although many pathogens involved in cird are reported to be associated with mild clinical disease, it is important to bear in mind that mixed infections can potentially be much more severe. patients should be monitored, because the condition may rapidly worsen. severe cases of cird with sudden death have been reported after infections with streptococcus equi subsp. zooepidemicus [8] . to date, no vaccines against crcov are available. vaccines against ccov are unlikely to protect against infection with crcov because of a low similarity in the spike proteins that are the major immunogenic proteins of coronaviruses. vaccines against other respiratory pathogens may not prevent cird, particularly in large populations because of the presence of other infectious agents. nevertheless, they have the potential to reduce the number of circulating pathogens if given to all dogs on entry. vaccines against canine distemper virus and cav are widely used, and this may account for the inability to identify either virus in a population with enzootic cird [28] . although no specific tests have been performed to determine the stability of crcov in the environment, other coronaviruses have been reported to remain infectious in respiratory secretions for more than 7 days [31] . thorough cleaning and disinfection of kennels after outbreaks of respiratory disease are therefore required. coronaviruses are inactivated by disinfectants commonly used for surface disinfection in kennels and veterinary practices. the role of fecal shedding and the potential transmission of crcov among dogs sharing common facilities, such as outdoor runs, have yet to be resolved. other generally recommended measures, such as washing one's hands after handling animals with respiratory disease should also help to reduce the spread of the virus. crcov has been detected in dogs up to 4 weeks after entry into a kennel. because the time of infection in those naturally occurring cases is not known, it is unclear how long crcov is being shed. after experimental infection of dogs with bcov, the virus was detected in a rectal swab after 11 days [29] . quarantine of newly arriving dogs, if feasible in training kennels or shelters, should therefore last for at least 2 weeks. group 1 ccovs, referred to in this article as ccovs, have previously been associated with mild gastroenteritis. according to their similarity to feline coronaviruses, they are divided into type i (related to feline coronavirus type i) or type ii (related to feline coronavirus type ii) [32] . although ccov has been isolated from the lung after experimental infections [33] , it was generally considered to be restricted to the gastrointestinal tract during naturally occurring infection. recently, an outbreak of a systemic fatal disease was described from which a type ii ccov was isolated [34] . although the dogs presented with vomiting, diarrhea, and neurologic signs, postmortem examination also revealed bronchopneumonia. ccov was detected in internal organs, including the lung, kidney, and brain. sequence analysis of the ccov isolate identified a mutation in open reading frame 3b, leading to a truncated nonstructural protein. it is not clear if this mutation is responsible for the extended tropism of this isolate [35] . further studies are required to determine the presence of type ii ccovs in cases of severe systemic disease and in cases of respiratory disease. crcov is a novel coronavirus of dogs distinct from ccov. it is present in north america, europe, and japan. crcov is frequently detected in dogs with clinical respiratory signs and may contribute to the cird complex. increased awareness of the existence of crcov and the development of routinely available diagnostic tests should enhance our knowledge of the presence of crcov in canine populations with and without respiratory disease. it is recommended to use pcr methods or serology on paired serum samples to diagnose crcov infections, because the sensitivity of virus isolation is low. identification of causative agents during outbreaks of respiratory disease in canine populations ought to be performed more frequently. this would help to determine the importance of individual viruses and bacteria, not only in the investigated population but in the cird complex as a whole. the etiology of cird is multifactorial and is likely to change continuously, because some pathogens are controlled by vaccination, although other infectious agents emerge to take their place. canine distemper: re-emergence of an old enemy sv-5-like parainfluenza virus in dogs association of canine adenovirus (toronto a 26/ 61) with an outbreak of laryngotracheitis (''kennel cough''): a preliminary report canine tracheobronchitis: isolation and characterization of the agent with experimental reproduction of the disease transmission of equine influenza virus to dogs bordetella and mycoplasma respiratory infections in dogs and cats mycoplasmas associated with canine infectious respiratory disease the association of streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease detection of a group 2 coronavirus in dogs with canine infectious respiratory disease studies on the epizootiology of canine coronavirus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus detection of coronavirus in cases of tracheobronchitis in dogs: a retrospective study from 1971 to 2003 isolation and sequence analysis of canine respiratory coronavirus complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event coronavirus as a possible cause of severe acute respiratory syndrome viruses and bacteria in the etiology of the common cold identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus avian infectious bronchitis virus coronavirus and pasteurella infections in bovine shipping fever pneumonia and evans' criteria for causation investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations survey of dogs in japan for group 2 canine coronavirus infection the prevalence of a group 2 coronavirus in dogs in japan serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus serological and molecular evidence that canine respiratory coronavirus is circulating in italy epidemiology of coronavirus respiratory infections longitudinal study of viruses associated with canine infectious respiratory disease the infectivity and pathogenicity of a group 2 bovine coronavirus in pups respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide do not correlate with bacterial isolation or clinical respiratory symptoms survival of severe acute respiratory syndrome coronavirus genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy canine coronavirus infection in the dog following oronasal inoculation canine coronavirus highly pathogenic for dogs molecular characterisation of the virulent canine coronavirus cb/05 strain key: cord-261867-6n0g3bz5 authors: evermann, james f.; ledbetter, eric c.; maes, roger k. title: canine reproductive, respiratory, and ocular diseases due to canine herpesvirus date: 2011-10-28 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2011.08.007 sha: doc_id: 261867 cord_uid: 6n0g3bz5 this review documents how clinical inquiry expands as our knowledge base about canine herpesvirus (chv) increases. we must understand the various forms of chv infection that may occur in the dog population. this has prompted the veterinary community to develop more sensitive diagnostic assays. chv is more common than we considered a decade ago. up to 70% of some high-risk dog populations have been infected with and are latent carriers of chv. recognition of the various forms of chv-induced disease, availability of diagnostic assays with increased sensitivity, and the formation of reliable biosecurity measures will allow for better control steps. immunosuppressive regimens of therapy for various dermatologic conditions, as well as dogs being treated for various cancers. this review will provide a brief overview of the reproductive aspects of chv disease and will then bring together the current literature, documenting the involvement of chv in adult dog respiratory and ocular diseases. consistent with the other alpha herpesviruses, chv has a predilection for pregnant dogs and neonatal puppies. [23] [24] [25] [26] early reports focused on the effects of chv on various reproductive parameters in the dog, in part due to the severity of the clinical symptoms and the profound pathologic effects. in the review by anvik, 12 acute neonatal viremia and systemic infection of naïve pregnant females were regarded as 2 of the most important disease outcomes of chv infection. the emphasis at that time was on the recognition of clinical symptoms for a rapid diagnosis. since there are no commercial vaccines currently available for the prevention of chv-induced disease, it has become paramount to understand the clinical features of chv infections (see table 1 ) and to incorporate this knowledge with sound management practices to minimize the effects on reproductive efficiency and puppy survival. 27 as was mentioned previously, this has been the primary focus of the earlier literature on chv infections. infection may occur during pregnancy or may be acquired by puppies during the first few weeks of life. the key feature during both of these phases is that the pregnant female and puppies are immunologically naïve to chv and therefore highly susceptible to disease. puppies may acquire the infection in utero, from passage through the birth canal, from contact with oronasal secretions of the dam, or contact shedders. humans may serve as fomites of the virus if attending to an adult carrier-shedder dog, and then proceeding to a nursery setting without proper disinfection. naïve neonatal puppies, younger than 1 week, are at highest risk of fatal systemic disease, while naïve dogs older than 3 weeks are relatively resistant to disease but can still become infected. [27] [28] [29] virus infection in naïve older dogs is generally acquired via aerosol, so that replication occurs in the nasopharynx tonsils and retropharyngeal and bronchial lymph nodes. 2,30 -32 this respiratory site will become an important aspect of the ecology of the virus when both respiratory and ocular clinical outcomes are covered in subsequent sections. although neonatal infections are regarded as the most common, in utero infection with chv may occur. infertility and abortion of stillborn or of weak pups has been reported. while the mortality rate usually approaches 100% for the fetal puppy, there may be no further clinical manifestations reported in the dam. 1, 5, 26 passive immunity acquired from the dam appears to be of primary biological importance in the survival of infected pups. 12, 27, [33] [34] [35] puppies that are nursing from chv-seronegative dams usually develop the fatal multisystemic disease, while puppies that suckle from chv-seropositive dams remain asymptomatic but still become infected. the chv is usually recovered from the oropharyngeal region in these disease-resistant pups. it is generally accepted that maternal antibody and/or immune lymphocytes acquired through the milk explain why naturally infected dams that have a diseased litter will usually give birth to normal litters on subsequent pregnancies. since chv is one of the few canine viral infections that can proceed to fatal disease and there is no commercial vaccine routinely available, it has become necessary for infection management to prevent reproductive disease. the literature has focused on 3 aspects of the virus and its relationship with host immunity and its carrier-spread dynamics within a population of susceptible dogs. the risk factors associated with chv infection and reproductive disease has been intensively studied over the past 5 years. 36, 37 the studies have used various diagnostic assays including serology, virus isolation, and polymerase chain reaction (pcr). these studies have provided valuable information on controlling chv-associated reproductive diseases (ie, infertility, abortion, stillbirths, and neonatal mortality). table 2 lists the 12 risk factors that were studied and whether there was an association with reproductive disease. of the 12 factors, 8 were identified as having a positive correlation with disease: breeding kennel, age, mating experience, cycle (stage), concurrent kennel cough, kennel size, breeding management, and hygiene. the underlying risks in the aforementioned factors are chv infection and an immune susceptible dog. this has led to strategies to naturally immunize (via contact with adult dogs) susceptible female dogs prebreeding, to screen female dogs for chv infection (by serology and/or pcr) prior to breeding, and to use a defined quarantine period for pregnant dogs with an unknown chv infection status. an age-risk, immunologically naïve-risk strategy has been used by clinicians and clients to focus on the most susceptible time periods for disease. this time encompasses the pregnant female during the last 3 weeks prior to whelping, and her puppies up to 3 weeks post whelping. 12, 27, 33 this understanding has constituted the rationale for the "6-week danger period." 12, 27 the primary contributing risk factors that allow for chv infection and disease are kennel size, hygiene, and kennel cough. all 3 of these are important in the spread and retention of chv in high-risk dog populations. while the controversy over chv being a significant contributor to the kennel cough syndrome has been an ongoing debate (see subsequent section on respiratory-ocular infections), it should be noted that chv was initially reported as a respiratory pathogen as early as it was a reproductive pathogen. 2 the data from ronsse and coworkers 22 support the contention that chv is primarily maintained and spread among dogs in a multidog environment as a respiratory infection. the disease outcomes of chv infections are age dependent. in naive puppies that are less than 1 month of age, natural and experimental infection with chv may be highly fatal. natural exposure of pups occurs by ingestion or inhalation of virus containing material. the primary replication sites are nasal mucosa, pharynx, and tonsil. systemic spread of the virus is enhanced by a cell-associated viremia. 29 -32 the pathology induced by chv in the lungs of newborn pups is depicted in figs. 1 and 2. experimental infection of older dogs (3 months or older) with chv has resulted in a mild rhinitis and pharyngitis. symptoms of tracheobronchitis were produced following experimental inoculation with chv isolated from naturally infected dogs. 38, 39 experimental infection of 5-to 12-week-old pups induced mild rhinitis and pharyngitis and virus replication was demonstrated in the upper respiratory tract. although chv has been isolated from dogs with upper respiratory disease, reproduction of "kennel cough" has only been rarely reported. thompson and coworkers 32 reported that aerosol exposure of 12-week-old dogs caused a necrotizing rhinitis, broncheointerstitial pneumonia, and multifocal alveolar necrosis. more severe disease can occur when chv infects dogs that are immunosuppressed. 9 a case of generalized chv infection in a 9-year-old dog with a normal immune system was documented recently (gadsden bj, langohr im, maes r. fatal herpesviral infection in an adult dog. submitted for publication, 2011). the most severe lesions were seen in the liver. the histologic lesions observed in the lung of this dog are presented in fig. 3 . infection rates, based on serologic studies, are high enough to explain entry of chv into multidog environments, either as an active infection or as the result of reactivation of latent virus in environments associated with natural, or pharmacologically induced immunosuppression. in belgium the seroprevalence in adult dogs was found to be 45.8%. 22 rijsewijk and colleagues 21 reported a seroprevalence of 39% in the netherlands. reading and field, 20 using an antibody detection elisa, found a seroprevalence of 88% in the united kingdom. in japan, the seroprevalence was recently reported to be 21.7%. 6 since chv is regarded as a weak immunogen, these antibody-based surveys are probably an underrepresentation of the true infection rate in the dog populations. 33 canine infectious respiratory disease (cird) is most commonly seen in rescue centers, boarding kennels, and veterinary hospitals. most of the affected dogs have a dry cough of limited duration. in complicated cases, bronchopneumonia is seen and can be fatal. multiple infectious agents can play a role in the induction of cird. canine parainfluenza virus and bordetella bronchiseptica are frequently involved. canine distemper and canine adenovirus type 2 (cav-2) have been associated with cird but are not routinely detected due in part to effective vaccines, and the population immunity is fairly high. canine influenza, canine respiratory coronavirus, and, most recently, canine pneumovirus, are emerging components of cird, which have added to the complexity of this disease syndrome. 18, 19, 40 although chv infections have been documented in multidog environments, its etiologic role in cird is still being assessed. during a 2-year longitudinal study of viruses associated with cird at a rescue center in the united kingdom, chv was found in 12.8% of the tracheal samples examined and in 9.6% of the lung samples. infections with chv were seen 3 to 4 weeks after entry and were associated with more severe respiratory signs. 18 the delay in detection of the virus by pcr was corroborated by the serologic data, which also indicated that chv infections occurred at a later time point. a possible explanation offered for its detection in more severe cases was the possibility that latent chv could have been reactivated as a result of the stress induced by a primary cird episode that was triggered by other viral or bacterial agents. the virus source was not determined. the authors speculated that genetically different chv strains would have been detected if the source of virus was the result of reactivation of latent virus from different dogs. it has been reported, however, that chv strains show very low sequence variability. 41 erles and brownlie 19 monitored dogs in 2 training centers in the united kingdom for 1 year. all dogs were vaccinated against cav-2, cpv-2, and leptospira interrogans. tonsillar swabs and serum samples were collected at entry and every 3 months thereafter. blood samples were collected at entry and every 4 weeks thereafter. most cird cases were observed in autumn and winter. most dogs were healthy at arrival and were in the kennel for at least 2 weeks before developing clinical signs. seroconversion to chv was detected throughout the year. the most logical explanation for the seroconversion pattern would be continuous introduction in the kennel by acutely infected dogs or reactivation of latent virus in the resident population. the authors concluded that while chv contributed to the cird, it was not an obligate pathogen in that environment, since some asymptomatic dogs also seroconverted. kawakami and colleagues 6 described an outbreak of infectious tracheobronchitis in japan accompanied by death in adult dogs. the only pathogen identified during the outbreak was chv. molecular testing led to the conclusion that a single strain was involved, with virulence characteristics that were only slightly higher than those of previously tested chv strains. as was the case in the study reported by erles and colleagues, 18 it was not clear whether the virus was introduced into the center in the form of acute infections or was the result of reactivation of latent infections in the resident population. regardless, the authors emphasized that there was sufficient amounts of immunosuppression in shelter populations to allow for chv to be a significant primary pathogen in that environment. ocular manifestations of chv infection may develop during both primary and recurrent infection and are dependent upon host age and immune status. in fetal and neonatal dogs with primary chv infection, severe intraocular lesions are frequently present concurrent with systemic viral disease. subclinical or mild recurrent ocular surface disease is typically observed in immunocompetent mature dogs. in immunosuppressed mature dogs, ocular lesions associated with chv infected are often more severe, persist for a longer duration, and may be refractory to treatment. primary chv infection occurring after in utero or early neonatal chv transmission (ie, first 2 to 3 weeks of life) is associated with a cell-associated viremia. hematogeneous dissemination of virus results in chv infection of intraocular tissues with severe clinical ocular manifestations. ocular disease is typically bilateral and becomes evident within a short period after the development of systemic disease in many, but not all, dogs. 1,3 panuveitis, retinitis, and optic neuritis with extensive monocular and neutrophilic infiltrates, edema, hemorrhage, and necrosis are observed histopathologically within the iris, ciliary body, choroid, retina, and optic nerve. 42 intranuclear viral inclusions are frequently detected during the acute inflammatory phase in uveal and retinal tissues. as the palpebral fissures do not open until 10 to 14 days postpartum in dogs, ocular changes may not be externally visible in young animals. in dogs with open eyelids, most clinically detectable ocular lesions are sequelae to panuveitis and include keratitis, corneal edema, aqueous flare, anterior synechiae, cataracts, and chorioretinitis ( fig. 4) . 42 reduced vision or blindness may result from various combinations of the ocular lesions. following the acute inflammatory stage of infection, developmentally mature tissues (eg, cornea, uvea) undergo varying degrees of necrosis, fibrosis, gliosis, and atrophy. 42 the canine retina is incompletely developed at birth and responds by a combination of necrosis, disorganization, and reorganization. retinal dysplasia, characterized by formation of retinal folds with rosette-like structures, and retinal degeneration are the final result. in dogs surviving neonatal chv infection, blindness, cataracts, optic nerve atrophy, retinal degeneration, and retinal dysplasia are frequent residual sequelae. 43 in contrast to fetal and neonatal dogs, ocular lesions associated with chv infection in mature dogs are typically restricted to the ocular surface with a variety of corneal, conjunctival, and eyelid lesions. 44 in immunocompetent dogs these lesions are frequently mild and self-limiting; however, they are a source of discomfort and their recurrent nature may be frustrating to clients. nonspecific clinical signs associated with chv ocular infection in mature dogs include blepharospasm, photophobia, and ocular discharge. blepharospasm and ocular pain are often disproportionally severe compared to that expected from the extent of ocular lesions. ocular discharge is initially restricted to epiphora, but becomes mucoid, mucopurulent, or serosanguineous with progression of infection. 7, 44 primary and recurrent ocular chv infection may be subclinical or associated with various combinations of blepharitis, conjunctivitis, keratitis, and corneal ulceration. 7,44 -46 in all published descriptions of naturally-acquired primary ocular chv infection, clinical lesions were bilateral; however, the severity and specific manifestations of chv infection were not always symmetrical between eyes of individual dogs. in most cases, primary ocular chv infection resolves spontaneously and without permanent ocular lesions; however, recovered dogs are at risk for developing recrudescent ocular disease associated with reactivation of latent chv. recrudescent chv ocular disease may present with either unilateral or bilateral lesions. recurrent chv ocular infection may occur in dogs with no identifiable risk factors; however, an immunocompromise state is present in most dogs. 7,44 naturally acquired recurrent chv ocular infection is reported in dogs with a variety of immunomodulating systemic conditions and receiving a variety of immunosuppressive therapeutics. systemic conditions included diabetes mellitus, immune-mediated thrombocytopenia, and lymphoma. immunosuppressive therapeutics included topical ocular corticosteroids, topical ocular cyclosporine, systemic corticosteroids, and a variety of antineoplastic chemotherapeutics (eg, cyclophosphamide, doxorubicin, vincristine). in many reported dogs, potentially immunosuppressive conditions were concurrently present with the administration of multiple topical and systemic immunosuppressive medications. blepharitis is occasionally present with ocular chv and may appear as focal or generalized eyelid erythema, edema, exudates, and crusting. regions of alopecia may be present. the blepharitis may represent self-trauma resulting from discomfort associated with conjunctival or corneal disease, or active viral infection of eyelid cutaneous epithelium as described for other dermal regions in dogs with chv infection. 46 conjunctivitis is the most frequently reported ocular lesion associated with both primary and recurrent chv infection 44, 47 and can be presented with conjunctival hyperemia, chemosis, and ocular discharge. ulceration of the conjunctival epithelium may occur and appears as flat, irregular, pale or pink regions on the conjunctival surface surrounded by regions of hyperemia. conjunctival ulcerations are readily detected with application of sodium fluorescein, rose bengal, or lissamine green stains. although the clinical features of chv conjunctivitis are often indistinguishable from other etiologies, conjunctival petechiae are frequently reported in dogs with chv infection (fig. 5) . 9, 44, 47 although not specific to chv infection, this clinical finding is uncommon with most other etiologies of conjunctivitis and should be considered suggestive of chv. ulcerative keratitis and nonulcerative keratitis are frequent lesions associated with primary and recurrent ocular chv infection. 7, 8, 47 a variety of clinical manifestations are observed in the cornea associated with chv infection and these likely represent a continuum along the progression of active corneal epithelial infection. punctate keratitis is the earliest detectable chv corneal ulceration and appears clinically as a fine stippling of epithelial loss. this subtle lesion is often clinically overlooked when examination is performed without the aid of magnification, but application of corneal vital stains (particularly rose bengal or lissamine green) facilitate detection. 47 as punctate ulcerations progress, they form the classic alphaherpesvirus corneal lesion of dendritic corneal ulcers. dendritic corneal ulcerations are strongly suggestive of chv infection in the dog. these linear, branching ulcers stain brightly with sodium fluorescein, rose bengal, and lissamine green (fig. 6) . 7, 47 prominent terminal end bulbs are a consistent feature of chv dendritic ulcers in the dog and can be used to differentiate chv corneal lesions from other potential causes of linear corneal ulcers that might appear clinically similar (eg, external trauma, cilia abnormalities, entropion). terminal end bulbs are club-shaped, rounded ends to the chv dendritic ulcer branches, and are not seen with other causes of linear corneal ulcers. coalescence of dendritic ulcerations may result in the formation of geographic corneal ulcers. 47 these appear as larger, irregular-shaped areas of corneal epithelial loss. in dogs with chv ulcerative keratitis, corneal ulcers are commonly located in discrete groups or linear arrangements on the corneal surface. unless complicated by secondary bacterial infection, chv corneal ulcers remain superficial and corneal stromal loss is not appreciable. nonulcerative keratitis is a less frequent lesion reported with chv ocular infection. 47 clinically, nonulcerative keratitis appears as a circumferential ring of cornea stromal neovascularization with epithelial and subepithelial leukocyte infiltrates in the peripheral cornea. nonulcerative keratitis may represent a resolution stage of active corneal epithelial disease. the largest published case series of primary chv ocular disease described an outbreak of chv infection a closed colony of young adult laboratory beagles. 47 in this group of 27 dogs, conjunctivitis was detected in 100% of dogs, ulcerative keratitis in 26% of dogs, and nonulcerative keratitis in 19% of dogs. corneal ulcerations were further subclassified by clinical appearance as punctate (7% of dogs), dendritic (19% of dogs), and geographic (4% of dogs). this report confirmed chv-associated ocular disease in group housed susceptible dogs, and provides an overview of the spectrum and relative frequency of ocular lesions associated with primary ocular chv infection in dogs. under experimental conditions, acquisition of primary chv infection by ocular surface inoculation consistently produces self-limiting conjunctivitis in immunocompetent mature dogs. 29, 46 this route of infection likely occurs frequently under natural conditions and has direct clinical relevance. 47 viral inoculation by other anatomic routes, such as the genital tract, is associated with inconsistent development of ocular disease. 48 clinical signs were manifested in both eyes, even when viral inoculation was unilateral, but the magnitude of conjunctivitis may not be symmetric between eyes. the clinical severity of ocular lesions peak approximately 7 to 10 days after infection and lesions slowly resolve over the following 2 weeks. histopathologic findings in dogs with acute experimental chv conjunctivitis include conjunctival epithelial necrosis, subepithelial lymphocyte and macrophage infiltration, and edema of the substantia propria. 28, 29 experimental induction of recurrent ocular chv infection was demonstrated by administering immunosuppressive dosages of systemic corticosteroids to latently infected dogs recovered from primary chv ocular infection. 8 recrudescent chv ocular disease was detected in 83% of immunosuppressed dogs in one study. 8 bilateral conjunctivitis or linear corneal ulcers developed as early as 3 days after initiating corticosteroid administration. the mean duration of detectable ocular disease was 8.6 days and was shorter than the experimental primary ocular chv infection in the dogs. cellular lesions observed by in vivo confocal microscopy in the dogs included conjunctival leukocyte infiltrates, corneal leukocyte infiltrates, abnormal corneal epithelial cell morphologies, and corneal langerhans cell infiltrates. subsequent research determined topical ocular corticosteroid administration does not result in recurrent chv ocular disease in latently infected dogs under experimental conditions. 15 in this study, topical ophthalmic prednisolone acetate (1.0% suspension) was administered 4 times daily for 28 days to both eyes of dogs with experimentally induced latent chv infection. viral shedding and recurrent chv ocular disease were not detected; however, crystalline corneal opacities developed in some dogs. these bilateral corneal lesions appeared clinically as subepithelial and anterior stromal punctate, white, refractile opacities within the central cornea. it was unclear if the crystalline corneal opacities were a nonspecific result of corticosteroid administration or influenced by prior chv corneal disease. in immunocompromised dogs, such as lymphoma who are receiving chemotherapy or dogs with autoimmune systemic disorders receiving long-term immunosuppressive therapy, relatively severe ocular lesions may develop during recurrent chv infection. 7, 9 these lesions include severe ulcerative conjunctivitis and extensive corneal ulceration that is refractory to treatment. development of viremia, systemic chv dissemination, and visceral hemorrhagic necrosis, similar to what is typically observed in fetal and neonatal dogs, has been reported in a mature dog with ocular chv infection while receiving chemotherapy for lymphoma. 9 in the reported dog, it was speculated that viremia and systemic chv disease developed secondary to localized ocular chv reactivation with an insufficient immune response to contain virus to the anatomic site of recurrent disease. recent evidence suggests chv ocular diseases in mature dogs are clinically underappreciated. a survey of dogs with idiopathic conjunctivitis determined chv was the most common viral etiology of conjunctivitis in mature, vaccinated dogs and was detected in ocular samples from approximately 17% of study dogs. 44 conjunctivitis is among the most common ocular diseases in dogs presented to veterinarians and, if these results are extrapolated to the general canine population, it implies chv ocular diseases occur commonly. 49 to determine the sites of latency of chv, miyoshi and colleagues 16 experimentally inoculated adult seronegative dogs via the intranasal (n ϭ 2), intranasal and intravenous (n ϭ 3), or intravaginal (n ϭ 3) routes with a strain of chv. although clinical signs were not observed, infectious virus was isolated from swabs until 4 to 6 days postinoculation. tissues were collected 2 to 4 months postinoculation and examined for the presence of latent viral dna. it was determined that the trigeminal ganglion (tg) was an important latency site for chv, regardless of the inoculation route. latency was detected also in lumbosacral ganglia of 2 of 3 dogs inoculated intravaginally, 1 of 2 dogs inoculated intranasally, and 1 of 3 dogs inoculated both intranasally and intravenously. abortion and stillbirths could also be associated with reactivation of latent chv, but the mechanism by which this takes place has not been investigated. retropharyngeal lymph nodes were another important latency site, since latency was detected in this tissue in 7 of 8 dogs. conversely, all attempts to demonstrate latency in peripheral blood lymphoid cells were negative. in humans, herpesviruses have been detected in the inner ear and are considered to play a role in vestibular dysfunction. parzefall and colleagues 50 reported on the prevalence of canine herpesvirus dna in the vestibular ganglia (vg) and vestibular labyrinth (vl) of 52 dogs that were included in their study. chv dna was detected in the vl of 17% of the dogs and in the vg of 19% of the dogs. although no attempt was made to differentiate between acute and latent infection, it is very likely that the pcr was detecting latent virus. interestingly, infection of the vg or vl was not always associated with infection of tg. since the vg, in contrast to the trigeminal and geniculate ganglia, do not have direct connection with sensory nerve endings on body surfaces, it remains most probable that there was primary infection of the tg or geniculate ganglia, with subsequent spread to the vg. burr and colleagues 13 examined tissues from 12 adult dogs that had been euthanized for various reasons. from each dog 12 tissues that have been associated with latency in other herpesvirus infections were examined. viral dna was detected in the organs of 9 of the 12 dogs. the tissues most commonly found to be positive were lumbosacral ganglia, tonsil, parotid salivary gland, and liver. based on the data, lumbosacral ganglia are an important site of latency and potential source of reactivated virus for venereal infections and infection of pups as they pass through the birth canal. finding of latent virus in tonsils and salivary glands points to the role of oronasal spread in the transmission of chv. it was noted that viral dna was detected in the trigeminal ganglia extracts of only 2 of the dogs. none of the 12 blood samples tested were found to be positive, indicting a lack of detectable viremia. the authors commented that chv is either totally absent from peripheral blood or that the level of infection is limited to 1 genomic copy per 2000 mononuclear cells. they also pointed out that basing the incidence of chv infection on serology only may lead to an underestimation of the true infection rate. the difficulty in detecting circulating chv in a kennel situation is highlighted in a study by ronsse and colleagues. 11 dogs in a breeding facility were followed for the duration of 1 reproductive cycle. a number of dogs seroconverted (negative to positive) to chv during this period. conversely, antibody-positive dogs became seronegative. the serologic data clearly indicate that chv was circulating in this kennel in the form of acute and/or reactivated form, primary infections. however, despite the fact that samples were taken at regular intervals, the results of pcr testing with a previously validated assay were uniformly negative both on all nasal and vaginal swabs and buffy coat samples. a possible explanation is that the shedding interval after reactivation is very short. even during acute infection, shedding of chv is limited to 2 to 6 days. latent chv has been reactivated by treatment with corticosteroids. okuda and colleagues 51 treated dams with a history of chv infection with 600 mg of prednisolone for 5 consecutive days. reactivation of latent chv infection was confirmed in 4 of 5 dams. infectious chv was recovered from nasal, oral, vaginal, and ocular secretions on the 5th to 21st days after initiation of treatment and also from nasal mucosa and tonsil tissues. these results indicate that latent chv infections develop frequently and that the latent virus may be reactivated, without clinical signs, in dogs with a history of chv infection. ledbetter and colleagues 8 investigated whether systemic administration of an immunosuppressive regimen of corticosteroids (3 mg/kg/day for 7 consecutive days) to experimental adult dogs would lead to reactivation and recrudescence. group1 dogs were latently infected and received corticosteroid treatment. group 2 dogs were latently infected and received a placebo. group 3 dogs were control dogs and received corticosteroid treatment. bilateral ocular disease, consisting of conjunctivitis and keratitis, was seen in 83% of the group 1 dogs between days 3 and 18 of the experiment. ocular shedding was detected in 50% of the group 1 dogs, and a 4-fold rise in antibody titer was detected in all dogs in group 1. none of the dogs in the control groups showed ocular disease, shed virus, or seroconverted. corticosteroidinduced reactivation is likely the result of enhanced expression of both viral and cellular genes. corticosteroid also lead to host immune response suppression, as discussed by the authors, the immunosuppression could be involved directly in the reactivation event, or indirectly in facilitating the spread of reactivated virus to peripheral tissues, leading to renewed replication at peripheral mucosal sites and potential transmission to susceptible animals that are in contact with the animal in which reactivation takes place. ledbetter and colleagues 15 also administered topical ocular prednisolone acetate or a placebo to mature dogs experimentally inoculated with chv via the ocular route and previously tested for reactivatable latency by systemic administration of an immunosuppressive dose of corticosteroids. the dogs were treated 4 times daily for a total of 28 days. the results of this study showed that topical ocular prednisolone at the concentration and treatment regimen used did not result in detectable reactivation of chv latency, based on a combination of recrudescent clinical signs, confocal microscopy findings, ocular infectious virus shedding, real-time pcr findings, and serologic response. a potential explanation for the data is that the concentration of topically administered corticosteroid that is absorbed systemically is insufficient to induce reactivation. malone and colleagues described a disseminated chv infection, which led to euthanasia, in an adult dog. 9 the dog had undergone chemotherapy for the treatment of generalized lymphoma. it was not clear whether generalized infection in this case was the result of enhanced susceptibility to chv as a result of immunosuppression or whether it was due to reactivation of a preexisting latent chv infection in this dog. amplification of target sequences by pcr method is currently the most common and most sensitive molecular diagnostic approach to the detection of chv in natural or experimentally infected animals. the pcr assays described initially were gel based, implying that the amplified products are visualized by uv illumination of ethidium bromide-stained agarose gels. miyoshi and colleagues 16 combined a nested pcr with southern blotting and showed that the detection limit of this combination was equivalent to 1 tcid 50 . schultze and baumgärtner 17 described nested gel-based pcr and in situ hybridization assays to diagnose acute chv infection in formalin-fixed paraffin-embedded tissues of 1-to 3-week-old puppies that were naturally infected. the specificity of the pcr products was confirmed by restriction endonuclease digestion. viral dna was detected in a variety of cell types, such as bronchiolar and alveolar epithelial cells, hepatocytes, renal tubular epithelial cells, neurons, fibrocytes, cardiac myocytes, and endothelial cells. this is in accordance with the previously described "pantropism" of chv. when paraffin-embedded tissues are used for pcr, it has to be kept in mind that the quality of the dna can be affected by several factors, such as the length of time between tissue removal and fixation, the presence of nucleases in the tissue, and the length of storage of the paraffin blocks. burr and colleagues 13 developed a gel-based pcr for chv and used it in conjunction with southern blotting to confirm the authenticity of the amplicons. they also assessed the pcr compatibility of each sample for chv pcr by first verifying that primers specific for a portion of the canine pancreatic lipase gene-amplified their target in each of the tissue extracts. the assay was capable of detecting approximately 14 genomic copies spiked into 1 g of placental dna and approximately 3500 copies when spiked into 0.2 ml of blood. erles and colleagues 18 described a gel-based pcr targeting a 494 -base pair region of a gene homologous to hsv-1 ul 37. reubel and colleagues 52 described a nested pcr that had a sensitivity that was 100 times higher than virus isolation. ronsse and colleagues 22 described the use of 2 pcr assays for chv. one of these assays had a sensitivity of 0.01 ccid 50 . the most sensitive and specific method currently available to detect chv dna is probe-based real-time pcr. a fluorogenic real-time pcr assay was described by reubel and colleagues 52 and reported to have a detection limit of 10 copies of viral dna. the first probe-based multiplex real-time pcr for chv was reported by ledbetter and colleagues. 8 very recently, decaro and colleagues 14 reported the development and complete validation of a probe-based real-time quantitative pcr for the detection and quantitation of chv dna in clinical samples. the assay was found to be very sensitive, since it could detect as few as 10 copies of the target per sample. in comparison with the gel-based pcr assay described by schulze and baumgärtner, 17 which was used in parallel, this assay has a 10-fold lower detection limit. specificity for chv was very high, as determined by lack of amplification of other canine viruses. the dynamic range was validated by successful amplification of a number of chv-positive samples from different geographic locations. reproducibility of the assay was determined by determining both intra-assay and interassay variability between the results obtained with samples containing variable amounts of target dna. both intra-assay and interassay variability, expressed as a coefficient of variation, were fairly low, were dependent on the target concentration, and were found to increase with decreasing target copy numbers. a potential pitfall of pcr assays is that the sample contains substances that are inhibiting the reaction, thus potentially leading to false-negative results. to control for this possibility, an internal control construct was spiked into each sample at known quantity and co-amplified. this way, any inhibition would be readily detectable from a decrease in the expected signal resulting from the amplification of this internal control. a relatively simple way to avoid inhibition was to prepare a 10-fold dilution of the sample. since it allows absolute quantitation, the assay was used to determine viral loads in tissues of pups that had died from acute infection and a vaginal swab collected from the dam. the viral load in the vaginal swab was 1.57 ϫ 10 3 copies/10 l. the highest viral load in tissues was 5.76 ϫ 10 9 copies/10 l, present in kidney homogenates. the authors concluded that, since it quantitates copy numbers over a wide range, this assay will be very useful not only for diagnostic purposed, but also for future pathogenesis studies and for the testing of the effect of antivirals on the replication of chv. the phrase "carrier animal" has been used extensively to describe an animal that harbors an infectious agent beyond the usual time allowed for the incubation phase of the infection and the acute and convalescent phases of clinical disease. 53 when it comes to the herpesviruses this is problematic since there are at least 2 phases that exceed those previously mentioned and are characterized by latency and exacerbation of clinical symptoms from latency. according to povey, a carrier animal may or may not shed virus in excretions or secretions, and shedding may occur continuously or intermittently. 53 as was noted in the preceding section, latency in its strict definition is the lack of viral transcription and translation, so no mature virus is being produced. a latently infected dog with chv would be defined as a carrier dog that is not shedding virus and would not be contagious to in-contact, susceptible dogs. exacerbation of the latent state to a replicative state would result in virus replication and shedding. the dog may have mild to severe clinical symptoms during this exacerbation phase. primary, systemic neonatal chv infection is associated with extensive viral shedding from numerous anatomic sites. high chv viral titers are detected in respiratory secretions, ocular discharge, saliva, and urine and on many mucosal surfaces (eg, genital, nasal, ocular, oral, pharyngeal, rectal, tracheal 4, 28 ). viral shedding may persist for up to 3 weeks in dogs that survive neonatal infection. viral shedding from infected neonates may serve to spread chv, either through direct contact or fomites, to littermates and other dogs. primary and recurrent chv infection in mature dogs is associated with mucosal viral shedding that it detectable by pcr assay or virus isolation. the duration and anatomic site of shedding vary markedly between dogs and infection episodes in individual animals. canine herpesvirus-1 shedding often occurs from multiple mucosal surfaces simultaneously and may be detected at sites anatomically distant to regions of overt clinical disease. reports of experimentally induced primary and recurrent chv infection suggest viral shedding during primary infection is prolonged and associated with higher viral titers than recurrent infection. 8, 45, 51, 54 there is an individual dog susceptibility to chv reactivation and shedding. latent chv infection can be reactivated, with induction of viral shedding, by short durations of corticosteroid administration in some dogs; however, other dogs are resistant to corticosteroidinduced viral reactivation. 8, 51, 54 when naturally infected mature bitches that previously aborted chv-infected pups where experimentally immunosuppressed by a 5-day course of systemic corticosteroid administration, chv was shed from the nasal, oral, ocular, and vaginal mucosa. 51 viral shedding could not be induced in all dogs. viral shedding was detected by virus isolation as early as 5 days, and as late as 20 days, after initiating corticosteroid administration. the duration of detected chv shedding ranged from 1 to 7 days in individual dogs. in a similar study 54 using 3-month-and 2-year-old dogs experimentally infected with chv by nasal and intravenous routes, chv reactivation and mucosal viral shedding were repeatedly induced by systemic corticosteroid administration. primary oronasal infection was associated with nasal chv shedding of approximately 2 weeks' duration. following recovery from primary infection, systemic corticosteroid administration induced viral shedding from the nasal, oropharyngeal, and genital mucosa. the onset of detectable shedding was between 5 and 9 days after initiating corticosteroid treatment and persisted for up to 32 days with marked variation between individual dogs. a second round of corticosteroid administration was administered 3 months later and again resulted in viral shedding in some, but not all, dogs. the duration of viral shedding was shorter in all dogs during the second experimental reactivation and was associated with a tendency for lower viral titers. in studies examining ocular chv infection, a similar pattern of viral shedding is reported. experimental primary ocular chv infection in mature dogs produced by direct ocular surface inoculation resulted in conjunctival viral shedding that persisted for 10 days after inoculation. 46 virus was detected in conjunctival samples by virus isolation and chv pcr, and viral titers peaked 5 days postinoculation. chv was inoculated into a single eye, but viral shedding was detected bilaterally in some dogs. following recovery from primary ocular infection, viral shedding was not detected over the subsequent 8 months. experimental recurrent ocular chv infection induced by systemic corticosteroid administration to dogs recovered from primary ocular infection again resulted in viral shedding. 8 ocular chv shedding was detected by pcr assay in 50% of dogs between 10 and 13 days after administering the first dose of corticosteroid. in comparison to primary ocular chv infection, ocular viral shedding associated with recurrent infection was briefer and viral titers in samples were lower. experimental primary chv genital mucositis in mature dogs, produced by intravaginal and intrapreputial chv inoculation, resulted in genital viral shedding that was detected by virus isolation for up to 20 days. 46 several dogs also developed nasal, pharyngeal, and conjunctival viral shedding during this period. canine herpesvirus tracheobronchitis induced by intranasal viral inoculation was associated with viral shedding for up to 18 days. 2 in the dogs with chv upper respiratory tract infection, viral shedding from the nasal mucosa was detected by virus isolation in all dogs and a some had concurrent tracheal and rectal viral shedding. the clinical ecology and epidemiology of chv can be summarized in table 3 . it basically starts with a series of questions that inquire into the status of the virus, the host, and the environment with which both are localized. 55 the critical question is whether chv infection and disease are of economic concern? as was mentioned earlier, the reproductive diseases associated with chv were the initial driving force behind the recognition of the economic and emotional effects upon dog owners. while the costs of chv-associated reproductive diseases have not been reported, it would be conceivable that a dam that loses an entire litter to chv would result in a loss of $10,000, since multiple puppies are involved. in cases of respiratory disease and ocular disease, the costs of treatment and long-term care of recurrent infections may exceed $1000 per case. the second question pertains to the zoonotic or public health risks associated with chv infections. the virus is species specific and there is no evidence to support its involvement in human disease. 26 the third question is the key to the persistence of chv in the canine population-where is the virus when not causing disease? this has been a key factor in understanding the virus and controlling it. the virus maintains itself in subclinical carrier dogs by way of latency. it may be exacerbated throughout life by stress, which results in mild to severe clinical symptoms that most commonly affect the respiratory and ocular systems. concurrent with these clinical episodes there is shedding from excretions and secretions to susceptible dogs. the 2 most susceptible age groups are pregnant chv naïve dogs and puppies of these dams (in utero, postnatal). the fourth question revolves around the epidemiology of chv once its infection occurs in the susceptible dog. the course of the infection to disease is variable and has been reviewed earlier under the contemporary clinical observations. one important aspect to reiterate here is the importance of immunity in controlling the infection-disease process in pregnant dams and their offspring during the postnatal period. early postnatal infection (3 weeks or less) results in high morbidity accompanied by high mortality. 34 later infection (3 weeks or later) results in low morbidity and very low mortality. however, it is usually the later postnatal infection that establishes the lifelong carrier state via latency. the fifth question is a natural extension of the sequence of clinical inquiry and addresses the control of chv, so that infection is minimized during diseasesusceptible periods and maximized during disease-resistant periods. as noted previously, shedding states are important in maintaining the virus infection on the population to attain a certain degree of population immunity. knowing when dogs are potentially contagious, and maintaining the 6-week barrier to infection, allows for maximum protection during this susceptible period. since there is no reliable vaccine available, kennel hygiene and biosecurity are essential. 34 therapy for neonatal chv infection is largely supportive and carries a poor prognosis for survival once clinical disease is manifested. 23 in instances where dogs survive neonatal chv infection, cardiac, neurologic, and ocular lesions may be permanent. elevating the environmental temperature of dogs in a litter after chv infection is diagnosed may provide some protection to uninfected pups. viral replication is reduced at elevated body temperatures and there are lower morbidity and mortality rates in dogs that are subsequently infected; however, this is ineffective for individual dogs if implemented after viral infection. 23 intraperitoneal injection of immune sera obtained from chv-seropositive dogs is described as a method to reduce mortality in an exposed litter, but it must be administered prior to infection to be most effective. 26 lactoferrin possesses in vitro antiviral activity against chv and inhibits cellular infection. 56 administration of lactoferrin to dogs at risk for infection could theoretically provide protection; however, this is not demonstrated in vivo. isolated reports of apparently successful therapy of neonatal chv infection with the antivirals vidarabine and acyclovir are described. acyclovir was administered orally as a 10-mg total dose per dog at 6-hour intervals until 3.5 weeks of age. 26 the pharmacokinetics and tissue distribution of intravenous, subcutaneous, and oral acyclovir were investigated in dogs. 57, 58 additionally, a sustained release buccal tablet form of acyclovir was evaluated in the dog. 59 acyclovir is bioavailable when administered orally to dogs and is widely distributed within tissues; however, target plasma concentrations and effective dosages for chv infection are currently unknown. [57] [58] [59] acyclovir toxicosis resultant from accidental ingestion is reported in dogs with dosages as low as 40 mg/kg and the routine clinical use of this, and other systemic antiviral medications, in dogs for chv infection requires further investigation of safety and efficacy. 60 the canine pharmacokinetics of newer-generation antiherpesviral drugs, including famciclovir, are reported. similar to acyclovir, safe and effective doses for dogs with chv infection are undetermined. 61 treatment of respiratory and genital chv infection is primarily symptomatic. unless complicated by secondary bacterial infection, these conditions are typically selflimiting and specific antiviral therapy is not reported. in contrast to respiratory and genital infection, there are detailed reports of the successful clinical management of ocular chv infection. in addition to nonspecific treatments to prevent secondary bacterial infection (topical ocular antimicrobials) and improve comfort (topical ocular atropine), antiviral therapy with 0.1% idoxuridine or 1% trifluridine ophthalmic solution was used. idoxuridine and trifluridine are nucleoside analogues, possess good anti-herpesvirus activity, and are well tolerated by dogs when applied topically as ocular formulations. trifluridine is available under the trade name viroptic, and idoxuridine can be acquired from compounding pharmacies. both antivirals are administered 6 to 8 times daily for the first 48 hours and then 4 times daily until resolution of clinical signs of active infection. cidofovir 0.5% ophthalmic solution is an alternative ophthalmic antiviral for chv ocular disease that is effective with twice daily administration (e.c. ledbetter, unpublished data, 2011) . this review has documented well that our level of clinical inquiry expands as our knowledge base about chv increases. while earlier studies focused on the reproductive effects of chv in susceptible pregnant dogs and neonatal puppies, it has become apparent that in order to control chv-related diseases that we must understand the various forms of chv infection that may occur in the dog population (fig. 7) . this has prompted the veterinary community to develop more sensitive diagnostic assays, such as pcr, in order to answer the questions, where is the virus when not causing disease, and what is its relationship with respiratory and ocular diseases in adolescent and adult dogs (1 year or older)? molecular and serologic studies have clearly demonstrated that we are dealing with an infection that is more common than we considered a decade ago. reports have indicated that up to 70% of some high-risk dog populations have been infected with and are latent carriers of chv. this is important for veterinarians to know as we confer with clients on the best management steps we can take to protect our at-risk populations. while pregnant chv-naïve dams and neonatal puppies born from a chv-naïve dam are considered at high risk for disease, we must also take into consideration dogs in kennels and rescue centers. it is these dogs that are at risk for either exposure-infection or stress-induced exacerbation of latent chv, which had been acquired at an earlier age. the manifestations of chv in adolescent and mature dogs may range from subclinical to severe respiratory and/or ocular diseases. the reports by malone and colleagues, 9 gadsden and colleagues (submitted, 2011), and ledbetter and colleagues 7,47 all indicate that chv can cause disease in older dogs and that it is not just a "puppy disease." recognition of the various forms of chv-induced disease, availability of diagnostic assays with increased sensitivity, and the formation of reliable biosecurity measures will allow for better control steps to be taken in dogs at-risk for infection and disease. clinical and pathologic features of a fatal viral disease of newborn pups experimental production of canine tracheobronchitis (kennel cough) with canine herpesvirus isolated from naturally 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response bsava manual of canine and feline reproduction and neonatology. gloucester (uk): british small animal veterinary association small animal pediatrics. the first 12 months of life infectious diseases of the dog and cat the role of neonatal canine herpesvirus infection in mixed infections in older dogs pathogenesis of canine herpesvirus in specific-pathogen-free dogs; 5-to 12-week-old pups the susceptibility of six-week of puppies to canine herpes virus canine respiratory viruses canine infectious tracheobronchitis canine herpesvirus respiratory infection canine herpesvirus infection: update on risk factors and control measures small animal pediatrics. the first 12 months of life protection of puppies against canine herpesvirus by vaccination of the dams reproductive effects of canine herpesvirus canine adenoviruses and herpesvirus herpesviruses of carnivores canine herpesvirus genomic analysis of a pneumovirus isolated from dogs with acute respiratory disease nucleotide sequence of glycoprotein genes b,c,d,g,h and i, the thymidine kinase and protein kinases and gene homologue ul24 of an australian isolate of canine herpesvirus canine herpes-induced retinal dysplasia and associated ocular anomalies lesions in puppies surviving infection with canine herpesvirus virologic survey of dogs with naturally acquired idiopathic conjunctivitis an atypical presentation for the first isolation of canid herpesvirus-1 in argentina experimental primary ocular canine herpesvirus-1 infection in adult dogs outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony genital disease in dogs caused by canine herpesvirus health status and population characteristics of dogs and cats examined at private veterinary practices in the united states naturally-occurring canine herpesvirus-1 infection of the vestibular labyrinth and ganglion of dogs virus reactivation in bitches with a medical history of herpesvirus infection experimental infection of european red foxes (vulpes vulpes) with canine herpesvirus persistent viral infection. the carrier state repeated canine herpesvirus (chv) reactivation in dogs by an immunosuppressive drug diagnostic medicine: the challenge of differentiating infection from disease and making sense for the veterinary clinician antiviral activity of lactoferrin against canine herpesvirus pharmacokinetics and bioavailability of acyclovir in the dog the disposition of acyclovir in different species a sustained release dosage form of acyclovir for buccal application: an experimental study in dogs accidental ingestion of acyclovir in dogs: 105 reports metabolic and pharmacokinetic studies following oral administration of famciclovir to the rat and dog the authors would like to acknowledge those clinicians and veterinary researchers who provided insights and recommendations for our understanding of chv pathogenesis and the management of chv; these include dr l. carmichael, dr m. appel, dr j. gorham, dr r. ott, dr a. hashimoto, dr a. sears, and dr m. spector. the technical support of a. mckeirnan and l. tanaka is greatly appreciated. the assistance of t. pfaff in preparing the word document was essential. this manuscript is dedicated to all the men and women who serve as dog handlers in roles of community protection, rescue operations, guide dogs, and national defense. key: cord-339759-us1spoxu authors: cornelis, i.; volk, h. a.; van ham, l.; de decker, s. title: clinical presentation, diagnostic findings and outcome in dogs diagnosed withpresumptive spinal‐only meningoen‐cephalomyelitis of unknown origin date: 2017-03-07 journal: j small anim pract doi: 10.1111/jsap.12622 sha: doc_id: 339759 cord_uid: us1spoxu objectives: to summarise clinical presentation, diagnostic findings and long‐term outcome for dogs clinically diagnosed with meningoencephalomyelitis of unknown origin affecting the spinal cord alone. methods: medical records were reviewed for dogs diagnosed with presumptive spinal‐only meningoencephalomyelitis of unknown origin between 2006 and 2015. results: 21 dogs were included; the majority presented with an acute (43%) or chronic (52%) onset of neurological signs. ambulatory paresis was the most common neurological presentation (67%). neurological examination most commonly revealed a t3‐l3 myelopathy, and spinal hyperaesthesia was a common finding (71%). a spinal cord lesion was visible in 90% of cases on magnetic resonance imaging. eighteen lesions (86%) showed parenchymal contrast enhancement and 17 lesions (81%) showed contrast enhancement of overlying meninges. all dogs were treated with immunosuppressive doses of glucocorticosteroids, sometimes combined with cytosine arabinoside. at time of data capture, 10/21 dogs (48%) had died or been euthanased because of the condition. overall median survival time was 669 days. clinical significance: meningoencephalomyelitis of unknown origin should be considered in the differential diagnosis of dogs presenting with a progressive myelopathy. magnetic resonance imaging features can possibly help to distinguish presumptive meningoencephalomyelitis of unknown origin from other more common spinal diseases. overall, long‐term survival is guarded, approximately 50% of dogs will die or be euthanased despite appropriate therapy. pure myelitis (inflammation of spinal cord parenchyma) or meningomyelitis (inflammation of spinal cord parenchyma and surrounding meninges) are rare diseases in small animals but occur most often in combination with inflammatory brain disease (tipold & stein 2010 ) . viruses [canine distemper virus (cdv), feline coronavirus], bacteria ( staphylococcus species, et al . 2009 , csebi et al . 2010 , dewey et al . 2016 . apart from infectious causes, non-infectious meningoencephalomyelitis including granulomatous meningoencephalomyelitis, pyogranulomatous meningoencephalomyelitis and steroid-responsive meningitis-arteritis (srma) are described (meric 1988 , griffin et al . 2008 , parry et al . 2009 , dewey et al . 2016 . current terminology implies that dogs clinically diagnosed with non-infectious inflammatory myelitis without positive infectious disease testing, not classified as srma or eosinophilic meningomyelitis, and not histopathologically confirmed with alternative diagnoses are categorised as having meningoencephalomyelitis of unknown origin (muo), equivalent to dogs diagnosed with meningoencephalitis of unknown origin. a clinical diagnosis of muo is typically made by a combination of clinical presentation, mr imaging of involved part of the central nervous system (brain/spinal cord), and results of cerebrospinal fluid (csf) analysis (griffin et al . 2008 ) . currently, only one study has focused specifically on the clinical presentation, diagnostic findings and outcome in dogs with meningomyelitis caused by a variety of underlying aetiologies (griffin et al . 2008 ). of 28 cases included, 15 dogs were diagnosed with muo. clinical signs were reflected by the affected spinal cord segments, and younger dogs, toy breeds, and hound breeds were suggested to be predisposed for meningomyelitis. although results of myelography, ct, and ct-myelography have been reported, little is reported about magnetic resonance imaging (mri) findings in dogs with muo of the spinal cord. the aims of this study were therefore to describe the signalment, clinical presentation, diagnostic findings, including results of mri and long-term survival in dogs diagnosed with presumptive muo of the spinal cord without concurrent clinical signs of intracranial involvement. the electronic medical database was searched between march 2006 and february 2015 for dogs diagnosed with "mua," "muo," "gme," "myelitis," "inflammatory spinal cord disease." dogs were included based on the criteria used by granger et al . ( 2010 ) , if they had (1) complete medical records available, (2) a complete neurological examination performed leading to a spinal cord localisation, (3) inflammatory csf analysis, (4) mri of the spinal cord and if (5) long-term follow-up information were available through revision of medical records or through contacting the referring veterinarian by telephone. dogs were excluded if (1) the clinical records or imaging studies were incomplete or not available for review, (2) dogs showed clinical or neurological signs of intracranial involvement at time of presentation, (3) they had a peracute onset of clinical signs that were not progressive after 12 to 24 hours, (4) they had signs of extradural or extradural/intramedullary spinal cord compression on mri and if (5) they had positive infectious disease titres or if clinical presentation, csf analysis or necropsy findings were suggestive of srma or eosinophilic meningoencephalomyelitis (>10% eosinophils in csf) (dewey et al . 2016 ) . typical clinical presentation for srma was considered to be a dog less than 2 years of age of a typical breed (boxer, beagle, bernese mountain dog, nova scotia duck tolling retriever, golden retriever, german shorthaired pointer) presenting with pyrexia and cervical hyperesthesia. csf analysis in srma typically reveals a predominantly neutrophilic pleocytosis (dewey et al . 2016 ) . dogs with histopathological confirmation of the disease [granulomatous meningo(encephalo)myelitis (gme) or necrotising meningo(encephalo)myelitis (nme) only needed to fulfil inclusion criteria (1) and (5). information retrieved from the medical records included breed, gender, age at diagnosis, body weight, results of neurological examination including neuroanatomical localisation, duration of clinical signs prior to diagnosis, results of complete blood count (cbc) and biochemistry profile, results of csf analysis including total nucleated cell count (tncc), white blood cell differentiation and total protein (tp) concentration, treatment received and outcome. duration of clinical signs prior to diagnosis (days) was classified as peracute (<2 days), acute (2 to 7 days) or chronic (>7 days). for dogs that had csf analysis performed, site of collection (cisternal or lumbar), tncc, tp and cytological differentiation were recorded. tncc was considered normal if there were <5 cells/mm 3 . protein concentration was considered normal for a cisternal collection if <0·25 g/l and for a lumbar collection if <0·4 g/l. the neurological status was classified from 0 to 5 according to the clinical examination (adapted from scott 1997 ): grade 0=neurologically normal; grade 1=spinal hyperaesthesia without neurological deficits; grade 2=ataxia, ambulatory para-or tetraparesis; grade 3=non-ambulatory para-or tetraparesis; grade 4=para-or tetraplegia with or with-out bladder control, and intact deep pain sensation; grade 5=para-or tetraplegia, urine retention or overflow, and deep pain sensation loss. possible neuroanatomical localisations included c1 to c5, c6 to t2, t3 to l3 or l4 to s3 spinal cord segments. dogs were diagnosed with a focal lesion if only one spinal cord segment was affected, and with a multifocal lesion if more than one spinal cord segment appeared to be affected on the neurological examination. magnetic resonance imaging mri was performed under general anaesthesia with a permanent 1.5-t magnet (intera, philips medical systems, eindhoven, the netherlands) and all images were reviewed by the corresponding author using osirix dicom viewer (osirix foundation, v.5.5.2 geneva, switzerland). sequences varied, but included a minimum of t2-weighted (t2w) [repetition time (ms) (tr)/ echo time (ms) (te), 3000/120] and t1-weighted (t1w) (tr/ te, 400/8) images of the affected spinal cord region in a sagittal and transverse plane. the t1w images were acquired before and after intravenous (iv) administration of paramagnetic contrast medium with a dose of 0·1 mg/kg gadoterate meglumine (dotarem, guerbet). if mr images of the brain were available, they were reviewed concurrently. variables recorded were lesion intensity on t2w and t1w images, lesion localisation and distribution, lesion length and parenchymal and/or meningeal contrast enhancement. lesion length was measured using osirix dicom viewer, and performed on sagittal t2w images for dogs that had focal lesions. lesion length was measured twice, and the mean value reported. to compensate for differences in body size, values were corrected with respect to the length of vertebral body of c6 (for cervical lesions) or l2 (for thoracolumbar lesions). vertebral body length was measured on t1w sagittal images. for all dogs, the specific treatment protocol was recorded. during hospitalisation, all dogs underwent daily at least one general physical and complete neurological examination by a board-certified neurologist or neurology resident. the results of the neurological examination as well as response to treatment (improvement, deterioration or static) were systematically recorded on the kennel sheets. follow-up information during hospitalisation was collected from the medical records, and later through medical records of re-examination visits or telephone contact with the referring veterinarian. a successful outcome was defined as the dog being ambulatory, with faecal and urinary continence and, according to the owners, without overt spinal hyperaesthesia. an unsuccessful outcome was defined as (1) deterioration in neurological status by one or more grades after diagnosis and treatment or (2) if the dog was not independently ambulatory, possibly with previously non-existing or worsening faecal and/or urinary incontinence, or was experiencing spinal hyperaesthesia as defined by the owner. data analysis was performed with the aid of a standard statistical software package (prism, graphpad software inc). numeric variables were expressed as median and interquartile ranges (iqr). values of p<0·05 were considered significant. survival analysis was performed using both a log-rank (mantel-cox) and gehan-breslow-wilcoxin test, resulting in median survival time (mst) calculation and a kaplan-meier survival curve. survival was defined as time from diagnosis to death or euthanasia, including whether this happened because of disease progression or due to unrelated causes, or time from diagnosis to last follow-up for dogs that were alive at time of data capture. dogs that died because of unrelated causes and dogs that were still alive at time of data capture were censored for survival analysis. twenty-one dogs were included in the study. represented breeds included french bulldog (n=2), jack russell terrier (n=2), lhasa apso (n=2) and one each of akita, bearded collie, boxer, bull mastiff, chihuahua, cross breed, english springer spaniel, giant schnauzer, labrador retriever, maltese terrier, rhodesian ridgeback, rottweiler, shih-tzu, west highland white terrier and yorkshire terrier. overall, median age at presentation was 56 months (10 to 128 months). thirteen dogs (62%) were male and eight (38%) were female. compared to the general hospital population between march 2006 and february 2015, there was no difference in sex distribution in the group of dogs with muo (fisher ' s exact test; p=0·075). median duration of clinical signs prior to diagnosis was eight days (ranging from 1 to 90 days). one dog (5%) presented with peracute, nine dogs (43%) with acute and eleven dogs (52%) with a chronic onset of neurological signs. thirteen (62%) and eight (38%) dogs were diagnosed with a focal and multifocal spinal lesion on neurological examination, respectively. for dogs with focal spinal lesions (n=13), three were diagnosed with a lesion affecting the c1 to c5 spinal cord segments, two with a lesion affecting the c6 to t2 spinal cord segments, six with a lesion affecting the t3 to l3 spinal cord segments and two with a lesion affecting the l4 to s3 spinal cord segments. at time of diagnosis, no dogs presented as grade 0; 2 dogs (10%) were grade 1; 14 (67%) grade 2 and 5 (24%) grade 3. no dogs were paraplegic or tetraplegic at presentation. pain on direct spinal palpation was present in 15 (71%) dogs. urinary retention was observed in two dogs (10%), and a combination of urinary and faecal incontinence was noticed in two dogs (10%). one dog (5%) developed seizures 669 days after diagnosis of muo. clinical findings of the 21 included dogs are summarised in table 1 . as required by the inclusion criteria, csf collection revealed pleocytosis in all cases. overall, median tncc was 209 cells/ mm 3 (ranging from 6 to 6000). tp measurement was performed in all but three csf samples, and was above reference values in 17/18 dogs (94%). the median tp concentration was 1·67 g/l (ranging from 0·21 to 16·3 g/l). cbc and serum biochemistry results were available in 16 dogs (76%). leucocytosis was only present in two dogs (10%) and lymphopenia was present in six dogs (29%). infectious disease testing based on serology and/or polymerase chain reaction on csf for cdv, t . gondii , and n . caninum was not performed in two (10%) dogs and was negative in the remaining 19 (90%) dogs. in the two dogs lacking infectious disease testing, full necropsy was performed, revealing gme. magnetic resonance imaging mri of the spinal cord was available in all cases, revealing a focal lesion in 15 dogs (71%), a multifocal lesion in four (19%) and no lesion was visible on sagittal t2w or t1w images in two (10%). lesion length was measured in the focal cases only. median lesion/vertebral body ratio was 4·8 (ranging from 0·6 to 10·9). all visible lesions were ill-defined, intramedullary, hyperintense on t2w images and isointense on t1w images (figs 1 and 2 ). lesions showed parenchymal contrast enhancement in 18 dogs (86%), and contrast enhancement of overlying meninges in 17 (81%). in the two cases in which no lesion was visible on sagittal t2w and t1w images, there was also no observable parenchymal contrast, but one dog only showed meningeal contrast enhancement. in two dogs (10%) intracranial images were contained within the field of view of the cervical spinal cord images (t2w transverse and sagittal images), revealing multiple t2w hyperintensities in the forebrain and/or brainstem. neither of those dogs had clinical or neurological signs of intracranial involvement at time of diagnosis. the first dog, a 56-month-old jack russell terrier, did not recover from general anaesthesia after diagnostic procedures, and full necropsy revealed gme. the second dog, a 123-month-old rhodesian ridgeback, developed seizures 669 days after diagnosis and was euthanased without further investigations. as required by the inclusion criteria, outcome was available in all dogs. as described above, one dog did not recover from general anaesthesia for mri of the spinal cord, and was censored for survival analysis. mean duration of hospitalisation was five days (ranging from 1 to 19 days), with 17 dogs (81%) showing improvement in neurological status within that period. one dog (5%) remained neurologically stable (no improvement nor deterioration), and three dogs (14%) showed deterioration of their neurological status. all dogs were treated with immunosuppressive doses of glucocorticosteroids immediately after diagnosis. this consisted doses of 0·3 to 0·5 mg/kg/day dexamethasone in nine dogs (43%) iv and 2 to 4 mg/kg/day oral prednisolone in 12 dogs (57%). fourteen dogs (67%) received additional treatment with cytosine arabinoside as a constant rate infusion (cri) of 200 mg/m 2 over eight hours in one dog (7%) and as four subcutaneous (sc) injections of 50 mg/m 2 every 12 hours for two consecutive days in 13 dogs (93%). twenty dogs (95%) survived to discharge. of these dogs, nine dogs (45%) were still alive at time of data capture. of these nine dogs, eight were neurologically normal according to the followup information and one dog still showed ataxia and ambulatory paraparesis. of the eight normal dogs, two were still receiving a dose of 5 mg/kg cyclosporine every 24 hours, one was receiving a dose of 50 mg/m 2 cytosine arabinoside every 12 hours for two consecutive days every nine weeks, one was receiving a dose of 0·2 mg/kg prednisolone every 24 hours, one was receiving doses of 1 mg/kg prednisolone every 24 hours and 50 mg/m 2 cytosine arabinoside every 12 hours for two consecutive days every four weeks, and three dogs were not receiving any treatment at time of data capture. the dog that was still showing neurological abnormalities was receiving doses of 0·5 mg/kg prednisolone every other day and 50 mg/m 2 cytosine arabinoside every 12 hours for two consecutive days every 5 weeks. for the 11/20 dogs (55%) that were dead at the time of data capture, three had died or were euthanased because of disease progression, six were euthanased because of acute neurological deterioration after initial neurological improvement and two were euthanased because of unrelated causes (complications after stifle surgery and development of aggression). dogs that showed acute neurological deterioration after initial improvement did so within a median of 171 days after diagnosis (ranging from 30 to 669 days). of those six dogs, one showed acute deterioration after discontinuation of prednisolone treatment and five were still receiving treatment doses of 1 mg/kg prednisolone every 24 hours, 0·5 mg/kg prednisolone every 24 hours, 2 mg/kg prednisolone every 24 hours and 2 mg/kg azathioprine every 24 hours or 50 mg/m 2 cytosine arabinoside every 12 hours for two consecutive days every seven weeks. overall, we can conclude that 10/21 dogs (48%) died or were euthanased because of muo. overall, the mst was 669 days (ranging from 1 to 2250 days) (fig 3 ) . confirmation from post-mortem examination was available in three dogs, revealing gme in two and necrotising meningomyelitis in one dog. all clinical data are shown in tables 1 and 2 . this study evaluated the clinical presentation, diagnostic findings and long-term survival in 21 dogs diagnosed with presumptive spinal muo. dogs had a median age of five years at time of pain on direct spinal palpation was present in 71% of dogs. spinal pain may reflect the involvement of the meninges, and/ or vertebrae (vertebral periosteum), and/or nerve roots or spinal nerves (da costa 2012 ). in the present study, the lesions showed meningeal contrast enhancement in 18/21 dogs, but there was no apparent association between spinal hyperaesthesia and meningeal enhancement on mri. mri of the spinal cord revealed no lesion on sagittal t2w and t1w images in 10% of dogs (n=2), which appears similar to the 7% described for the brain in dogs with muo (granger et al . 2010 ). in the retrospective study of griffin et al . ( 2008 ) , only one dog with meningomyelitis had mri performed, revealing no abnormalities. based on these findings, muo cannot be ruled out based on unremarkable mri findings. the first dog was a 42-month-old bullmastiff with a 1-month history of slowly progressive t3 to l3 spinal cord lesion. after diagnostic procedures, the dog was treated with oral prednisolone but continued to deteriorate and was euthanased after 6 days. no post-mortem examination was performed. the second dog was a 136-monthold bearded collie with a 1-week history of a progressive multifocal spinal cord neuroanatomical localisation (t3 to s3 spinal cord lesion). the dog showed improvement on treatment with prednisolone and cytosine arabinoside (see table 1 ) after diagnostic investigations, and was still alive without current treatment 1100 days after diagnosis. both dogs had inflammatory csf analysis (increased tncc and tp concentration). for both dogs, vascular, degenerative or neoplastic spinal cord lesions cannot be excluded. as both dogs had a progressive disease course, a vascular (ischaemic) lesion seemed less likely. a neoplastic lesion cannot be excluded, although this seems rather unlikely in the bullmastiff considering his very young age. the second dog had a lymphocytic pleocytosis on cfs analysis, but no signs of lymphoma on microscopical examination, although no specific test for clonality was performed. all mri-observed lesions were extensive, ill-defined, intramedullary, hyperintense on t2w images and isointense on t1w images. other spinal conditions, including acute non-compressive nucleus pulposus extrusions (annpe) and ischaemic myelopathy (im), are also associated with intraparenchymal hyperintensities on mri. however, these conditions are associated with other clinical and additional mri characteristics, which could potentially aid in differentiating between these conditions (cardy et al . 2015 , fenn et al . 2016 ). according to cardy et al . ( 2015 ) , in dogs presenting with spinal cord dysfunction, im [most commonly fibrocartilagenous embolic myelopathy (fcem)] and annpe are typically characterised by a peracute onset of non-progressive clinical signs and affected dogs do not commonly demonstrate overt spinal hyperaesthesia at time of admission. this is in contrast with the clinical presentation of dogs with spinal muo, which was characterised by an acute onset of progressive and mainly symmetrical neurological deficits, with pain on spinal palpation or manipulation in 86% of dogs (cardy et al . 2015 ) , which is comparable with the 71% of dogs presenting with spinal hyperaesthesia in the current study. although csf analysis in dogs with im is most often within normal limits, affected dogs can demonstrate an increased tp concentration and mild neutrophilic or mixed cell pleocytosis with a median tncc of 12 cells/ µ l (de risio et al . 2007 ) . a marked pleocytosis with a median tncc of 209 cells/mm 3 was seen in the current study, although this conclusion should be treated with caution because csf pleocytosis was one of the inclusion criteria. to conclude, the presentation of a dog with an acute or chronic onset of a progressive and painful t3 to l3 myelopathy in combination with an extensive, ill-defined, intramedullary lesion plus parenchymal and/or meningeal contrast enhancement on mri, and marked pleocytosis on csf analysis, can be presumptively diagnosed with spinal muo. the importance of differentiating between these conditions is highlighted by the differences in treatment and prognosis between dogs with presumptive muo and dogs with annpe or im. a previous study demonstrated that short tau inversion recovery (stir) hyperintensities in the cervical epaxial musculature of dogs with meningoencephalomyelitis had a sensitivity of 78% and a specificity of 92% in predicting inflammatory csf results (eminaga et al . 2013 ). in the current study, stir images were unfortunately only available in 3/21 cases. adding this sequence to the protocol in dogs with presence of a focal or multifocal, illdefined t2w intramedullary hyperintensity might be considered in the future. several studies have evaluated survival times of dogs diagnosed with muo (granger et al . 2010 , coates & jeffery 2014 . overall, dogs with muo appear to have a guarded prognosis. a large meta-analysis of dogs with muo revealed an overall reported mst of 240 to 590 days in 96 dogs treated with corticosteroids plus any other immunosuppressive protocol, compared to a mst of 28 to 357 days for 43 dogs receiving corticosteroids alone (granger et al . 2010 ). in the current study, dogs with presumptive spinal muo had a mst of 669 days (2 years), but ultimately, 48% of dogs died or were euthanased because of the disease, indicating a more guarded long-term prognosis. limitations of this study are the small sample size and retrospective character, which limited standardisation of patient assessment and treatment. although all dogs were treated with glucocorticosteroids, it cannot be excluded that specific differences in treatment may have influenced outcomes. despite including cases over a relative large period and from a busy referral hospital, only 21 dogs were found through our record search. this could indicate that spinal muo should be considered a rare disorder, which is in agreement with previous reports (cardy et al . 2015 ) , suggesting that muo represents approximately 6% of all spinal disorders in dogs. presumptive spinal muo can be diagnosed in any type of dog of any age that is presented with signs of acute or chronic, possibly painful, myelopathy. although clinical signs can vary, affected animals most typically present with ambulatory paraparesis and ataxia, localising to t3 to l3 spinal cord segments. mri typically reveals an extensive, ill-defined and intramedullary lesion that appears hyperintense on t2w images and isointense on t1w images. most lesions showed parenchymal contrast enhancement and/or enhancement of the overlying meninges on post-contrast t1w images which can possibly differentiate dogs with muo from other more common spinal diseases. in 10% of cases, no lesion was visible on sagittal t2w and t1w images. almost 50% of dogs died or were euthanased because of muo, with a mst of 669 days. none of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper. clinical reasoning in canine spinal disease: what combination of clinical information is useful? perspectives on meningoencephalomyelitis of unknown origin vertebral osteomyelitis and meningomyelitis caused by pasteurella canis in a dog -clinicopathological case report spinal pain magnetic resonance imaging findings and clinical associations in 52 dogs with suspected ischemic myelopathy neurodiagnostics . in: practical guide to canine and feline neurology stir muscle hyperintensity in the cervical muscles associated with inflammatory spinal cord disease of unknown origin inter -and intraobserver agreement for diagnosing presumptive ischemic myelopathy and acute noncompressive nucleus pulposus extrusion in dogs using magnetic resonance imaging . veterinary clinical findings and treatment of non-infectious meningoencephalomyelitis in dogs: a systematic review of 457 published cases from meningomyelitis in dogs: a retrospective review of 28 cases canine meningitis -a changing emphasis imaging diagnosis -necrotizing meningomyelitis and polyarthritis hemilaminectomy for the treatment of thoracolumbar disc disease in the dog: a follow-up study of 40 cases inflammatory diseases of the spine in small animals key: cord-278224-sq7tokbx authors: protopopova, alexandra; hall, nathaniel j.; brown, kelsea m.; andrukonis, allison s.; hekman, jessica p. title: behavioral predictors of subsequent respiratory illness signs in dogs admitted to an animal shelter date: 2019-10-23 journal: plos one doi: 10.1371/journal.pone.0224252 sha: doc_id: 278224 cord_uid: sq7tokbx individual variability is evident in behavior and physiology of animals. determining whether behavior at intake may predict subsequent illness in the animal shelter may influence the management of dogs housed at animal shelters and reduce overall disease. while normally associated with mild disease and low mortality rates, respiratory disease nevertheless poses significant challenges to the management of dogs in the stressful environment of animal shelters due to its highly infectious nature. therefore, the aim of the study was to explore whether behavior at intake can predict subsequent occurrence and progression of upper respiratory disease in dogs at animal shelters. in a correlational study, 84 dogs were assessed throughout their stay at a city animal shelter. the dogs were subjected to a behavioral assessment, 1 min in-kennel behavioral observations across two observation periods, and the collection of urinary cortisol:creatinine (c:c) ratio. the occurrence and progression of upper respiratory disease was monitored through repeated clinical exams (rectal temperature and the occurrence of nasal and ocular discharge, and presence of coughing and sneezing). a basic pls path regression model revealed that time in the shelter (estimate = .53, p < .001), and sociability (estimate = .24, p < .001) and curiosity scores (estimate = .09, p = .026) were associated with increased illness. activity and anxiety scores, however, were not associated with illness. urinary c:c, taken on the first full day, did not predict subsequent illness when accounting for time. limitations included attrition of dogs, a small percentage receiving vaccinations, and continuous and non-systematic rotation of dogs in the kennels. understanding if behavior can predict subsequent illness may improve shelter management practices, and in turn, result in improved live-release outcomes. respiratory disease, while normally associated with mild symptoms and low mortality rates, poses significant challenges to the management of dogs in the highly stressful environment of plos animal shelters due to its highly infectious nature [1] . canine infectious respiratory disease complex (cirdc), also known as kennel cough complex, is composed both of bacterial and viral agents associated with low mortality but high prevalence (canine adenovirus-2, canine parainfluenza virus, canine respiratory coronavirus, canine herpesvirus, canine influenza, mycoplasma bronchiseptica, and mycoplasma cynos) and agents associated with high mortality though much lower prevalence (canine distemper virus and streptococcus equi subsp. zooepidemicus [1] [2] [3] ). cirdc signs include ocular and nasal discharge and cough [1] . the highly infectious nature of the complex has implications for animal shelters due to the need to isolate symptomatic animals, inability to neuter infected animals, and the reluctance of potential adopters to bring infectious animals into their homes, where they may have other dogs. associated veterinary care for symptomatic animals also taxes limited shelter resources [4] . finally, canine distemper and strep zoo, though less prevalent than the others in the complex, can be associated with significant loss of life in a crowded shelter [5] . the outcome of these challenges is that many shelter dogs exhibiting signs of cirdc may be euthanized rather than placed for adoption if there are budget and/or time constraints [6, 7] . not only does the shelter provide an ideal setting for disease transmission, with dogs housed in close proximity to each other, but the stress of the shelter environment likely reduces the immune system's ability to respond to microbial challenge [1, 8] . differential susceptibility to various pathogens has been well established in young dogs and pregnant females [9] as well as individuals who are immunocompromised due to an already established disease (e.g., feline immunodeficiency virus [10] ). however, other less obvious factors, such as increased stress, may increase susceptibility in the shelter. the shelter environment presents an array of psychosocial stressors for dogs, resulting in increased activity of the hypothalamic-pituitary-adrenal (hpa) axis, as indicated by elevated cortisol levels in animals in that environment compared to animals in pet homes, at least in the first few days after admittance [11] . the hpa axis is the pathway that is responsible for the activation of the stress response in animals. an environmental stressor triggers the hypothalamus to release corticotropin-releasing hormone (crh) and arginine vasopressin, which, in turn, stimulate the production of adrenocorticotropin hormone (acth) in the anterior pituitary. the release of the latter hormone stimulates the release of corticosterone or cortisol from the adrenal cortex into the blood stream, depending on the species [12, 13] . the high levels of cortisol (or corticosterone) inhibit further production of the crh and acth resulting in a negative feedback loop (see [14] for a discussion in shelter dogs). cortisol levels negatively correlate with the levels of secretory immunoglobulin a (s-iga) in dogs [15] . yet s-iga plays a central role in the mucosal immune system, the body's primary defense against infection of the respiratory system [16] . individual variability is evident in behavior and physiology of human and non-human animals; individuals tend to cope with stressors in systematic and consistent ways [17] . correlations between behavior and physiological ability to cope with environmental stressors, such as disease and parasite infection, have been demonstrated in a wide variety of species. capitanio, medoza, and baroncelli [18] found that rhesus macaques that were high in the sociability trait showed a more rapid decrease in plasma cortisol concentrations, a higher igg response, and a lower viral load when challenged with a simian immunodeficiency virus. pigs that spent more time struggling when flipped over on their backs for a brief amount of time, have been found to have a higher concentration of cortisol and lower immune function [19] . free-roaming tom cats that were higher in aggression had a higher viral load of feline immunodeficiency virus [20] . trapped norway rats, who had a higher presence of wounds (as an indicator of aggression), also showed a higher level of hantavirus infection, and in turn, higher infection status males showed elevated serum testosterone and corticosterone concentrations, among other differences in neurotransmitters [21] . the activity-exploration profiles of siberian chipmunks, as measured by a standardized test, predicted the numbers of ticks present on the animals; tick load increased with space use [22] . more recently, the predictive effect of behavior on immune function under chronic stress conditions has been extensively explored in cattle [23, 24] . temperamental cattle, those that display shorter latency to exit and higher velocity when exiting their enclosure, have been shown to have a higher cortisol concentration and a weaker immune response to pathogens [23] . the predictive nature of behavior on immune function or disease status in dogs has not received much attention. however, early prediction of illness in a shelter environment may lead to higher life-saving through improvements in population management. to decrease overall disease within an animal shelter, experts recommend removing sick animals from the population as well as differential treatment of those that are more likely to succumb to disease [25] . thus, characterizing dogs on intake into high and low-risk categories may decrease overall incidence of disease in animal shelters as well as protect those that are more susceptible. recently, corsetti et al. [26] have suggested that dogs displaying a bold personality were less susceptible to diseases in the animal shelter. the researchers assessed 28 dogs for one month at the shelter. the behavior of the dogs was assessed using standardized personality tests, including a t-maze and a novel object test. the complex "boldness" trait was statistically derived from scores from several other tested traits (e.g., activity, attentiveness, dominance, and sociability). the aim of the study was to extend the work of corsetti et al. by exploring whether behavior at intake can predict the subsequent development of cirdc in dogs surrendered to animal shelters. adult dogs of unknown breed (n = 84; those that appeared to be approximately 1 year of age and older; 61% male) housed at the lubbock animal shelter, a city shelter in lubbock, texas, were enrolled in the study from february through november, 2016. the shelter is an openadmission shelter, which admits owner-surrendered, stray, and confiscated animals. the dogs which are available for adoption are placed into a separate kennel area, with the remaining dogs in a different area. for this study, only dogs that were not in the adoptable area were enrolled. dogs were excluded from the study due to the presence of sign of illness (e.g., nasal discharge, coughing, etc.) on day 1, pronounced aggression towards the experimenters, obvious injury, and mothers with litters. dogs were group housed, with some exceptions, in 1.6m x 1.2m x 1.9m steel kennels with cement siding and floors. a guillotine door divided the two parts of each kennel. occasionally the guillotine door was raised and the dogs were given access to both of the kennels. dogs who were dog-aggressive, as evidenced by fighting when group housed, were placed into the kennel alone. the kennels contained a water and food bowl. staff cleaned the kennels and fed the dogs daily. dogs were not routinely taken out of their kennels. occasionally the kennels had a towel or blanket in them. the front two rows of kennels were for male dogs and the middle two rows were for female dogs. the last row of kennels was used for aggressive, pregnant/lactating or injured dogs. there were drainage grates directly in front of the kennels and a cement walkway in between rows of kennels. eleven dogs (13%) received vaccinations (combined canine distemper virus, hepatitis, parvovirus, and parainfluenza administered subcutaneously, and bordetella administered intranasally) after intake (one dog each on days 1 and 2, two on day 3 , one each on days 6, 7, 8, 9, and 10, and two on day 11) . however, no systematic programs to vaccinate dogs on intake were present at the time of the study in the shelter, with an approximate >80% unvaccinated (at intake) dogs present at the shelter during the study. the new dogs were placed into kennels with existing unvaccinated dogs, thus, it is very likely that the animals were already exposed to pathogens prior to developing immunity even for the few dogs that were vaccinated on day 1. moreover, efficacy of vaccination against cirdc is variable, and the most significant predictive factor in whether a dog contracts cirdc may be the number of days in the shelter, rather than vaccination status [5, 27] . while the housing practices, husbandry, and outcome decisions were beyond our control and strictly at the shelter staff's discretion, our study procedures were approved by the texas tech university institutional animal care and use committee (#15064-09). dogs were handled gently, with care and respect, and we tried to be the best part of their otherwise stressful day. withholding vaccination from all dogs would have made our results easier to interpret. however, dogs are routinely vaccinated in shelters with multivalent vaccines that include highly effective protection against lethal diseases such as distemper virus and parvovirus. for this reason, withholding vaccination for the purposes of this study was not deemed ethical. at the time of the study, the animal shelter had poor disease management practices, including poor sanitation, poor medical care, no vaccination at-intake, overcrowding, and continuous rotation of dogs in the kennels. since the time of our study, welfare improvements have been made, including intake vaccinations, improved medical care and sanitation practices. dogs were enrolled in the study the day after they arrived at the shelter (arrival day was coded as day 0, and data collection began on day 1) . the dogs' intake id number, intake date, and kennel tag number were recorded. there were two cohorts of dogs: those for whom data collection occurred on monday, wednesday, friday and those for whom data collection occurred on tuesday, thursday, and saturday. on day 1 to day 14 (a total of seven observations), the dogs were videotaped in their kennel for 1 min using a kodak pixpro spz1, while one or two experimenters stood passively in front of the kennel. the behaviors during the in-kennel videos were coded at a later time. this short observation period was previously used to detect behavioral differences across kenneled dogs in the animal shelter environment [28, 29] . on days 1 through 14, using a slip lead, the dogs were led outside into a 34m x 26m fenced yard and the dog's health was assessed. the yard contained synthetic grass and concrete with a large window looking into the yard from inside the shelter. on day 3, prior to the health assessment, a behavioral assessment was conducted and saliva and urine were collected. if insufficient volumes of these samples were obtained, the sample collection was repeated for day 5 or 6. saliva samples were collected prior to the health exam using inert polymer swabs (salivabio children's swab, salimetrics, carlsbad, ca, usa) held in the dog's mouth for 60 s, but were lost due to human error during analysis; therefore, no data are reported. health observation. at least two researchers were present to conduct the health assessment. the presence of coughing and sneezing, and nasal and ocular discharge were noted during approximately 1 min observations of the dog in the kennel (these signs were noted as important through conversations with several experienced shelter veterinarians). the operational definitions of these categories are listed in table 1 . the dog's body condition score (purina body condition system [30] ) with a range of 1-9 was recorded. on few occasions, the dog was too sick or distraught to exit their kennel; in that case, the health assessment occurred inside the kennel. while one experimenter briefly and gently restrained the dog (when necessary), the second obtained a rectal temperature twice. if the two values differed by more than 0.1˚c, the temperature was taken a third time. the dogs were fed dog treats (pup-peroni1 dog snacks, big heart pet inc., san francisco, ca, usa) throughout the health assessment as a distraction. dogs that consistently refused to allow for the collection of rectal data were excluded from further procedures and data analysis; three dogs refused several times during their stay, resulting in ten missing time points (out of 403 time points total). on 22.6% of observations, the two observers collected data independently in order to calculate inter-observer agreement. cohen's kappa (κ) was calculated to determine agreement between the two observers in their score determination for the condition of the nose, the eyes, and the presence of coughing and sneezing. there was high agreement for all four measures, κ nose condition = .84 (95% ci, .78 to .89), κ eye condition = .73 (95% ci, .63 to .80), κ coughing = .88 (95% ci, .65 to 1), κ sneezing = .86 (95% ci, .75 to .98). behavioral assessment. following the collection of 1 min video clips of in-kennel behavior, the videos were coded on all behaviors listed in table 2 . videos were coded using a partialinterval coding procedure with 5 s time bins, in which an occurrence or non-occurrence of each behavior was noted. the behavioral dependent variables were derived by taking the proportion of the time bins in which a behavior occurred. a portion of the videos (24%), selected at random, were double coded. inter-observer agreement was calculated by adding the number of agreements by interval, dividing by the total number of intervals, and multiplying by 100. the inter-observer agreement was calculated for each behavior independently by summing all agreements of whether or not a behavior occurred in that interval, dividing by the sum of agreements and disagreements, and multiplying by 100. the average agreement across behaviors was 99% (sd = 0.01%; min: 95% for "leaning on wall," max: 100% for "out of sight," "chasing tail," "lying down," "cowering," "tucking tail," "growling," "howling," and "leg lift"). the behavioral assessment was modified from hennessy et al. [31] to contain three components to measure activity, sociability, and boldness/curiosity. a 1m x 1m square was marked off using adhesive measuring tape in the outdoor yard. the dogs were first allowed a few minutes to habituate to the area as well as to toilet (urine was collected at this time; see below for details). the first component of the test consisted of the researcher allowing the dog to remain alone, unrestrained, in the outdoor yard. the researcher videotaped the dog through the window for 2 min. the second component of the test included the researcher kneeling in the center of the 1m x 1m square for 3 min. if the dog had two or more paws inside the square, the researcher would pet and praise the dog using the hand closest to the body of the researcher to allow for the dog to escape at any moment. a second researcher videotaped the interaction through the window. the third part of the test involved the placement of a clear plastic tub, with a remote-control car inside, placed within a 1m x 1m square on the side of the play yard. one researcher stood approximately 1.5 m away from the car and controlled it using a remote table 2 . operational definitions of all of the behaviors that were observed during the in-kennel observation period. front of kennel located between front of cage, and up to and including the midpoint of the visible kennel located between back wall of kennel, and up to, but not including, midpoint of the visible kennel. not visible from the front of the cage, behavior cannot be defined facing forward head is oriented such that the observer is able to see more than the side profile of face likely eye contact with the eyes of the observer head is oriented such that the observer is not able to see more than the side profile of face moving forward distance between the dog and the observer is decreased distance between the dog and the observer is increased both front paws make contact with the cage door that does not include lunging orients towards tail repeatedly (more than 3 times) and continuously repeatedly (more than 3 steps) locomoting around kennel in fixed route supported upright with all four legs to continuously and erratically drive the car inside the tub. the other researchers stood on the opposite side of the pen and videotaped the interaction for 2 min. these videos were coded on the behaviors listed in table 3 and additionally on the corresponding behaviors listed in table 4 . the videos were coded using the partial-interval coding procedure with 5 s time bins, in which an occurrence or non-occurrence of the behavior was noted. the behavioral variables were derived by taking the proportion of the time bins in which a behavior occurred. a portion of the videos (29%), selected at random, were double coded. inter-observer agreement was calculated by adding the number of agreements by interval, dividing by the total number of intervals, and multiplying by 100. inter-observer agreement was calculated for each behavior independently. the average agreement across behaviors was 99% (sd = 0.01%; min: 97.3% for "walking," max: 100% for "jumping on fence," "howling," "tail tucked," "cowering," "cowering," "body trembling," "grab car," and "play bow"). urinary cortisol:creatinine ratio. the urine was collected using a clean plastic vial. immediately following collection, urine was labeled and placed in a cooler with ice packs. following the collection of the urine for the day, the samples were taken to a secured freezer in the texas tech university animal and food sciences building. twenty dogs did not urinate when taken outside, resulting in 64 of dogs containing urinary c:c data. the urine was shipped, in dry ice, to an independent texas a&m veterinary medical diagnostic laboratory (college station, tx, usa) for analysis. a cortisol radioimmunoassay from mp biomedicals (mp biomedicals, llc, santa ana, ca, usa) was utilized. urine was extracted with dichloromethane using 0.5 ml of urine with 1 ml of solvent. the procedure involved first mixing for 5 min followed by 5 min of rest. following rest, the sample was dried down under nitrogen (50 μl per tube) and 25 μl of stripped canine serum was used to the quantification standard of the assay kit (25 μl of standard, control, and target serum). health coding. the health observations (table 1) were coded to provide a quantifiable severity of the observation as an "illness score". the coding scheme for translating observations to a numerical score is presented in the same table. the median rectal temperature was coded as a numerical value. behavior coding and filtering. when considering each behavioral variable for each observation period, 254 behavioral variables were scored across the study period. we implemented a variable quality selection procedure. first, we removed all variables in which fewer than 8 dogs (~10% of all dogs) exhibited across the study period. this filter retained 201 variables. second, we removed all variables that showed little variability across dogs. variables with a standard deviation of less than .01 were removed, leaving a total of 141 behaviors. because the in-kennel behaviors were evaluated at multiple time points, we further restricted our analysis to use the behavioral variables from the first two observation days to predict health observations across the 14 study days. all data are available in the supplementary materials; however, for the aims of this study to predict health outcomes, we restricted our analysis on the first two observation periods (day 1 and day 3). only 56 behavioral variables remained for exploration. path analysis. to identify whether temperament influenced overall health, we conducted a path analysis using the plspm package in r [32] . to test the hypothesis that curiosity, sociability, anxiety, and activity may impact illness risk, the coded behaviors were categorized into latent variables by the first author based on previous research by hennessy et al. (see table 5 [31, [33] [34] [35] [36] ). each behavioral indicator variable was included as a reflective indicator of the latent variable. for the classification of behaviors not listed in the previous study, we attempted to group similar behaviors into established categories. for example, "gazing" and "proximity" to car were grouped with "approach" to car. in-kennel behaviors were interpreted taking into account previous research that showed that some behaviors correlated strongly together and were emitted by dogs when a person was actively interacting with them through the kennel [29] . previous research has shown that "back of kennel" is highly correlated with "front of kennel" and can be considered in unison; the same phenomenon occurs for "facing forward" and "facing backwards". combined, these can be labeled "back and forth facing or motion" as was done in protopopova et al. an example of this phenomenon can be demonstrated by observing a dog pacing back and forth in the kennel. because "gazing", "jumping on cage", "barking", "whining", and "wagging tail" increased in previous research when a person actively solicited attention, these may be considered as part of sociability [29] . hekman and colleagues previously found that panting and lip licking were positively correlated with salivary cortisol concentrations, indicating stress [37] . thus, we included "panting" and "licking self" into the "anxiety" latent variable. "leaning on the wall" has previously been found to correlate with a long length of stay at the shelter [29] , which may indicate some form of distress. therefore, we elected to place this behavior into the "anxiety" latent variable; however, we recognize that this table 4 . operational definitions of the additional behaviors that were observed in the sociability and the curiosity components during the behavioral assessment. was a subjective decision. "barking" and "proximity to the experimenter" during the boldness/ shyness test were logically grouped into the "anxiety" latent variable as they may have indicated distress of the dog as a result of the toy car; again, we recognize that this was a subjective decision. all activity-related behaviors were grouped into the "activity" latent variable. an initial model was fit and the loadings, cross loadings, cronbach's alpha, and dillon-goldstein's rho were checked to evaluate the unidimensionality of the temperament and health variables. indicator variables that were poorly correlated with their respective latent variable (cronbach's alpha & dillon-goldstein's rho < .4) were removed from consideration. the model was re-evaluated and temperament and health variables were assessed for unidimensionality with a raised criterion of .5 for cronbach's alpha and dillon-goldstein's rho. the remaining indicator variables were deemed acceptable for inclusion. to evaluate whether the latent temperament variables were associated with health, we proposed a basic structural model in which curiosity, sociability, anxiety, activity, and time in the shelter independently predicted illness (fig 1) . details of this model are described in the results. table 6 shows the attrition from the study. dogs were euthanized (83%), sent to the adoption floor (7%), returned to owner (6%), or died in their kennel (3.5%). most dogs were available for the behavioral assessments through the first 3-4 days; however, only 19 dogs remained by day 14. during this time, health became progressively worse. fig 2 shows the change in the health observations across time. health steadily worsened as indicated by increases in the severity of the illness observation scores. in addition, temperature increased indicative of fever. fig 3 shows the mean rectal temperature across time as well as the 95% confidence interval (boot-strapped confidence intervals obtained via packages ggplot2) [38] . the dotted line indicates the threshold for fever (39˚c). at study initiation, the 95% confidence interval was well below the fever threshold. however, by day 8 through the end of the study, the 95% confidence interval overlapped with a fever threshold. together, these results clearly indicate the development of illness and systematic increase in severity across the study period. through our established exclusion criterion for relatedness of a behavioral variable to the latent variable, 32 variables were with cronbach's alpha and dillon-goldstein's rho < .4. in a table 7 and table 8 shows latent variable loadings. to evaluate whether activity, sociability, anxiety, curiosity, and time in the shelter were related to the illness score, we conducted a basic pls path regression model in which our 5 latent variables were tested for association with illness. fig 4 shows the hypothesized path model with regression coefficients. table 9 shows the model estimates and statistical significance. overall, sociability, curiosity and time in the shelter were significantly associated with illness. as expected, as time in the shelter increased, illness scores did also (estimate = .53, p < .001). increases in sociability scores (estimate = .24, p < .001; fig 5) and curiosity (estimate = .09, p = .026) were associated with increased illness. activity and anxiety, however, were not associated with illness. the mean c:c ratio was 18.4 x 10 −6 (sd = 11.2 x 10 −6 ). due to missing c:c ratio data (19/83; 23% missing), c:c ratio data were excluded from the pls path regression. to evaluate whether table 6 . https://doi.org/10.1371/journal.pone.0224252.g002 the c:c ratio was associated with illness, a linear mixed model with dog id as a random effect and c:c ratio and time in the shelter as fixed effects indicated that the c:c ratio was not associated the illness score, although time in the shelter was (see table 10 ). to further explore in a reduced sample, whether the c:c ratio was associated with the latent behavioral variables, we computed a cross-correlation matrix between c:c ratio and our latent variable scores from our pls path model. c:c ratio was slightly negatively correlated with sociability (r = -.22), indicating that more sociable dogs had lower c:c ratios. however, due to the large number of correlations and reduced sample size for this analysis, we did not compute p-value to interpret statistical significance. lastly, the cross-correlations indicate sociability and curiosity were positively correlated (r = .42), suggesting these variables may be related and perhaps a more complex path analysis may be worth exploring in future studies with a larger sample size. a correlation matrix was constructed with c:c ratio, time at the shelter, standardized illness, sociability, and curiosity scores. time in the shelter and the standardized illness score had a moderate correlation of .52. sociability and curiosity scores had a moderate correlation of .42. sociability had a moderate correlation with the standardized illness score of .35. standardized curiosity and illness scores had a lower correlation of .20. c:c ratio had a lower negative correlation of -.23. no correlation was present between sociability and time in the shelter, curiosity and time in the shelter, and c:c ratio and illness score (table 11 ). in support of previous research, we found that time in the shelter was positively associated with the incidence of illness symptoms. across time, each sign of upper respiratory illness (coughing, sneezing, ocular and nasal discharge, and fever) became more severe. whereas increases in all signs of illness were already evident as early as the third day in the shelter, by two weeks, the average dog in this animal shelter had a fever, colored nasal discharge, clear ocular discharge, and half of dogs were coughing and/or sneezing. this data supports previous research that found that the risk of coughing increased by 3% each day [39] . out of the four behavioral components, only sociability and, to a much lesser extent, curiosity, but not activity or anxiety was associated with illness. dogs who had higher standardized scores in both sociability and curiosity in the first few days after intake, were more likely to have higher illness scores. sociability consisted of tail wagging and jumping on the cage when a person came up to the kennel and jumping, leaning on, and staying in proximity to the person during the sociability test. curiosity consisted of paying attention to the remote-controlled car (approaching, retreating from, gazing at, and staying in proximity to the car) during the boldness test. the decision to label these behaviors as "curiosity" rather than "boldness" was arbitrary and was informed by subjective opinion by the authors that the dogs' behavior was more closely in line with the human concept of curiosity (i.e., information seeking) rather than boldness (e.g., a willingness to take risks). furthermore, in our study, we did not assess for repeatability and thus are limited in the interpretations of our data in terms of personality or temperament literature. according to visual analysis of the data, a clear positive linear relationship was evident between the standardized sociability and illness scores. however, the relationship appeared less clear between standardized curiosity and illness scores, with some potential outliers driving the positive correlation. it is also noteworthy that sociability and curiosity were moderately correlated, suggesting a potential underlying trait or that a more complex path model might be suggested for future larger studies. previous research has suggested that the various behavioral components may be part of a greater whole. for example, svartberg and forkman [36] suggested that various traits, such as sociability and exploration, among others, may be related to a single higher-order dimension. corsetti and colleagues [26] have also combined the individual traits of activity, attentiveness, dominance, and sociability to differentiate dogs into proactive and reactive coping styles. however, they found that dogs displaying the proactive style (higher boldness, higher sociability) had a lower incidence of illness; our current results seem to be contrary to this previous data. however, corsetti et al. [26] did not find any statistically significant predictors when assessing individual traits, such as boldness, activity, sociability, or anxiety; the lack of statistically significant correlations among individual traits to illness ). urinary c:c ratio, taken on the first full day, was not associated with subsequent illness in the animal shelter. previous research suggests that coping style has a link with the responsiveness of the hpa axis. the proactive coping style has previously been found to correlate with low cortisol responses in dogs [40, 41] . and in fact, we did find that cortisol had a low negative correlation with the sociability component. interestingly, the same negative correlation of cortisol and sociability (but to conspecifics) was found in rhesus macaques [18] . however, instead of the proactive temperament protecting the dogs from illness (through the reduction of cortisol), we found a positive association between sociability and illness. as the correlation between cortisol and sociability was low, this finding may be a type i error, and no true relationship may exist between the two in this population; alternatively, the relationship between proactive coping style and hpa reactivity may differ in this species or in this environment. our data may fit the risk-of-parasitism (rop) hypothesis, which suggests that animals that exhibit bold or exploratory behavior encounter more parasites or pathogens (e.g., [42] ). the probability of encountering a parasite increases mechanically as the animal engages in exploratory behavior. for example, pumpkinseed sunfish that exhibited a bold temperament were more likely to have a higher parasite load [42] . tom cats who exhibited a more dominant and bold temperament were also more likely to be infected with feline immunodeficiency virus [20] . norway rats with higher testosterone were more likely to engage in fighting and more likely to have a hantavirus infection [21] . wood mice that were infected by nematode exhibited more locomotion [43] . similarly, in our study, dogs that were more curious and social may have encountered more infected surfaces, thus were more exposed to pathogens than dogs that were not curious nor social. however, during the time of the study, the animals were typically group housed in relatively small kennels with continuous rotation of animals and no sanitation prior to new arrivals. therefore, the already very high risk of transmission in this particular shelter reduces the likelihood that the rop hypothesis accounts for the entirety of these results. nevertheless, this hypothesis remains a viable candidate for the explanation of the found phenomenon, and more data and experiments are required. an additional hypothesis has been put forth to explain whole-animal differences in immune function, the pace-of-life-syndrome (pols) hypothesis. the pols hypothesis has originally been used to differentiate different species by their "pace of life," or metabolic and reproductive evolutionary strategy [44, 45] . for example, some species may prioritize reproduction but not immune function or longevity. this strategy may be regarded as "live fast, die young." in contrast, some species may prioritize longevity and immune function instead of reproduction -the "live slow, die old" strategy [45, 46] . recently, the pols hypothesis has been utilized to explain whole animal differences within a single species [47] . in fact, such differences have been previously suggested in dogs [48] . thus, it is possible that intra-species differences in dogs may also follow these two evolutionary strategies, with one strategy prioritizing immune function and the other prioritizing reproduction. perhaps dogs that are curious and social are utilizing the "live fast, die young" strategy, and are thus not prioritizing immune function. in fact, due to dogs' reliance on human influence, perhaps human-directed sociability is a strategy for dogs to ensure medical care; thus, by putting more resources into sociability, fewer resources are needed for immune system function. chersini, hall, and wynne [49] suggested that dogs may utilize human intervention for their survival; people rate pups at weaning as most desirable, and this is also the time when dams leave their pups to fend for themselves. with pup survival being only around 70% in free-ranging situations, human involvement becomes crucial to the dog [50] . while intriguing, our current data are not adequate for supporting or refuting this hypothesis. in order to provide support, future data need to show that social and curious dogs also have higher litter size, higher basal metabolic function, and shorter lives. in addition, future data would need to show that people will spend more money or effort on healthcare of social dogs. according to the path analysis model, the outcome illness variable consisted of rectal temperature, the presence of nasal discharge, and coughing. sneezing and ocular discharge did not load well onto the illness factor; however, it is noteworthy that both also increased with time, suggesting that they may be associated with the later part of disease progression, or with a distinct disease process. another important consideration is the potential paradoxical effect in which a positive correlation maybe observed across individuals but a negative correlation observed within individuals [51] . thus, although we observed positive relationships with sociability and curiosity, the correlation at the individual level maybe negative, such that when a dog is more sociable than its typical average, it may be less likely to develop an illness. the present study, however, is limited in its ability to detect such effects due to our limit of only two data points from the first two observations per dog. had we taken more longitudinal data for dogs that stayed for longer periods, this would have been an interesting analysis. several limitations were present in the current study. due to human error, we were not able to assess the immune function of the dogs directly. future research may need to verify the effects of temperament on immune function itself, rather than relying on the indirect measure of subsequent illness. however, in the shelter environment, the predictive nature of temperament remains to be meaningful, regardless of the underlying mechanism. a second limitation was that 12 (14%) of the animals received vaccinations against pathogens that contribute to cirdc, the target disease complex. vaccination on intake can reduce disease incidence in shelters, probably through stimulation of innate immunity. only a subset of these dogs (n = 9; 10%), however, received them during the data collection phase. of the 9 dogs that were vaccinated during the data collection phase, 6 showed signs of illness post-vaccination (s1 fig). it is likely that even the vaccinated dogs were still exposed to pathogens prior to vaccination. nevertheless, future research may circumvent this issue by administering a vaccine challenge to all and measuring the immune response directly. this might also circumvent the problem of animals having unknown prior vaccination histories. a third limitation was that dogs were continuously rotated through the kennels, thus resulting in different groups of dogs per kennel at the different observation times. this shelter procedure made it difficult to assess the risk-of-parasitism hypothesis as well as generally made it difficult to account for the effect of conspecifics during kenneling. finally, we also found high rates of attrition from the study. this limits the longitudinal sample size. to try to limit the effects of attrition, we focused our analysis on early prediction of illness and therefore utilized predictors obtained from the first three days of entering the study only, allowing us to have information on the predictors for all dogs. further, our predictor of interest was health, and unfortunately, many dogs were becoming sick early on, with many dogs showing illness in the first week, while we still retained many of the dogs. nonetheless, it's unclear to what degree the data maybe censored due to euthanasia before developing an illness or going up for an adoption. expanding on the current sample size would be an important follow-up study. regardless of the biological and/or evolutionary mechanism by which dogs with certain temperaments were more susceptible to illness, these data are important for the establishment of predictive models in the applied animal shelter environment. providing knowledge about which dogs are more susceptible to illness, would allow shelter staff to manage the dog population more effectively. a suggestion may be to treat highly social animals as immunocompromised and manage these individuals similarly to nursing moms and puppies. current best-practices suggest housing immunocompromised individuals in a different location away from the general population and taking additional care with disease transmission in these rooms [52] . however, further applied research is needed in order to develop behavioral screening assessments that would adequately predict subsequent illness. to summarize, we found a positive relationship between some temperament traits of dogs, namely sociability and curiosity, and subsequent signs of cirdc illness in the animal shelter environment. due to significant dropout of participants, however, we were not able to observe whether individual variability had a similar relationship. further, we did not find any effect of urinary c:c ratio on subsequent illness. these data are contrary to previous pilot data that suggest that proactive temperaments may protect dogs from subsequent illness. explanations for our data may include the risk-of-parasitism and pace-of-life syndrome hypotheses. future research is needed to differentiate between these two hypotheses as well as develop predictive models for use in animal sheltering. supporting information s1 file. r file of raw data and analyses. the file contains all raw data and all analyses conducted in the statistical software r. practical overview of common infectious disease agents. shelter medicine for veterinarians and staff, second edition canine infectious tracheobronchitis (kennel cough): a status report canine infectious tracheobronchitis short review: kennel cough treatment strategies. shelter medicine for veterinarians and staff, second edition infectious disease management in animal shelters strategies to reduce the euthanasia of impounded dogs and cats used by councils in victoria factors associated with high live release for dogs at a large, open-admission, municipal shelter. animals effects of repeated petting sessions on leukocyte counts, intestinal parasite prevalence, and plasma cortisol concentration of dogs housed in a county animal shelter immune system development in the dog and cat epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental united states and canada and possible mode of transmission using hypothalamic-pituitary-adrenal measures for assessing and reducing the stress of dogs in shelters: a review. applied animal behaviour science how do glucocorticoids influence stress responses? integrating permissive, suppressive, stimulatory, and preparative actions the concepts of stress and stress system disorders: overview of physical and behavioral homeostasis effects of sheltering on physiology, immune function, behavior, and the welfare of dogs a possible stress marker in dogs mucosal immunity: implications for vaccine development personality dimensions in nonhuman animals: a cross-species review the relationship of personality dimensions in adult male rhesus macaques to progression of simian immunodeficiency virus disease effects of housing and individual coping characteristics on immune responses of pigs bold attitude makes male urban feral domestic cats more vulnerable to feline immunodeficiency virus elevated testosterone and reduced 5-hiaa concentrations are associated with wounding and hantavirus infection in male norway rats personality, space use and tick load in an introduced population of siberian chipmunks tamias sibiricus interactions between temperament, stress, and immune function in cattle natural variations in the stress and acute phase responses of cattle. innate immunity university of florida maddie's shelter medicine program. management of disease outbreaks in animal shelters bold personality makes domestic dogs entering a shelter less vulnerable to diseases a placebo-controlled trial of two intranasal vaccines to prevent tracheobronchitis (kennel cough) in dogs entering a humane shelter the effects of exercise and calm interactions on in-kennel behavior of shelter dogs in-kennel behavior predicts length of stay in shelter dogs development and validation of a body condition score system for dogs behavior and cortisol levels of dogs in a public animal shelter, and an exploration of the ability of these measures to predict problem behavior after adoption tools for partial least squares path modeling (pls-pm) the effects of dog obedience training and behavioural counselling upon the human-canine relationship behavioral traits detected in shelter dogs by a behavior evaluation species-specific differences and similarities in the behavior of hand-raised dog and wolf pups in social situations with humans personality traits in the domestic dog (canis familiaris) salivary cortisol concentrations and behavior in a population of healthy dogs hospitalized for elective procedures ggplot2-elegant graphics for data analysis epidemiology and ecology of h3n8 canine influenza viruses in us shelter dogs three different coping styles in police dogs exposed to a short-term challenge behavioural and physiological responses of domestic dogs (canis familiaris) to agonistic growls from conspecifics shy-bold continuum in pumpkinseed sunfish (lepomis gibbosus): an ecological study of a psychological trait apodemus sylvaticus infected with heligmosomoides polygyrus (nematoda) in an arable ecosystem: epidemiology and effects of infection on the movements of male mice performance, personality, and energetics: correlation, causation, and mechanism integrating animal temperament within ecology and evolution. biological reviews personality and the pace-of-life syndrome: variation and selection on exploration, metabolism and locomotor performances slow pace of life in tropical sedentary birds: a common-garden experiment on four stonechat populations from different latitudes the pace of life under artificial selection: personality, energy expenditure, and longevity are correlated in domestic dogs dog pups' attractiveness to humans peaks at weaning age. anthrozoos comparative social ecology of feral dogs and wolves a simple method for distinguishing within-versus between-subject effects using mixed models strategies for management of infectious diseases in a shelter we thank the lubbock animal shelter staff for allowing us to conduct the study. we also thank the following undergraduate students, without whom we could not have conducted the study: kerbey jacobs, francine camara kaercher, sadie bowling, sarah huerta, julianna maynard, jocelyn banschbach, morgan rowland, and priscilla rubio. we thank the dogs, especially who have died during this study; they are not forgotten. writing -review & editing: alexandra protopopova, nathaniel j. hall, kelsea m. brown, jessica p. hekman. key: cord-026021-b8vtmr9h authors: hohenhaus, ann e. title: blood transfusion and blood substitutes date: 2011-06-22 journal: fluid, electrolyte, and acid-base disorders in small animal practice doi: 10.1016/b978-1-4377-0654-3.00031-7 sha: doc_id: 26021 cord_uid: b8vtmr9h nan ann e. hohenhaus blood transfusions have many things in common with fluid therapy. like crystalloid and colloid solutions, blood products are not used to treat disease; they are supportive therapies given to correct deficiencies in the patient until the underlying disease process can be treated. for example, a red blood cell transfusion is given to replace red blood cells lost as a result of a traumatic laceration. the transfusion of red blood cells increases the oxygen-carrying capacity of the blood, allowing for surgical repair of the laceration; it is not the primary treatment for hemorrhage. likewise, sodium chloride is used to replace sodium, chloride, and water in a dehydrated patient with hypoadrenocorticism until adrenal hormones can be replaced. the use of both blood transfusions and fluid therapy must be carefully assessed before inclusion in a patient's treatment plan, and the veterinarian should evaluate the risk/benefit ratio for each patient. volume overload, electrolyte disturbances, and transmission of infection can occur from administration of pathogen-contaminated blood products or fluids. 31, 66, 130 despite the potential negative effects of transfusion, most veterinarians view it as lifesaving therapy, allowing the transfusion recipient to receive other necessary treatments such as surgery, chemotherapy, or medical care. 56 three major differences exist between the more commonly used fluids and blood products. the differences between crystalloid or colloid solutions and blood products are their immunogenicity, availability, and cost. the immunogenicity of blood products stems from the proteins and cellular material in the blood. because crystalloid solutions lack proteins and cellular material, they are not considered immunogenic; however, certain colloid solutions such as hydroxyethyl starch have been reported to cause acute anaphylaxis in rare instances in humans. 96 the mechanism of this reaction is unknown. limited availability differentiates blood products from crystalloid and colloid solutions. crystalloid and colloid solutions are readily available because they can be manufactured according to market demand. only a living animal can produce blood, and the donor's physiologic capability limits production. the small number of commercial canine and feline blood banks providing a convenient source of blood for the veterinary practitioner further limits availability of blood for transfusion (box 24-1). furthermore, blood products require a more regulated storage environment and have a significantly shorter shelf life than crystalloid or colloid solutions, making blood a less convenient product to store and use in a veterinary hospital. nearly 20 years ago, veterinarians estimated costs associated with transfusions, but an exact analysis of cost is lacking. in 1992, the estimated cost of a 500-ml whole blood transfusion ranged from $25 to more than $300. 56 the cost of 500 ml of lactated ringer's solution is about $1. despite the fact that the first documented transfusion was given to a dog in 1665 by richard lower at oxford university, veterinary transfusion medicine scientifically and technologically lags behind its counterpart in human medicine. 76 information in this chapter is based on veterinary studies whenever possible. when none is available, currently accepted guidelines from human medicine will be applied to the veterinary patient. the purpose of this chapter is to provide the reader with the following: 1. a basic understanding of the theory of blood component therapy 2. information on the technical aspects of obtaining blood for transfusion 3. suggestions for the administration and monitoring of transfusions 4. a description of the clinical applications of a veterinary blood substitute blood is the body's largest connective tissue. when collected from the donor, it contains all the elements of blood: red blood cells, white blood cells, platelets, coagulation factors, immunoglobulins, and albumin. whole blood can be transfused into the recipient as it is collected from the donor, but it is neither a specific therapy nor an economical use of blood. the optimal method of preservation of blood for transfusion is to separate whole blood into its component parts. appropriate use of blood components not only conserves the products but also allows the most specific and safe product to be administered for each patient. when blood components are used instead of whole blood for transfusion, two dogs can benefit from 1 unit of whole blood. a plasma transfusion counteracts the anticoagulant effects of rodenticide intoxication in one dog, and red blood cells from the same donor provides enhanced oxygen-carrying capacity in a second, anemic dog. component transfusions also have been used in cats, but preparation of components is more difficult because of the small volume of blood collected from donor cats. 23, 53, 68, 99 production of components is not feasible for most veterinary practices. most will purchase their blood inventory because they lack the time and equipment to recruit donors, and collect and process whole blood into components. blood components predominate in the inventory of commercial blood banks, requiring veterinarians to become familiar with their usage. the technical aspects of component production are not included in this chapter but can be found elsewhere. 85, 99 a brief summary follows. preparation of blood components from whole blood requires that the blood from the donor be collected into the anticoagulant-containing bag of a multibag plastic blood collection system. the whole blood then is separated into packed red blood cells (prbcs) and plasma by differential centrifugation in a refrigerated blood bank centrifuge, and the plasma is transferred into one or more of the attached satellite bags via the sterile tubing linking the bags. the bags are separated, and prbcs are stored in a refrigerator and plasma is stored in a freezer. blood collected into glass bottles is not amenable to centrifugation and cannot be processed into components. additionally, storage of canine blood in a glass bottle results in lower levels of 2,3-diphosphoglycerate and adenosine triphosphate (atp) than blood stored in plastic bags; consequently, plastic bags are the preferred storage container for blood. 28 the most commonly used blood products, their indications, and suggested dosages are described later. the dosage of a blood product depends on the physical state of the patient and the response of the patient to the treatment: in essence, the treatment is "to effect." whole blood is the blood collected from the donor plus the anticoagulant. in veterinary medicine, no standards have been established for the volume of blood that constitutes 1 unit. when a human blood collection system is used for dogs, 450 ae 45 ml of blood is collected and combined with 63 ml of anticoagulant, and often is designated as 1 unit. whole blood contains red blood cells, clotting factors, proteins, and platelets and is the product most commonly transfused into dogs and cats. 56 once whole blood is refrigerated, the white blood cells and platelets become nonfunctional. as a starting point, the dosage for whole blood is 10 to 22 ml/kg. prbcs are the cells and the small amount of plasma and anticoagulant that remains after the plasma is removed from 1 unit of whole blood. if 450 ml of blood are collected, the volume of prbcs obtained is approximately 200 ml. because the plasma has been removed, the total volume transfused is less than 1 unit of whole blood but contains the same oxygen-carrying capacity as 450 ml of whole blood. in cats, the increase in packed cell volume (pcv) after transfusion of 1 unit of prbcs has been shown to be equivalent to the increase after transfusion of 1 unit of whole blood. 68 prbcs are used only to treat clinically symptomatic anemia because they do not contain platelets or clotting factors. red blood cell transfusions are administered to cats for a variety of reasons. data on 126 cats administered whole blood or prbcs indicated 52% were transfused for blood loss anemia, 38% for erythropoietic failure, and 10% for hemolytic anemia. 68 similar reasons for transfusion of cats have been reported elsewhere. 22, 126 dogs more commonly are transfused for blood loss anemia (70%) with 14% to 22% being transfused for hemolytic anemia and 8% to 14% for erythropoietic failure. 17, 64 the initial dosage of prbcs is 6 to 10 ml/kg, and transfusion is continued until the clinical signs of anemia resolve. fresh frozen plasma is the plasma obtained from whole blood plus the anticoagulant solution, which is frozen within 8 hours of collection. when whole blood is centrifuged to produce plasma and prbcs, the anticoagulant segregates with the plasma fraction. fresh frozen plasma contains all clotting factors, which, if frozen at à30 c in a blood bank freezer, maintains activity for 12 months. 121 fresh frozen plasma maintained in an upright freezer at à20 c maintains clotting factor activity for 6 months. when frozen, the plastic storage bag becomes brittle and if not carefully handled can crack, rendering the plasma unusable. for this reason, plasma is stored in special boxes to protect the plastic bag and must be handled carefully before transfusion. if plasma is thawed, and not transfused, it can be refrozen within 1 hour of thawing without loss of coagulation factor activity. 131 fresh frozen plasma has been used to treat a wide variety of clinical patients. a retrospective analysis of fresh frozen plasma usage in dogs identified multiple indications for administration of fresh frozen plasma, including replacement of coagulation factors, albumin, a 2 -macroglobulin, and immunoglobulin despite the recommendation that fresh frozen plasma should not be used as a source of albumin, for volume expansion, or nutritional support. 75, 87 calculations suggest 45 ml/kg of plasma would need to be given to increase albumin serum concentration by 1 g/dl. 120 in cases of coagulation factor deficiencies, plasma should be given to effect (i.e., until active bleeding ceases). 70 for the treatment of coagulation disorders, 6 to 10 ml/kg is the recommended starting dosage. multiple doses may be required to control bleeding because of the short half-life of clotting factors, especially in patients with disseminated intravascular coagulation. normalization of previously abnormal coagulation tests can be used as a guide for discontinuation of plasma therapy. cryoprecipitate is prepared by thawing fresh frozen plasma at 0 c to 6 c. a white precipitate forms, the liquid plasma is removed after centrifugation, and both aliquots are refrozen. the cryoprecipitate is a concentrated source of von willebrand's factor, fibrinogen, and factors xiii and viii (antihemophilia factor). it is useful in the treatment of deficiencies of these clotting factors and is handled in the same manner as fresh frozen plasma. two studies have shown cryoprecipitate to be the blood product of choice for the treatment of von willebrand's disease because it concentrates the larger, more hemostatically active von willebrand's multimers into a smaller volume than fresh frozen plasma. 23,108 a preliminary study suggests cryoprecipitate corrects the hypocoagulable state associated with disseminated intravascular coagulation when administered at a dose of 5 to 7 ml/kg. 117 cryoprecipitate is equivalent to fresh frozen plasma for the treatment of hemophilia a. the dose is 1 unit per 10 kg body weight. 84 cryo-poor plasma is the supernatant plasma removed from the cryoprecipitate. cryo-poor plasma contains factors ii, vii, ix, and x, which make it useful for the treatment of rodenticide intoxication. storage and handling of cryo-poor plasma is similar to fresh frozen plasma. the initial dose is 1 unit per 10 kg of body weight. platelet-rich plasma is prepared from fresh whole blood by centrifugation at a slower rate than is used for production of prbcs and plasma. 85 the platelets are suspended in a small amount of plasma to facilitate transfusion. storage of fresh platelets is impractical outside a blood bank because of their requirement for storage at 20 c to 24 c in special plastic bags and continuous agitation. 2 transfused platelets are rapidly destroyed in human patients with immune-mediated thrombocytopenia, and because immune-mediated thrombocytopenia is a common cause of profound thrombocytopenia in dogs, most cases of thrombocytopenia-mediated hemorrhage may not be amenable to successful platelet transfusion. if a platelet transfusion is given, the dose is the platelets collected from 1 unit of whole blood per 10 kg of body weight. cryopreserved canine platelets are collected from a single donor via plateletpheresis, and the manufacturer reports one bag contains 1 â 10 11 platelets preserved in dimethyl sulfoxide (dmso). 57 the bag also contains a small amount of fresh frozen plasma. efficacy data on this product have not been published, but the manufacturer recommends this product be used for the treatment of immune-mediated thrombocytopenia. the dose is 1 unit of frozen platelets per 10 kg of body weight. according to the manufacturer, anticipated increase in platelet count is 20,000/ ml 1 to 2 hours posttransfusion. because the product contains dmso, it must be infused slowly to prevent bradycardia. cryopreserved canine platelet concentrate was compared with fresh platelet rich plasma in the laboratory. 48 this study identified decreases in platelet number and function as a result of the freeze-and-thaw process. platelet number decreased 59% compared with the manufacturer's reported platelet count and platelets demonstrated multiple features of activation. the impact of cryopreservation on platelet function and number in vitro has not been studied. the use of serum has been recommended for the treatment of kittens and puppies with failure of passive transfer. kittens treated with 5 ml subcutaneously or intraperitoneally three times in 24 hours achieved immunoglobulin g (igg) concentrations comparable to kittens receiving colostrum. 72 treatment of puppies with 22 ml/kg of serum given orally or subcutaneously at birth did not result in equivalent igg and iga concentrations when serum-treated puppies were compared with nursing puppies. 90 igm was higher in the puppies treated with subcutaneously administered serum. human albumin is a concentrate of albumin derived from pooled human plasma. homology between canine and human albumin is approximately 79%, and human albumin is antigenic in dogs. 30, 80 previous human albumin transfusion does not appear to be required for production of antibodies in dogs. 80 hypoalbuminemia predicts a negative outcome in several canine diseases; consequently, the ability to correct hypoalbuminemia by albumin transfusion would be a medically desirable intervention. 1, 20 because canine albumin was not previously available, human albumin has been used in dogs. two retrospective studies have evaluated transfusion of human albumin to critically ill dogs. 81, 115 one associated improved albumin levels and blood pressure with human albumin administration and did not report serious adverse events. 81 the second concluded the serious nature of the diseases treated with precluded recognition of complications of the transfusion. 115 a recent study performed in normal dogs has identified serious adverse events suggestive of anaphylactic and fatal type iii hypersensitivity reactions. 25 transfusion of dogs with human albumin should be undertaken with great caution especially because lyophilized canine albumin is available. 58 human intravenous immunoglobulin (hivig) is a highly purified preparation of immunoglobulin g, obtained from large pools of donated human plasma. the manufacturer provides the product as a lyophilized powder, which is reconstituted before transfusion. sporadic availability of hivig limits its use, as does its high cost. estimates indicate the cost of the drugs alone may be as high as $3000 to treat a 20 kg dog. 129 reconstituted hivig is infused over 6 hours. most report a single administration of the drug at a dosage of 0.5 to 1.0 g/kg, but in some cases the dose is administered three times on 3 consecutive days. 8, 9, 113, 129 because of its immunomodulatory properties, transfusion of hivig has become more common in veterinary patients. 94 the two major diseases treated with hivig have been immune-mediated hemolytic anemia (imha) and immune-mediated thrombocytopenia (itp), but hivig has also been used to treat some immune-mediated dermatologic disorders as well. 8, 9, 113, 129 randomized, controlled prospective studies of glucocorticoids with and without hivig for the treatment of imha and itp have been published. 8, 129 the itp study demonstrated reduction in platelet recovery time without a concurrent increase in associated charges in the group randomized to receive glucocorticoids and hivig. the imha study did not show an advantage to treatment with hivig and glucocorticoids compared with glucocorticoids alone, but the study was underpowered to distinguish a difference between the two treatment groups. administration of hivig to normal dogs promoted a hypercoagulable state, but in clinically ill dogs causality of thromboembolism is difficult to determine given the complexity of diseases undergoing hivig transfusion. 100, 116 the most convenient source of blood for a veterinary clinic is a commercial blood bank. currently, there are only a limited number of commercial veterinary blood banks in the united states, and they cannot adequately supply all the small animal practices in the country with blood (see box 24-1). veterinary school blood donor programs may serve as an additional source of blood for the practitioner. 56 because of the limited supply of blood from commercial animal blood banks, small animal practitioners typically borrow a donor from an employee or maintain a blood donor on the premises. 56 borrowing a donor from either an employee or a client is a frequently used, if less convenient, option and is less expensive than maintaining an in-hospital donor. maintaining a donor on the premises is advantageous because they are readily available for donation and their health status and disease exposure can be controlled, but the expense associated with feeding, housing, and caring for a blood donor is significant. 54 volunteer blood donor programs have replaced many on-site blood donors. 14, 59 collecting blood from stray animals is unsafe because infectious disease exposure and health status are unknown. identification of donor dogs and cats before blood is needed is essential to allow blood type to be determined and the health status of the donor to be assessed before blood collection, thus ensuring the safety of the blood being transfused. the american college of veterinary internal medicine has published recommendations on infectious disease screening for canine and feline blood donors as a consensus statement. 124 the recommendations have been incorporated into the following sections. for nearly 60 years, the best blood donor was believed to be a large, quiet dog not requiring anesthesia during blood collection. 78 the current recommendation is unchanged. a canine blood donor weighing more than 27 kg can safely donate 450 ml of blood in one donation, allowing collection of blood into commercially manufactured blood collection bags designed to facilitate sterile processing of components. dogs weighing 27 kg or more have been shown to consistently donate 1 unit of blood for 2 years at 3-week intervals. 92 dogs selected as donors also should have an easily accessible jugular vein to facilitate venipuncture. prior pregnancy does not exclude female dogs from donation because pregnancy does not induce alloantibodies. 12 greyhounds have been promoted as ideal blood donors because of their gentle disposition, high hematocrits, and lean body type, which simplifies blood collection. 45 many greyhounds are euthanized because of poor racing performance, and these dogs are available from racetracks, breeders, and rescue organizations. 36 blood banks choosing greyhounds as blood donors should be aware of certain breed idiosyncrasies that will impact on the management of a greyhound donor. the greyhound idiosyncrasy most important in transfusion medicine is the high red blood cell count, pcv and hemoglobin concentration, and low white blood cell counts and platelet count compared with mixed breed dogs. 91, 109 greyhounds in florida have a 46% seroprevalence of babesiosis. 111 because the geographic origin of greyhounds serving as blood donors cannot always be determined, all greyhounds being screened as donors should have serologic testing for babesia canis performed, and dogs with positive titers should be excluded as donors. greyhounds with negative titers against b. canis should have b. canis polymerase chain reaction (pcr) performed, and if the test is positive, the dog should be excluded as a donor. in addition to the tendency of greyhounds to be asymptomatic carriers of b. canis, some other breeds of dogs should be used cautiously as blood donors because they are known to be asymptomatic carriers of infectious organisms transmitted by transfusion. american pit bull terriers and staffordshire bull terriers recently have been recognized as carriers of babesia gibsoni. 10, 77 use of these dogs as blood donors should be restricted to those dogs that are seronegative and pcr-negative for b. gibsoni. leishmaniasis has been identified in american foxhounds and transfusion of leishmania infantum-infected blood from american foxhounds resulted in clinical leishmaniasis in transfusion recipients. 47, 89 all potential foxhound donors should be screened for leishmania sp. determination of blood type is critical to the selection of a blood donor dog. although seven canine blood groups or blood type systems have received international standardization, typing sera are available for only five types (box 24-2). red blood cells can be negative or positive for a given blood type, except for the dog erythrocyte antigen (dea) 1 system, which has three subtypes: dea 1.1, 1.2, and 1.3. canine red blood cells can be negative for all three subtypes (a dea 1-negative blood type) or positive for any one of the three subtypes. naturally occurring alloantibodies occur infrequently and without previous sensitization from transfusion do not appear to cause transfusion incompatibility in the dog 51 (see . a new canine red blood cell antigen, dal, has recently been described. 11 this antigen appears to be common in the general canine population and lacking in dalmatians. transfusion with dal-positive blood, induced an anti-dal antibody producing multiple incompatible crossmatch tests. dogs producing anti-dal antibodies are at risk for hemolytic transfusion reactions. the blood type of the ideal canine blood donor is not uniformly agreed on among transfusion experts. of the five blood groups for which typing sera are available, a transfusion reaction has been attributed to antibodies against dea 1.1 induced by a dea 1.1-positive transfusion in a dea 1.1-negative recipient and to an antibody induced by a dea 4-positive transfusion in a dea 4-negative dog. 40, 83 in theory, red blood cells expressing dea 1.2 can sensitize a dea 1.2-negative transfusion recipient, resulting in an acute hemolytic transfusion reaction if a second transfusion of dea 1.2-positive blood is given. in a laboratory setting, antibodies against dea 1.2 have been reported to cause transfusion reactions, but clinical reports of hemolytic transfusion reactions mediated by anti-dea 1.2 antibodies are lacking. dea 7 is believed to be structurally related to an antigen found in common bacteria. a naturally occurring antibody against dea 7 has previously been described in 20% to 50% of dea 7-negative dogs, but recently revised down to 9.8% of dogs. 51 this antibody may result in accelerated removal of dea 7-positive cells from a dea-negative donor with anti-dea 7 antibodies. 51, 102 based on this information, the recommendation has been made to select donors that are negative for dea 1.1, 1.2, and 7. others suggest the donor dog should also have red blood cells positive for dea 4 to be designated as a universal donor. 50 the recent description of a transfusion reaction attributed to antibodies against dea 4 in a dog with dea 4-negative red blood cells calls into question this recommendation. 119 ninety-eight percent of dogs are dea 4-positive, making it easy to find donors of this blood type. the importance of dea 3 and 5 and dal in blood donor selection remains to be determined. one other feature that should be considered before selection as a blood donor is the dog's plasma von willebrand factor concentration. von willebrand's disease is the most common inherited coagulopathy in dogs and has been reported in many breeds of dogs and in dogs of mixed breeding as well. because of the high frequency of this disease in the canine population, plasma from a canine blood donor will likely be used to transfuse a dog with von willebrand's disease-induced hemorrhage, and a donor with a normal concentration of von willebrand's factor is essential to replace the deficient coagulation factor. the physical requirements for a feline blood donor are similar to those for a canine donor. the ideal feline donor is a large cat, more than 5 kg of body weight, with a pleasant disposition. easily accessible jugular veins facilitate collection of blood, and choosing a shorthair cat decreases the clipping required before phlebotomy. it is essential to determine the blood type of potential donors. one feline blood group system has been identified with three blood types: a, b, and ab (see box 24-2) and recently a new common red blood cell antigen, mik has been identified. 6, 128 unlike dogs, cats have naturally occurring alloantibodies. type a cats have naturally occurring alloantibodies against type b cells and type b cats against type a cells. 38 cats of blood type b have strong hemagglutinating antibodies of the igm type against type a cells, and cats of blood type a have weak hemolysin and hemagglutinating antibodies of the igm and igg type against type b cells. the clinical significance of these alloantibodies is threefold in transfusion medicine. first and most importantly, a cat may have a transfusion reaction without sensitization from a previous transfusion; second, type a kittens born to a type b queen are at risk for neonatal isoerythrolysis 21 ; and third, the antibodies are useful in determining the blood type of a cat. mik appears to be a common red blood cell antigen. only a few cats lacking mik have been identified and they all produce anti-mik alloantibodies. donors of both type a and type b blood must be available because there is no universal donor in cats. incompatible transfusions result in shortened red blood cell survival and potentially death in the transfusion recipient; therefore the serologic compatibility between recipient and donor must be determined before every transfusion in cats. 38 donors of type a blood are easy to find because more than 99% of the domestic cats in the united states are type a. 42 the prevalence of domestic cats with type b blood varies geographically. in the united states, the western states have the highest percentage of type b cats, 4% to 6%. 42 australia has the highest reported percentage of type b cats in their domestic cat population, 73%. 6 in europe, the frequency of blood type b in domestic cats varies from 0% in finland, 14.9% in france, and 24.6% in turkey. 3, 41 some purebred cats have a higher frequency of type b in their population. 39 the british shorthair, devon rex and turkish van have been reported to have the highest proportion of type b individuals, approximately 50% to 60%. 4 the siamese, oriental shorthair, burmese, tonkinese, american shorthair, and norwegian forest cat breeds have not been reported to have any members with type b blood. blood type ab is extremely rare, occurring in 0.14% of cats in the united states and canada. 46 fortunately, a type ab donor is not required to successfully transfuse a type ab cat. blood from a type a cat is adequate. screening blood donors for infectious diseases transmitted by blood transfusion is an integral step in maintaining a safe blood supply. infectious disease screening of canine and feline blood donors varies within the different geographic regions of the united states and with the breed of the blood donor. an american college of veterinary internal medicine consensus statement, developed by a committee consisting of members of the infectious disease study group and the association of veterinary hematology and transfusion medicine should serve as the guideline for donor screening. 124 organisms infectious to dogs and known to be transmitted by blood transfusion include b. canis, b. gibsoni, haemobartonella canis, and leishmania sp. 31 124 dogs should not donate if they are ill or have fever, vomiting, or diarrhea; using donors with these clinical signs has resulted in yersinia enterocolitica contamination of human units of blood. 32 organisms infectious to cats and known to be transmitted by blood transfusion include: feline leukemia virus (felv), feline immunodeficiency virus (fiv) bartonella henselae, anaplasma phagocytophilum, ehrlichia spp. and neorickettsia spp., and the organisms formerly classified as haemobartonella sp. (mycoplasma haemofelis and candidatus mycoplasma haemominutum). 33, 49 potential donor cats should be screened for felv and fiv. because the prepatent period for felv infection can be 3 months, cats being considered as donors should be screened monthly for felv for 3 consecutive months. testing for fiv antibodies can be performed simultaneously. bartonella henselae is an emerging feline infectious disease and has been transmitted to cats by infected blood. 69 the use of cats with positive serology or cultures for b. henselae as blood donors is controversial and eliminating these cats from the donor pool is the safest approach. testing for hemoplasma should include both light microscopy and pcr, and infected cats should be eliminated from the donor pool. 33 screening of donor cats for feline infectious peritonitis (fip) is problematic because there is no reliable test to identify the fip-causing coronavirus. feline blood donors should be screened for infection with cytauxzoon felis and the agents causing feline ehrlichiosis if they reside in or are known to have traveled to endemic locations. a safe blood supply begins with healthy blood donors. all blood donors should undergo a complete physical examination each time they donate blood. complete and differential blood counts, biochemical profile, and fecal examination should be performed annually. donor cats and dogs with exposure to the outdoors or to ectoparasites should be routinely screened for infectious disease. blood donors should be tested for heartworms, treated for ectoparasites, and vaccinated for the diseases on the schedule recommended for pets residing in the geographic region of the blood bank. because the ideal feline blood donor lives in an indoor environment and is not exposed to other cats, the author believes vaccinations against felv, fiv, and fip are unnecessary in donor cats. exposure to the outdoors or to fleas approximately doubles the prevalence of hemoplasma infections in donor cats and restricting access to the outdoors, fleas, and other cats can prevent most infectious diseases in feline blood donors. 49 strict aseptic technique must be used during the blood collection process to prevent contamination of blood with microorganisms. whenever possible, solutions and equipment used for the collection process should be single-use products to prevent inadvertent contamination of blood. 55 after clipping the hair over the venipuncture site, the skin is surgically scrubbed. the ideal skin preparation regimen is yet to be determined in animals; however, in human blood donors, a 30-second, 70% isopropyl scrub followed by a 2% iodine tincture resulted in better skin surface disinfection than alcohol followed by chlorhexidine or green soap. 44 the phlebotomist wears sterile surgical gloves and performs venipuncture without touching the scrubbed area. several different solutions are available to anticoagulate and preserve blood for transfusion and species-specific storage times are listed (table 24-1) . anticoagulants provide no nutrients to preserve red cell metabolism during storage. blood collected in anticoagulants should be transfused immediately. anticoagulant-preservative solutions have been designed to provide nutrients to maintain red blood cell function during storage. one common anticoagulant solution for preservation of canine red blood cells, citrate phosphate dextrose adenine (cpda-1), is found in commercially prepared, multiple-bag systems. maximal storage time for feline blood in cpda-1 has yet to be determined but may be as long as 35 days. 15 acid citrate dextrose or anticoagulant citrate dextrose (acd) formula b can be used to store either canine or feline blood. 29, 79 it can be purchased in 500-ml bags and placed in syringes for collection of blood. cat and dog red blood cells maintain adequate viability following storage in acd for 30 and 21 days, respectively. additive solutions are contained in a multibag system containing citrate phosphate dextrose (cpd) or citrate phosphate dextrose 2 (cpd-2) as the anticoagulant. the additive solution is contained in a bag separate from the main bag and is added to prbcs after the plasma is removed. additive solutions that have been evaluated in dogs are adsol (fenwal laboratories, baxter health care corporation, deerfield, ill.) and nutricel (miles pharmaceutical division, west haven, conn.). 123, 125 additive solutions have not been evaluated for storage of feline blood, but are sometimes used as storage media for feline red blood cells. white blood cells are responsible for some adverse effects of transfusion and donot contribute to transfusion efficacy(see "adverse effects of transfusion"). an integral filter to remove white blood cells from whole blood is incorporated into some blood bag systems. one system has been evaluated using canine blood and effectively removed white blood cells without affecting red blood cell viability. 13 98 the choice is strictly a matter of personal preference and skill. acepromazine is not recommended because it causes hypotension and platelet dysfunction. the flow of blood into the bag can occur by gravity or suction. blood collected by suction does not have a greater rate of hemolysis than that collected by gravity flow, and it can be collected more rapidly. 27 suction collection of blood is facilitated using a vacuum chamber manufactured by the animal blood resources international (stockbridge, mich.). this device requires an external vacuum source during collection of blood. it is unusual to find a feline blood donor that does not require sedation during blood donation. the author prefers a combination of ketamine (10 mg) and diazepam (0.5 mg) intravenously for cats, whereas others recommend using midazolam, and isoflurane or sevoflurane. 98, 114 if the sedative agent is to be given intravenously, a peripheral vein (cephalic or medial saphenous) should be used to preserve the jugular veins for blood collection. no commercially available system is manufactured for the collection of blood from cats because of the small volume of blood that can safely be withdrawn from a cat. typically, anticoagulant can be withdrawn from a blood bag port using a syringe. it is placed in one or two large syringes (25 to 60 ml) depending on the volume of blood to be collected (see table 24 -1). a large (19gauge) butterfly needle is used for jugular venipuncture so that if a second syringe of blood is to be collected, the full syringe can be removed and the second syringe connected without a second venipuncture. by the definition of the american association of blood banks, this is an "open" system, and blood collected in this manner should not be transfused more than 4 hours after collection. 118 alternatively, a standard blood collection bag containing cpda-1 can be used. all cpda-1 is expelled from the bag except for the amount remaining in the tubing. feline blood is collected directly into the bag. 93 a commercially available vacuum system can be used for collecting blood from cats, but some authors find this system less satisfactory than the syringe method. 63, 98 selection and transfusion of compatible blood is one component of the process to provide a safe and efficacious red cell transfusion. with the identification of a new red blood cell antigen in both dogs and cats, recommendations for appropriate compatibility testing before the first transfusion are a currently being revaluated. because each unit of red blood cells is antigenically distinct, the recipient may form antibodies after transfusion of any unit of blood. the immune system will take a minimum of 5 days to make antibodies against transfused red blood cells; therefore, a crossmatch should be performed if more than 4 days elapse between transfusions. performing a crossmatch will not prevent an immune reaction to subsequent transfusions; it can only identify those units of blood with potential to cause acute hemolytic transfusion reactions. because of the lack of clinically significant preformed alloantibodies in the dog, blood typing and crossmatching are not routinely performed before the first transfusion. when dea 1.1-positive blood is transfused, ideally it would be given to a dea 1.1-positive recipient to prevent sensitization of a dea 1.1-negative dog. dea 1.1 status can be determined by using the typing systems described below. crossmatching should be performed before any subsequent transfusion to identify a compatible unit of red blood cells. blood typing or crossmatching is not required before transfusion of canine plasma. previously, blood typing was considered adequate pretransfusion testing before administration of red blood cells or plasma to cats. blood typing prevented administration of type b blood to a type a cat and vice versa. however, transfusion of a-b mismatched blood results in decreased red blood cell survival or a potentially fatal, acute hemolytic transfusion reaction. 38 blood typing will not identify the mik antigen and its naturally occurring alloantibody; however crossmatching will detect the anti-mik antibody and crossmatching may become the preferred compatibility test for all feline transfusions to prevent incompatible transfusions due to anti-a, anti-b, or anti-mik alloantibodies. determining a-b blood type in the cat has been simplified by the availability of in-clinic typing systems (figure 24-1) . a special situation with regard to blood typing and crossmatching exists in cats. when blood typing is unavailable, crossmatching will administration of an incompatible transfusion due to a, b, or mik alloantibodies. when crossmatching is performed with a known type a donor, an incompatible major crossmatch strongly suggests the potential recipient is a type b or a mik negative cat because of the naturally occurring alloantibodies in these cats. if cat plasma is administered, it should be the same blood type as the recipient because plasma will contain anti-a, anti-b, and anti-mik antibodies. crossmatching the donor to the recipient cat will prevent a reaction because of a, b, or mik alloantibodies. the person administering the blood should pay careful attention to the blood bag label before transfusion. the most common reason for an acute hemolytic transfusion reaction in human patients is clerical error-the wrong unit of blood is released from the blood bank or a unit of blood is given to a patient who was not intended to receive a transfusion. 110 in veterinary medicine, it is crucial to confirm that the blood comes from the correct species of blood donor in addition to being typed and matched to the patient requiring a transfusion. the contents of the bag also should be examined for normal color and consistency. bacterially contaminated blood often appears brown or purple because of deoxygenation, hemolysis, and formation of methemoglobin. 55, 65 blood and plasma can be administered using several routes. most commonly, blood is given intravenously. the diameter of the catheter used for transfusion is important in determining the rate of blood flow because blood flows more slowly through a small catheter; however, small diameter catheters have not been associated with increased risk of hemolysis during transfusion. 118 the intraosseous route can be used successfully for administration of blood and plasma. 88 in normal dogs, 93% to 98% of red blood cells administered through an intraosseous catheter are found in the peripheral circulation within 5 minutes. 24 this rapid and simple method is especially useful in animals with vascular collapse and in extremely young puppies and kittens. special intraosseous catheters are available, but a spinal needle, bone marrow aspiration needle, over-the-needle catheter, or even an ordinary hypodermic needle can be used. sites for the placement of the intraosseous catheter include the trochanteric fossa of the femur, the medial tibia, and the iliac crest. blood flows very rapidly through an intraosseous catheter, and rate of administration should be monitored closely. plasma can be administered intraperitoneally in emergency situations, but red blood cells are slowly and poorly absorbed when administered by this route, and it is not recommended for red blood cell transfusions. a blood transfusion administration set is required for administration of red blood cells or plasma to remove blood clots and debris, which form during storage and which could cause embolism. the filter typically used in veterinary medicine is 170 mm in size. for smallvolume transfusions, an 18-mm filter attached to intravenous tubing is useful. an 18-mm filter does not work well for large-volume transfusions because it rapidly becomes obstructed with debris, and transfusion rate slows. the risk of an air embolism is increased when blood is collected into glass bottles. a blood administration set does not remove air from stored blood; accordingly, glass bottles are not recommended for collection and storage of blood. the american association of blood banks explicitly states that medications should not be added to blood or components. 118 in addition, no fluid should be added to blood excep. 0.9% sodium chloride when it is necessary to decrease the viscosity of prbcs. fluids containing calcium such as lactated ringer's solution may overcome the anticoagulant properties of citrate, resulting in coagulation of the blood. solutions such as 5% dextrose in water are hypotonic and may induce hemolysis. the recommended rate of transfusion of red blood cells depends on the status of the recipient. in massive hemorrhage, the transfusion should be given as rapidly as possible. in a normovolemic, stable transfusion recipient, some clinicians recommend a rate of 0.25 ml/ kg for the first 30 minutes, after which the rate is increased if no reaction is seen. 112 in patients with heart disease, a rate of 4 ml/kg/hr should not be exceeded. 45 transfusion rates of 10 ml/kg/hr, 4 ml/kg/hr, and up to 60 ml/kg/hr were used to transfuse red blood cells to cats with normovolemia, cardiovascular dysfunction. and hypovolemic shock, respectively. 126 plasma can be given more rapidly (4 to 6 ml/min). 67 whatever the rate chosen, it should be rapid enough to complete the transfusion within 4 hours of initiation because of the risk of bacterial growth in blood maintained at room temperature for a prolonged period. control of blood product delivery rate can be accomplished by use of infusion pumps to deliver a preset volume over a specific period of time. the use of infusion pumps must be limited to devices approved for use with blood because some infusion pumps can result in hemolysis of red blood cells as a result of excessive pressure. 107 because blood does not contain any antibacterial agents, it must be refrigerated until used to retard bacterial growth and maintain red blood cell viability. if the clinical status of the animal requires that the transfusion be given over a period greater than 4 hours, the blood can be split into smaller units with a transfer bag. one portion of the blood is transfused while the other is returned to the refrigerator until the first half of the transfusion is completed. in patients with cardiac disease at risk for volume overload, the risk can be further minimized by use of prbcs, which require infusion of a lower volume than whole blood. warming of blood before transfusion has been recommended to prevent hypothermia in the transfusion recipient. warming of blood probably is only necessary if a large volume of blood is to be given or if the recipient is a neonate. for adult animals receiving a single unit of blood, the blood can be administered directly from the refrigerator. warming blood has the potential for excessive heating, causing red blood cell membrane damage and hemolysis or promoting bacterial growth if contamination is present. blood warming devices that use dry heat, radio waves, microwaves, or electromagnetic energy are available, but cost often is prohibitive. refrigerated human blood can be warmed quickly by admixing it with warm (45 c to 60 c) 0.9% saline in a ratio of 1:1 without damage to red blood cells. 61 this method has not been tested for dogs or cats. once blood is warmed to 37 c, it deteriorates rapidly and, if not used, should be discarded. fresh frozen plasma must be thawed before transfusion. a method for thawing canine fresh frozen plasma in a microwave oven has been described, but the author has found this unsatisfactory because of uneven heating by household microwave ovens. 60 plasma can be thawed at room temperature, and if the thawing time needs to be shortened, the plasma can be placed into a plastic bag and thawed in a 37 c water bath. the plastic bag is necessary to prevent contamination of the infusion ports in the water bath. if thawed and not used within 1 hour, it maybe refrozen with out loss of anticoagulant activity. 131 plasma should be used within 4 hours of thawing or discarded. transfusion recipients should be monitored during transfusion to allow early detection of a transfusion reaction. rectal temperature, heart rate, and respiratory rate should be recorded every 10 minutes during the first 30 minutes and then every 30 minutes thereafter. the patient should be monitored for vomiting, diarrhea, urticaria, and hemoglobinuria or hemoglobinemia. changes in vital signs or clinical status may indicate a transfusion reaction. patients developing volume overload will become tachypneic or dyspneic, and tachycardic. massive transfusions (1 blood volume in 24 hours) have been reported in both dogs and cats. 16, 62, 97 patients receiving massive transfusions of stored blood may develop specific abnormalities. consequently, patients receiving massive transfusions should be monitored for changes in serum potassium, ionized calcium, and ionized magnesium concentrations, as well as hypothermia and coagulation abnormalities. 16, 62, 97 an adverse effect of transfusion or transfusion reaction consists of the range of immunologic and metabolic changes that occur during or after administration of a blood product. four classes of adverse effects of transfusion have been described (box 24-3). acute transfusion reactions occur during or within a few hours after a transfusion, and delayed transfusion reactions occur after the completion of the transfusion. the delay may be hours to years. reports describing adverse effects of transfusion in dogs and cats are limited to case reports and retrospective series.* acute immunologic transfusion reactions occur because antibodies that elicit an immune response are present in the plasma of either the donor or recipient. the sequelae of an acute immunologic transfusion reaction are rapid, often irreversible, and sometimes fatal. current theories on the pathogenesis of acute hemolytic transfusion reaction in humans propose that hemolysis induces the release of cytokines, such as tumor necrosis factor, interleukin 1 (il-1), il-6, and il-8, complement, endotheliumderived relaxing factor (nitric oxide), and endothelin, resulting in the clinical syndrome of disseminated intravascular coagulation, shock, and acute renal failure. 9 the pathophysiology of acute hemolytic transfusion reaction in dogs and cats must differ in some manner from that described in humans because acute renal failure is not reported to be a feature in dogs and cats. 5, 37, 40, 132 the best example of an acute hemolytic transfusion reaction in veterinary medicine is the administration of type a red blood cells to a type b cat. in the recipient cat, naturally occurring alloantibodies and complement bind to the transfused red blood cells and cause hemolysis. clinical signs described in cats having an acute hemolytic transfusion reaction include fever, vomiting, lethargy, icterus, and death. 5 results of laboratory testing often show a positive coombs test, rapidly declining pcv, and increasing serum bilirubin concentration. dogs experiencing an acute hemolytic transfusion reaction show clinical signs similar but not identical to those observed in cats. most affected dogs exhibit fever, restlessness, salivation, incontinence, and vomiting. some dogs develop shock, and an occasional dog experiences acute death. plasma and urine hemoglobin concentrations increase within minutes of transfusion. incompatible cells are cleared from circulation in less than 2 hours. dogs whose red blood cells lack the dea 1.1 antigen that have previously been sensitized by transfusion of dea 1.1-positive cells are at the greatest risk for an acute hemolytic transfusion reaction. 40 other acute immunologic transfusion reactions reported in dogs and cats include nonhemolytic fever and urticaria. 17, 53, 64, 120 in humans, nonhemolytic fever is a result of antibodies against donor white blood cells, and urticaria occurs as a result of antibodies-against donor plasma proteins. nonhemolytic febrile transfusion reactions do not require treatment, but antipyretics may be used if the patient is uncomfortable (table 24 -2). urticaria is the most common reaction to plasma transfusion in dogs. 120 if urticaria caused by plasma administration is diagnosed, it should be treated with short-acting corticosteroids and antihistamines. the plasma transfusion then may be restarted at a slower rate and the recipient carefully observed. delayed immunologic transfusion reactions are classified as delayed hemolytic, transfusion-induced immunosuppression, posttransfusion purpura, and graft-versus-host disease. these reactions are not preventable by crossmatching or blood typing. delayed hemolytic transfusion reactions invariably occur in persons who have been previously sensitized to allogenic red blood cell antigens by transfusion or pregnancy. even though compatible blood is given to a patient, the recipient may develop antibodies against any one of the hundreds of red blood cell antigens present on the transfused cells. an anamnestic response to the antigens on the transfused red blood cells results in a delayed hemolytic transfusion reaction that occurs 7 to 10 days after a transfusion and is a well-described complication of red cell transfusion in humans. it has not been reported in dogs, but there is no reason it could not occur. fever is the most common sign of a delayed hemolytic transfusion reaction in humans. icterus also may be noticed 4 to 7 days after a transfusion. the only delayed immunologic transfusion reaction that has been reported in veterinary medicine is posttransfusion purpura occurring in a previously transfused dog with hemophilia a. 122 five to eight days after subsequent transfusion, thrombocytopenia and petechiation were evident. blood collected during a thrombocytopenic episode was positive for plateletbound igg, indicating an immune mechanism for platelet destruction. acute immunologic acute nonimmunologic transfusion reactions are caused by physical changes in the red blood cells during collection, storage, or administration. improper collection of blood can result in an adverse reaction to transfusion. collection of blood from an inadequately screened donor can result in transmission of bacteria, spirochetes, or protozoa and eventually clinical signs of the associated disease in the recipient. transfusion of blood contaminated by bacteria can cause shock, which is managed with volume expansion and pressor agents, as well as empirical antibiotic administration based on results of a gram stain. endotoxic shock results from transfusion of blood heavily contaminated with endotoxin-producing bacteria. clinical signs in cats transfused with blood contaminated by bacteria include collapse, vomiting, diarrhea, and acute death, but most cats did not exhibit clinical signs after receiving bacterially contaminated blood. 55 hypotensive shock developed in a dog that received a b. canis-infected transfusion. 18 during storage, the atp content of red blood cells decreases, and some cells undergo hemolysis resulting in leakage of potassium out of the cells into the storage medium. the increase in potassium in the storage medium is a contributing factor in the development of hyperkalemia in patients receiving large volume transfusions of stored blood. a large-volume transfusion of stored blood can cause hyperkalemia, but this is rare unless the patient has renal failure or preexisting hyperkalemia. 62 hyperkalemia in a transfusion recipient is as it would be in any patient with hyperkalemia. the transfusion should be discontinued and 0.9% nacl administered because 0.9% nacl does not contain added potassium and will facilitate renal excretion of potassium. intravenous administration of insulin, followed by administration of 50% dextrose and frequent monitoring of blood glucose and potassium concentrations until serum potassium concentration normalizes, is all that is necessary. physical damage (such as freezing or overheating) to red blood cells during storage causes hemolysis. while being transfused, the patient exhibits hemoglobinuria and hemoglobinemia without evidence of other signs of an acute hemolytic transfusion reaction, such as fever, vomiting, or collapse. during storage of blood, formation of clots or introduction of air into the bag may occur, resulting in embolism during transfusion. a rare adverse event associated with transfusion is an embolism. venous air embolism causes sudden onset pulmonary vascular obstruction, a precordial murmur, hypotension, and death as a result of respiratory failure. administration of large-volume transfusions can result in multiple adverse events. ionized hypocalcemia or ionized hypomagnesemia can result from the citrate used as an anticoagulant complexing with calcium or magnesium, and lead to myocardial dysfunction and potential cardiac arrest and tetany. 66 routine empirical administration of calcium to transfusion recipients cannot be recommended because of the risk of hypercalcemia and increased myocardial irritability, but animals with ionized hypocalcemia resulting from large transfusion should be treated with calcium gluconate or calcium chloride to effect. 26 hypothermia is common after large-volume transfusion in veterinary patients, and use of warming blankets should be instituted whenever possible. dilution of coagulation factors by large-volume transfusion of coagulation factor-depleted stored blood results in prolongation of coagulation times. in dogs receiving large-volume transfusions, prolongation of coagulation times is associated with a poor prognosis. 62 administration of fresh frozen plasma is indicated to correct the coagulation abnormalities. any transfusion can cause circulatory overload. dogs and cats with chronic severe anemia or compromised cardiac and pulmonary systems are at greater risk for circulatory overload and pulmonary edema than are those without cardiopulmonary disease. dogs and cats developing volume overload from transfusion are treated with oxygen supplementation, diuretics, and vasodilators. improvement should be seen within 1 to 2 hours. in humans, human immunodeficiency virus, hepatitis virus, and cytomegalovirus infections are documented as late effects of transfusion. one late complication of transfusion described in veterinary medicine is hemochromatosis. 104 a schnauzer received blood transfusions every 6 to 8 weeks for 3 years to treat chronic anemia. hemochromatosis was confirmed by necropsy when the dog was euthanized because of progressive liver disease. when an acute transfusion reaction is suspected, immediate intervention is critical because of the life-threatening nature of acute transfusion reactions. in all animals suspected of having some form of acute transfusion reaction, the transfusion should be stopped and samples of patient blood and urine obtained for baseline evaluation of biochemical, hematologic, and coagulation values. the unit of blood should be inspected to ensure it is from the appropriate species and is the intended unit based on the crossmatch or blood type. a gram stain and bacterial culture of the blood remaining in the blood bag should be submitted to the laboratory. urine can be visually inspected to determine the presence or absence of hemoglobin. rectal temperature of the recipient should be compared with the pretransfusion value. a transfusionassociated fever is defined as an increase in 1 f over the pretransfusion temperature. 118 the cardiovascular system should be monitored by electrocardiogram and blood pressure measurement. immediate evaluation of serum ionized calcium and potassium concentrations are critical, but certain electrocardiographic changes serve as surrogate markers of hypocalcemia (long qt-interval with a normal heart rate) or hyperkalemia (decreased height of p waves, loss of p waves, or widening of the qrs complex with large t waves) if rapid measurement of serum electrolyte concentrations cannot be obtained. venous access and blood pressure should be maintained by an infusion of a crystalloid solution such as lactated ringer's solution or 0.9% nacl. intravenous administration of short-acting glucocorticoids may suppress some of the mediators of acute hemolytic transfusion reactions and lessen the clinical progression, but their efficacy in transfusion reactions has not been evaluated in veterinary patients. when the evaluation of a patient with a suspected transfusion reaction suggests that an acute hemolytic transfusion reaction is occurring, the blood typing and crossmatching must be repeated to determine whether a laboratory error is responsible for the reaction. in patients with fever, without evidence of hemolysis, the transfusion may be restarted if the gram stain is negative for bacterial contamination. it is important to recognize the late effects of transfusion and not mistake them for another disease process. delayed transfusion reactions usually are managed with supportive care. the only specific treatment for a delayed transfusion reaction consists of treating a transfusionacquired infection appropriately. a special effort is not necessary to prevent transfusion reactions. by simply following the transfusion guidelines discussed here with reference to donor selection, blood typing, blood storage, and administration, most transfusion reactions can be prevented. crossmatching should be included in the guidelines for providing a safe blood transfusion. major and minor crossmatches detect antibodies in the plasma of the donor or recipient capable of causing an acute hemolytic transfusion reaction; however, a transfusion reaction may still occur despite a compatible crossmatch. crossmatching does not prevent sensitization to red blood cell antigens, which may result in a hemolytic reaction during future transfusions because it detects only antibodies that are currently present in the donor or recipient. it should be performed routinely in veterinary clinics either by a commercially available gel tube method (dms laboratories, inc., flemington, n.j.) or by the tube method. a tube crossmatch is described below. performing a crossmatch is an intimidating but simple procedure once all the equipment is assembled (box 24-4) . several descriptions of the procedure have been published, all of which describe the same basic procedure with minor variations. 14, 35, 103 not all protocols recommend the use of phosphate-buffered saline; others have an additional step at the end using species-specific coombs reagent to increase test sensitivity, and some recommend that tubes be incubated at 4 c, 37 c, and 42 c. the following is the protocol the author uses: 1. obtain edta-anticoagulated blood from the recipient and the potential donor or the tube segments of blood from the units being considered for transfusion. 2. centrifuge both donor and recipient blood for 5 minutes at 1000 g. 3. using pipettes, remove the plasma, and save in separate labeled tubes. 4. wash the red blood cells by adding phosphate-buffered saline to the red cells to fill the tube. resuspend the red cells in the saline by tapping the bottom of the tube with a finger. 5. centrifuge the red cells and saline for 5 minutes at 1000 g. pipette off saline, and discard. 6. repeat step. 4 and 5 twice. 7. after the third washing of the red cells in saline, resuspend the red cells to a 3% to 5% solution. it will appear bright cherry red. 8. for each potential donor, mix two drops of recipient plasma and one drop of donor red cell suspension for the major crossmatch. mix gently. 9. for each potential donor, mix two drops of donor plasma and one drop of recipient red cell suspension for the minor crossmatch. mix gently. 10. for the recipient control, mix two drops of recipient plasma and one drop of recipient red cell suspension. mix gently. 11. incubate the tubes at room temperature for 15 minutes. 12. centrifuge the tubes for 15 seconds at 1000 g. 13. observe the plasma for hemolysis. 14. resuspend the centrifuged cells by shaking gently. 15. observe the red blood cells for agglutination. interpretation. hemolysis or agglutination in a crossmatch indicates transfusion incompatibility. the degree of agglutination is graded 0 to 4þ (box 24-5 and figure 24 -2). units of blood that are incompatible should not be used. if all available units are incompatible, the least reactive unit should be chosen. when the recipient control shows hemolysis or agglutination, the crossmatch cannot be interpreted. this is common in patients with hemolytic anemia. multiple methods of blood typing dogs and cats have been described, including tube tests, typing cards, slide tests, immunochromatography, and gel tubes. 43, 106 a reference laboratory can perform blood typing for dea 3, 4, 5, and 7 and the recently described dal and mik of dogs and cats, respectively. in clinical situations in the united states, the commercially available blood typing cards for feline types a, b, and ab are commonly used (dms laboratories, inc., flemington, n.j.). however, when results indicate type ab, the results should be interpreted with caution as the card typing method commonly gives false positive results as type ab when the cat is actually type a. 7 any cat typed as ab should be confirmed by a second typing method. gel tube typing tests, available in europe, but not currently available in the united states, give accurate results when used in cats of type a, b, and ab. a simple immunochromatography method of blood typing has recently become available in the united states. 57 a paper strip impregnated with anti-a and anti-b monoclonal antibodies is placed in a red blood cell solution, allowing the cells to migrate up the strip and bind to the antibodies. results are rapidly available and easily interpreted. when using any blood equipment for performing crossmatch ) is an ultrapurified, polymerized hemoglobin of bovine origin (13 g/dl) in a modified ringer's lactate solution with a physiologic ph (7.8). the hemoglobin polymers range in molecular mass from 65 to 500 kda, with an average of 200 kda. the viscosity is low compared with blood (1.3 and 3.5 centipoise, respectively), and the solution is isosmotic (300 mosm/kg) with blood. the concentration of methemoglobin, the inactive form of hemoglobin, is 10%. oxyglobin can be stored at room temperature or refrigerated (2 c to 30 c) for up to 3 years. its intravascular half-life is dose dependent (18 to 43 hours, at a dosage of 10 to 30 ml/kg), as measured in healthy dogs. it is expected that more than 90% of the administered dose will be eliminated from the body in 5 to 7 days after infusion. the oxygen half-saturation pressure (p-50) of oxyglobin is greater than that of canine blood (38 vs. 30 mm hg, respectively). this increase in p-50 facilitates off-loading of oxygen to the tissues. the hemoglobin is packaged in the deoxygenated state in an overwrap that is impermeable to oxygen. complications of severe anemia result from poor oxygenation of tissues. restoration of adequate tissue oxygenation typically is achieved by administering a blood transfusion. improvement in the clinical signs of anemia results from a corresponding increase in hemoglobin concentration, which in turn increases the arterial oxygen content of the blood. the increased oxygen content of the blood supplied by oxyglobin also relieves the clinical signs of anemia. two prospective randomized trials have evaluated oxyglobin for the treatment of anemia. 95, 133 one was a multicenter clinical trial for dogs with moderate to severe anemia (pcv, 6% to 23%). 95 sixty-four dogs in need of blood transfusion were studied, including those with anemia caused by blood loss (n ¼ 25), hemolysis (n ¼ 30), or ineffective erythropoiesis (n ¼ 9). dogs in both groups were monitored for a decrease in hemoglobin concentration or deterioration in physical condition at which time they received additional oxygen-carrying support. if additional oxygen-carrying support was needed, oxyglobin-treated dogs received prbcs (n ¼ 1), and untreated control dogs received oxyglobin (n ¼ 19). treatment success was defined as the lack of need for additional oxygen-carrying support for 24 hours. the success rate in the 30 treated dogs (95%) was significantly greater than the success rate in the 34 control dogs (32%). this difference between treated and control dogs was significant, regardless of the cause of anemia. the other trial randomized 12 dogs with severe anemia (pcv ¼ 10% to 20%) secondary to babesiosis to receive either 20 ml/kg of oxyglobin, or packed red blood cells. 133 blood gas, acid-base, and blood pressure were objective measures of response to treatment. similar overall improvements were seen in both the oxyglobin and prbc transfusion groups. although oxyglobin is approved only for use in dogs, other species have been infused with the solution. oxyglobin administration to cats has been retrospectively evaluated. 34, 127 the median dosage was approximately 10 to 11 ml/kg/24 hours. oxyglobin has also been administered to other species to increase oxygen carrying capacity: mallard duck, miniature horse, and serval cat. 73, 82, 101 one published dosage for birds is 5 ml/kg iv or interosseously for the treatment of shock and for the treatment of shock in small mammals, 2 ml/kg as a 10 to 15 minute intravenous bolus followed by a continuous rate infusion at 0.2 to 0.4 ml/kg/hr. 74 because it lacks the antigenic red blood cell membrane, oxyglobin is not only useful in multiple species, but it eliminates some of the pretransfusion testing required with red blood cell transfusions. blood typing and crossmatching are not necessary because the red blood cell membrane, which is the major cause of transfusion incompatibility, has been removed during the manufacturing process. repeated dosing of oxyglobin was reported in both feline retrospective studies. 34, 127 no allergic reactions were reported. a laboratory study of repeated dosing in dogs showed antibodies to oxyglobin did form, but those antibodies did not decrease binding of oxygen to oxyglobin and did not result in systemic allergic reactions. 52 adverse effects of treatment with oxyglobin are similar in dogs and cats. after treatment, a transient discoloration (yellow, brown, or red) of the mucous membranes, sclera, urine, and sometimes skin occurs. overexpansion of the vascular volume may occur, especially in normovolemic animals. rates of administration greater than 10 ml/kg/hr in anemic, clinically ill dogs sometimes resulted in increased central venous pressure, with or without pulmonary edema or other respiratory signs of circulatory overload. pleural effusion and pulmonary edema were found commonly in cats given oxyglobin, but evidence was insufficient to directly link either to the administration of oxyglobin. 34, 127 cats recently administered blood transfusions or having underlying cardiac disease appear to be more likely to develop pleural effusion and pulmonary edema. low infusion rates are recommended in these cats (<5 ml/kg/hr). in the canine clinical trial, vomiting occurred in 35% of the treated dogs. diarrhea, fever, and death also were seen in approximately 15% of oxyglobin-treated dogs; however, an association with oxyglobin or the underlying disease could not be determined. these findings were most common in dogs with immune-mediated hemolytic anemia that received oxyglobin. the presence of oxyglobin in serum may cause artifactual changes in the results of serum chemistry tests. interference by oxyglobin depends on the type of analyzers and reagents used but is not typical of hemolysis. 18, 86 blood samples for analysis should be collected before infusion. a list of valid chemistry tests by analyzer is included in the product labeling. results of any clinical chemistry test performed on serum containing oxyglobin should be interpreted with consideration of the validity of the test. in general, all tests using colorimetric techniques are invalid, but other methodologies also show some interference. no interference is seen with hematologic or coagulation parameters except when optical methods are used for measuring prothrombin time and activated partial thromboplastin time. dipstick measurements (ph, glucose, ketones, protein) of urine are inaccurate when gross discoloration of the urine is present. the urine sediment is not affected. chronic enteropathies in dogs: evaluation of risk factors for negative outcome room temperature storage and cryopreservation of canine platelet concentrates blood type a and b frequencies in turkish van and angora cats in turkey frequencies of blood type a, b and ab in non-pedigree domestic cats in turkey blood transfusion reactions in the cat the ab blood group system in cats erythrocytic pyruvate kinase deficiency and ab blood types in australian abyssinian and somali cats a prospective, randomized, double-blinded, placebo-controlled study of human intravenous immunoglobulin for the acute management of presumptive primary immune-mediated thrombocytopenia in dogs treatment of severe immune-mediated thrombocytopenia with human iv immunoglobulin in 5 dogs serosurvey of anti-babesia antibodies in stray dogs and american pit bull terriers and american staffordshire terriers from north carolina canine dal blood type: a red cell antigen lacking in some dalmatians lack of evidence of pregnancy-induced alloantibodies in dogs use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability problems in veterinary medicine. philadelphia: jb lippincott storage of feline and canine whole blood in cpda-1 and determination of the posttransfusion viability massive transfusion and surgical management of iatrogenic aortic laceration associated with cystocentesis in a dog canine red blood cell transfusion practice 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red blood cells after vacuum facilitated collection evaluation of preservatives and containers for storage of canine blood use of biochemical measures to estimate viability of red blood cells in canine blood stored in acid citrate dextrose solution with and without added ascorbic acid adverse reactions suggestive of type iii hypersensitivity in six healthy dogs given human albumin hypotensive shock syndrome associated with acute babesia canis infection in a dog transfusion acquired yersinia enterocolitica survival of mycoplasma haemofelis and "candidatus mycoplasma haemominutum" in blood of cats used for transfusions use of a hemoglobin-based oxygen-carrying solution in cats: 72 cases problems in veterinary medicine. philadelphia: jb lippincott where to get blood donors? acute hemolytic transfusion reaction in an abyssinian cat with blood type b transfusion of type-a and type-b blood to cats frequency and inheritance of a and b blood types in feline breeds of the united states an acute hemolytic transfusion reaction caused by dog erythrocyte antigen 1.1 incompatibility in a previously sensitized dog frequencies of feline a and b blood types in europe geographical variation of the feline blood type frequencies in the united states comparison of various canine blood-typing methods evaluation of donor skin disinfection methods blood transfusion therapy: an updated overview blood type ab in the feline ab blood group system seroprevalence of antibodies against leishmania spp among dogs in the united states assessment of a dimethyl sulfoxide-stabilized frozen canine platelet concentrate anaplasma phagocytophilum and species of bartonella, neorickettsia and ehrlichia in cats used as blood donors in the united states canine blood groups and their importance in veterinary transfusion medicine incidence of canine serum antibody to known dog erythrocyte antigens in potential donor population absence of immunopathology associated with repeated iv administration of bovine 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of small animal blood component administration haemobartonella canis infection following splenectomy and transfusion use of adult cat serum to correct failure of passive transfer in kittens comparison of fluid types for resuscitation after acute blood loss in mallard ducks (anas platyrhynchos) emergency care and managing toxicoses in the exotic animal clinical indications for use of fresh frozen plasma in dogs: 74 dogs a treatise on the heart on the movement and colour of the blood and on the passage of the chyle into the blood babesia gibsoni infection among dogs in the southeastern united states the blood and plasma bank posttransfusion viability of feline erythrocytes stored in acid citrate dextrose solution serum antibodies against human albumin in critically ill and healthy dogs the use of 25% human serum albumin: outcome and efficacy in raising serum albumin and systemic blood pressure in critically ill dogs and cats use of a bovine hemoglobin preparation in the treatment of 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cord-318061-xe8lljz0 authors: overgaauw, paul a.m.; vinke, claudia m.; van hagen, marjan a.e.; lipman, len j.a. title: a one health perspective on the human–companion animal relationship with emphasis on zoonotic aspects date: 2020-05-27 journal: int j environ res public health doi: 10.3390/ijerph17113789 sha: doc_id: 318061 cord_uid: xe8lljz0 over time the human–animal bond has been changed. for instance, the role of pets has changed from work animals (protecting houses, catching mice) to animals with a social function, giving companionship. pets can be important for the physical and mental health of their owners but may also transmit zoonotic infections. the one health initiative is a worldwide strategy for expanding collaborations in all aspects of health care for humans, animals, and the environment. however, in one health communications the role of particularly dogs and cats is often underestimated. objective: evaluation of positive and negative one health issues of the human–companion animal relationship with a focus on zoonotic aspects of cats and dogs in industrialized countries. method: literature review. results: pets undoubtedly have a positive effect on human health, while owners are increasing aware of pet’s health and welfare. the changing attitude of humans with regard to pets and their environment can also lead to negative effects such as changes in feeding practices, extreme breeding, and behavioral problems, and anthropozoonoses. for the human, there may be a higher risk of the transmission of zoonotic infections due to trends such as sleeping with pets, allowing pets to lick the face or wounds, bite accidents, keeping exotic animals, the importation of rescue dogs, and soil contact. conclusions: one health issues need frequently re-evaluated as the close human–animal relationship with pet animals can totally differ compared to decennia ago. because of the changed human–companion animal bond, recommendations regarding responsible pet-ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conforming the biology of the animal are required. education can be performed by vets and physicians as part of the one health concept. the one health initiative or concept is a worldwide strategy that recognizes that public health is connected with animal health and the environment. it concerns multidisciplinary collaboration between physicians, veterinarians, environmental scientists, public health professionals, wildlife experts, and many others [1, 2] . with a multisectoral and transdisciplinary approach, public health threats can be better monitored and controlled. the resulting synergism enhances the knowledge of how diseases, known as zoonotic diseases, can be shared between animals and people with the goal of this article is based on an existing post-graduate course for veterinarians, vet technicians, family doctors, midwifes, and specialists such as pediatricians which explores healthy human-animal relationships. the existing evidence-based knowledge contained in this course has been actualized by performing a literature search to add new relevant publications. a literature search was conducted through 2 march 2020, using the national library of medicine's pubmed for the terms "one health" and "companion animals"; "pet ownership"; "households" and "pets"; "dogs" or "cats" or "pets" and "mental" or "physical health" or "children"; "animal assisted therapy"; "dogs" or "cats" and "nutritional problems" or "overweight" or "obesity" or "homemade" or "raw meat diets"; "dogs" or "cats" and "behavior problems" or "aggression" or "fear" or "anxiety" or "abnormal repetitive behavior"; "dogs" or "cats" and "breeding" or "genetic problems"; "dogs" or "cats" and "zooanthroponoses"; "pets" and "anthropomorphism"; "dogs" or "cats" or "exotic animals" or "rescue dogs" or "soil" and zoonoses. for some topics the internet was accessed and used as reference if additional information was not available as scientific publication. the authors selected articles that described pivotal and novel insights in the different topics. all searches were carried out without filters. the titles of all found articles were screened for relevance to the topic, and appropriate titles were assessed and selected based on their abstracts. if a selected article was a review, it was read and relevant citations were used to find primary literature on the subject. additional studies were found using the bibliographies of selected articles. occasionally, reviews were directly used as sources, mostly to convey background information that is not in the core focus of this article. original articles in english and different national languages (dutch, german, french, spanish, if available) were included. specific searches were made for citations dated after the year 2000 to ensure more recent literature on the topic had not been missed. a pet or companion animal is an animal that lives in or around the house and is fed and cared for by humans. until the 1960s, pets were mainly kept as utility animals, for example as draft dogs or watch dogs or for pest control when it comes to cats. due to major changes that have taken place in society since the second world war, such as increased leisure time and prosperity, but also individualization of humans, animals are nowadays kept as pets and are regarded by many owners as valued family members, e.g., over 90% in the united kingdom [13] . pet ownership is still increasing in many industrialized countries and these animals are more often considered a member of the family [5] . even in china, a country where pets were banned in urban areas until 1992, pet ownership has grown quite rapidly in the major cities. the rate of pet ownership of all usa households increased from 57.6% to 59% in 2018. dogs continue to dominate in popularity among american households. approximately 38% of households nationwide owned a dog, bringing the population of pet dogs to nearly 77 million, while 25% of households owned cats, with a total population of 58 million [14] . in 2018, an estimated 80 million european households owned at least one pet animal; 24% of households owned dogs and 25% owned cats [15] . there are 85 million pet dogs and 104 million pet cats in europe which is a 15% increase of dogs within 8 years (74 million dogs in 2010) and a 22% increase of cats (85 million in 2010) [16] . pet cats in europe thereby are more popular than dogs. an explanation of the higher popularity of cats may be the number of single-person households in the eu that rose on average by 2.0% per annum between 2006 and 2016 to 32.5%, and the growth of two-family households grew by 1% to 31% in this period [17] . also, dual-earner families are widespread as a result of quite a steep growth in female employment over the past two decades [18] . when animals live with humans, they too benefit from human interaction. over the past decades, animal welfare has evolved to recognize that animals are sentient beings capable of experiencing positive and negative emotions. the social and ethical dimensions of animal welfare, which are concerned with how human society morally regards and treats non-human animals, are also increasingly being recognized [19] . in dutch law, the intrinsic value of kept animals is expressly incorporated and used as a guiding ethical principle that forms the basis of any further legislation. the intrinsic value is thereby defined that animals are sentient beings that can feel pain and discomfort. therefore, they should be kept free of stress, pain, disease, hunger, thirst, and should be able to show natural behavior known as the five freedoms of r. brambell 1965 [20] . in industrialized countries animal keepers, their owners, are legally required to provide such circumstances and can be prosecuted if they infringe the law. of course, in the field there is animal abuse, negative animal welfare conditions, and animal diseases. however, in general, caretaking for companion animals is nowadays performed at a high level. new insights into animal behavior has had its influence on the general public. for example, owners are aware or are told by vets and pet shops that rabbits should not be kept alone but at least in pairs due to their need for social contact [21] . there are various reasons to keep pets, such as love, warmth, and companionship. companion animals have an important emotional value, and promote the socialization of the lonely elderly because they facilitate additional contact with people. pets form a goal in life, reduce stress, and ensure that the owner keeps physically active. around 95% of dog owners and 93% of cat owners expressed that owning their pet makes them happy and 44% of owners selected this as one of the reasons they got their pet in the first place [22] . however, the function of companion animals consists of more than just providing a socializing being. studies show other benefits of having a pet, such as the positive effect on individuals' mental and physiological health status. most research addressing the health benefits of pet ownership show reductions in distress and anxiety, decreases in loneliness and depression, and increases in physical condition [23] . the positive benefits to human health from interacting with animals, focusing on the companion animal, have also be described with the term "zooeyia" [24] . in fact, 63% of owners agreed that having a pet makes them physically healthier, with dog owners more likely to agree, most probably because dog owners exercise more (85% agreeing, compared to 41% of cat owners). besides, 84% of owners agreed that having a pet makes them mentally healthier. expressed reasons are the non-judgmental nature of their pets, their playfulness, or physical contact [22] . another demonstrated positive influence is the blood pressure and heart rate lowering effects that occurs when stroking a friendly-looking dog or even being in the presence of a friendly animal, while it is not necessary to own a pet to obtain these stress-moderating benefits [23] . many studies have demonstrated the association between pet ownership and cardiovascular health and dog owners appear to have a significantly greater chance of survival after a heart attack compared to people without pets [25] [26] [27] . pets can therefore play an important role in reducing absenteeism and visits to family doctors or the hospital [28] . it has been estimated that pet ownership saved australia $1 billion in 1994 [29] , while it may reduce the use of the national health service (nhs) in the uk to the value of £2.45 billion per year [30] . dogs also play an increasing role as co-therapist or as supporter for people with psychological or physical disabilities. the benefits of these animal-assisted activities are improved mood, decreased physiological distress, depression, dementia, and loneliness [31, 32] . examples include resident or visiting dogs in prisons, nursing homes [33] , mental institutions, and hospitals where they can reduce patient anxiety in a hospital emergency department [34] , reduce pain perceptions in children after surgery [35] , or calm young patients at a pediatric dental clinic [36] . since dogs have extremely sensitive noses, they are used for several purposes such as tracking, bomb detection, and search and rescue. in recent years, canine olfaction has also been more recognized as a diagnostic tool for identifying pre-clinical disease status, such as diabetes (ketones), different forms of cancer, and infections from biological media samples [37] . animal-assisted therapies can act as co-therapies to facilitate psychotherapy or to provide specific types of therapeutic interventions such as improving motor skills or behavior [23] . such interventions were effective in improving the state of children or adults with or at risk of developing mental disorders such as attention deficit hyperactivity disorder (adhd), post-traumatic stress disorder (ptsd), or autism spectrum disorder (asd) [38] [39] [40] , and for the treatment of ptsd in military veterans [41, 42] . assistance or service animals are trained to perform tasks for the benefit of individuals who have disabilities such as hearing loss, physical disabilities, emotional disabilities, seizure disorders, or diabetes [23] . finally, a wide range of emotional health benefits from childhood pet ownership has been identified, particularly for those suffering from low self-esteem and loneliness. there is evidence of an association between pet ownership and educational and cognitive benefits, increased social competence, social networks, social interaction, and social play behavior [43, 44] . significantly less absenteeism from school through sickness among children who live with pets has also been reported [13] . having a dog or cat in the house during the first year of life may protect against childhood asthma and allergy [45, 46] . it can therefore be concluded that companion animals contribute significantly toward the public health, but also increasingly, the health of individually challenged persons through animal-assisted interventions [47] . providing companion animals with feed by humans, has been considered an advantage for companion animals in their relationship with humans. however, the feeding practices can also have a negative impact on companion animals [48, 49] . obesity in cats and dogs is a disease which is rapidly increasing with significant and lifelong implications for animal welfare. although no universally accepted definition of canine and feline obesity exists, the american veterinary medical association defined obesity being more than 30% above the ideal weight of an animal. overweight is defined as 10%-20% above the ideal weight. using body condition scores, it has been estimated that in the united states, 54% of dogs and 59% of cats are obese or overweight. a study in the united kingdom reported 65% of adult dogs and 37% of juvenile dogs as being obese or overweight [50, 51] . overweight dogs are more likely to be diagnosed with, e.g., urinary tract diseases. obese and overweight dogs are at risk developing orthopedic disorders and hypothyroidism [52, 53] . obese cats are at higher risk for developing urinary tract disease, diabetes mellitus, and neoplasia [54, 55] . other diet-related problems in companion animals can be caused by the changed feeding behavior of humans, e.g., by providing companion animals with bone and raw feed (barf) or vegan diets. risks for companion animals associated with barf or vegan diets are the presence of microbial hazards, insufficient nutrition, and in raw meat diets the presence of risk materials like thyroid tissue. through contact with their animals, it is possible that risks could even develop for owners. there could be an increased risk of human salmonellosis because of the presence of salmonella spp. in the diet which can spread to humans through diet leftovers or by contact with animal feces. recently a review was published on the risks of barf feeding [56] . the authors concluded that the data for the nutritional, medical, and public health risks of raw feeding are fragmentary, but they are increasingly forming a compelling body of formal scientific evidence. publications were found reporting the presence of escherichia coli o157, salmonella typhimurium, campylobacter spp., and antibiotic resistant bacteria in the feed. nutritional problems, such as calcium/phosphorous imbalances and specific vitamin deficiencies [57] are also reported. moreover, homemade diets are inherently susceptible to nutritional imbalances and deficiencies [58] . awareness about climate change, public health and animal welfare has incited a major change in dietary choices among many individuals. the number of vegans in the world keeps growing, even quadrupling from 150,000 to 600,000 individuals between 2014 and 2019 in affluent countries such as the uk [59] . the popularity of veganism goes beyond the scope of the human diet, as more people are interested in the possibility of feeding their companion animal a vegan diet than ever before. to create animal-free complete cat food requires replacing nutrients in animal-based materials with plant-based materials. different sources are used such as corn, rice, peas, soy, potato, and different oils and seeds. any further nutrients that are missing from plant-based materials, such as taurine and carnitine, are replaced with synthetically produced versions [60, 61] . feeding trials using vegan animal food are either not performed due to testing costs or kept private due to the highly competitive vegan pet food market [62] . additionally, they reported testing 24 vegetarian diets for cats and dogs and found that one was lacking protein and six did not meet all amino acid concentration requirements. vegan animal food may not contain meat, but it does contain grains, soy, and corn. plant-based products, such as grains, can be a source of health problems because of the presence of mycotoxins, for example [63] . warm, humid storage conditions can lead to the formation of mycotoxins such as aflatoxins, produced by the fungi aspergillus flavus and aspergillus parasiticus. many regular animal feeds also contain plant-based products, therefore the negative impact of feeding vegan diets to companion animals, especially obligate carnivores such as the cat or the ferret, seems therefore more related to diet insufficiency than to microbial health risks. additionally, addressing behavioral problems, the "free" provision of food might fulfil the consumptive part of feeding behavior of our companion animals but does not fulfil the appetitive phase. especially this phase of feeding patterns can have consequences for the companion animals' mental health and may result in behavioral problems if appetitive physical and mental challenges remain chronically absent in the human-animal relationship. in the human-companion animal bond, pets may develop abnormal behavior, including excessive aggression, fear and anxiety, or even abnormal repetitive behavior. abnormal repetitive behavior (arbs) were first noticed in zoo-, shelter-, and laboratory animals: all animals housed under stimulus-poor conditions and with limited space. however, companion animals can develop arbs as well, if the individual's adaptive capacity is exceeded due to, e.g., a lack of social contact, physical exercise, mental challenges, and in uncontrollable and unpredictable environments (e.g., separation, mistreatment, or inadequate application of cages). arbs can either be classified in stereotypies or compulsive disorders [64] with stereotypies generally defined as unvarying repetitive behavior patterns with no obvious goal or function [65] . the terminology of compulsive disorders is preferably chosen for repetitive behavior patterns that are goal-directed and show variability in the repetitive (motor) patterns [66, 67] . under chronic conditions without possibilities to adapt (cope), companion animals may develop stereotypies or compulsive disorders like, e.g., tail chasing, polyphagia, compulsively self-directed licking and/or biting the coat [68] , or feather pecking in parrots [69] . self-directed patterns can result in serious degrees of alopecia, lick granuloma, or even self-inflicted injuries (auto-mutilation) with a risk of infection. two main reasons underlie the development of arbs in our companion animals. first of all, a lot of companion animals are social species eager for social contact. in the human-companion animal bond, the need for social contact with either conspecifics and/or humans [70, 71] can remain unfulfilled if owners work from nine to five, five days a week with the pet staying alone at home on a daily basis. on the other hand, a cat which is originally a solitary hunter with a complex dynamic social structure may start overgrooming or house soiling in the presence of another cat in the territory. such situations might occur in multi-cat households, in the presence of neighboring cats, and in in-stable grouped housing conditions in shelters [72, 73] . secondly, most companion animals are species that are eager for mental and physical challenges on a daily basis. the lack of foraging opportunities, the appetitive phase of feeding behavior [74] might be another reason for the possible development of arbs in companion animals. foraging is often regarded as a high priority behavior [75, 76] , i.e., an internally motivated behavioral pattern that should be performed, or otherwise may induce a state of chronic stress, which may result in behavioral pathology like arbs as described in many other animal species [77] [78] [79] . foraging patterns may include walking, running, jumping, nose pushing, digging, and overseeing the area, all active patterns that imply the daily need for physical exercise and mental challenges in most of our companion animals. nonetheless, our pets mostly, if not always, get their food for free with minimal foraging challenges, except for going out 3-5 times a day. for some individuals (and especially some dog breeds, e.g., malinois, border collies, and pit bull terriers) [80, 81] , situations and contexts with limited challenges can make them more vulnerable to the development of arbs. as well as arbs, other problematic behavior may develop in our companion animals, and the prevalence of some of this behavior is even higher than that of arbs, for example excessive interspecific and/or intraspecific aggression, fear, and anxiety. at what moment, and which type of problem behavior may develop, depends on the intermingled factors of, e.g., genetics, early life experiences (maternal-child bonding, weaning, socialization [82] ), daily environment, and multiple factors in and around the human-companion animal bond. the history of breeding animals goes back to a time when humans and animals shared each other's habitat. dogs originally have been selectively bred to support human needs, such as hunting, herding, obedience, guarding, rescuing, and for companionship. this artificial selection has generated a large number of dog breeds, displaying a large variation of behavior, size, head shape, coat color, and coat texture [83, 84] . unfortunately, in the last 100 years, intensive selection for extreme looks and a narrow gene pool of many breeds has interfered in the genetic make-up of dogs, leading to unfavorable anatomy (extreme large, or extreme "teacup" small), and genetic predisposition to numerous health, welfare, and behavioral problems [85] . over 700 inherited disorders and traits have already been described in the domestic dog [86] . one type of dog with a distinct dysmorphology is the brachycephalic dog. brachycephalic dogs are characterized by a large head and round face due to a shortened muzzle, a high and protruding forehead, and widely spaced large eyes. these facial features fit the concept of baby schema ("kindchenschema") proposed by konrad lorenz [87] . infantile (cute) faces are biologically relevant stimuli for rapidly and unconsciously capturing attention and eliciting positive or affectionate behavior, including the willingness to care [88] . the appeal of brachycephalic animals has led to specimens that are the so-called "over-typed" dogs and cats with a too short nose, excessively protruding eyes, too straight angulations, etc. breeding animals with this type of severe skull and muzzle abnormalities leads to physical and physiological hardship and limits their natural behavior [89, 90] . this violates their integrity and is a big risk for their welfare. selectively breeding animals in order to express specific traits does not only alter existing animals, but also creates new ones, turning animals into an instrument for human use [91] . the bambino sphynx cat is an example of so called "mutant breeding", where breeders deliberately stack in two steps the recessive inheriting mutations, which leads to hairlessness in sphynx cats, on the dominant inheriting (lethal) mutation responsible for the shortened legs of the munchkin cat. the lack of hair in combination with short legs interferes with the normal physiology of the cat with regard to the manner of movement, thermoregulation, and skin health. one may argue that artificial selection in exchange for money, status, or aesthetic reasons violates the animal's dignity and integrity [92] . conclusively, artificial selection for excessive traits can have direct consequences for individual health and welfare, may obstruct and prevent a pet from fulfilling its behavioral needs, and conflicts with the current moral way of thinking on animal dignity and integrity. pet animals are not only perceived in 80%-90% as family members or partners but are almost treated like humans. in one study, up to 62% of owners agreed with the statement "my dog is more important to me than any human being" [93] . this kind of behavior is the result of the attribution of human cognitive processes and emotional states to animals, such as feelings of happiness, love, or guilt. people believe that animals have awareness, thoughts, and feelings. this behavior is called anthropomorphism, personification, or humanization and can also be applied to plants, gods, or objects. anthropomorphism appears to be caused by the perceived similarity between humans and animals and the extent to which people have developed an affectionate bond with their dogs and cats [94] . human empathy provides the basis for the attribution of empathy to other animals, as well as attributions of the communicative ability of other animals [95] . anthropomorphistic behavior can be harmless, such as talking to pets, which many owners do and one of the reasons for this may be the unique ability of humans to recognize facial expressions. talking to pets is also found to be linked to social intelligence [94] . however, it can lead to animal welfare problems when the feelings of owners no longer match the needs and the intrinsic values of their animal. examples of this are designer dog clothes, animal perfumes, and jewelry, thought the use of protective coats in colder climates for small, short-haired breeds, e.g., chihuahuas, is considered useful. the large number of obese pets can also be partly attributed to anthropomorphism. studies from north america, europe, and australia to determine what proportion of animals, mainly dogs, are overweight or obese reported prevalences of between 22%-44% [96] . in 2018, an estimated 60% of cats and 56% of dogs in the usa were overweight or obese [50] . when the owner takes a treat with coffee, it is believed that the dog should also get it. even chocolate treats are given, when these are potentially fatal for dogs and cats. this also applies to a good meal that is shared with the pet. dog owners who did not consider obesity to be a disease, maybe because the facial features of their pets fit the concept of baby schema [87] , were more likely to have obese dogs [97] . an often-unrecognized risk for pets is reverse zoonotic disease transmission, the so-called zooanthroponosis. a review on this subject reported 56 articles dealing with human to animal disease transmission [98] . most of the articles dealt with bacterial pathogens but also viral, parasitical, and fungal pathogens were studied in these publications. animals reported to have been infected or inoculated with human diseases included wildlife, livestock, companion animals, and other animals or animals not explicitly mentioned. the majority of the studies focused on human to wildlife transmission with an emphasis on mycobacterium spp. for companion animals, mrsa-infection was especially reported but m. tuberculosis, influenza a, and candida albicans were also discussed. for all groups of animals, microsporum spp. and trichophyton spp. were identified as infectious agents originating from humans [99] . recent publications report a different kind of zooanthroponosis: the transmission of high-risk, multidrug-resistant pathogens from humans to animals [100] . a major issue mentioned is the transmission of high-risk clones of extended-spectrum beta-lactamase (esbl) producing bacteria including escherichia coli, enterobacter cloacae, and klebsiella pneumonia [100, 101] . the transmission of carbapenem-resistant ndm-5 producing e. coli from previously hospitalized humans to dogs has also been suggested [102] . transmission of hospital acquired antibiotic resistant bacteria from human patients to their pets has been confirmed, such as the vim-2 producing pseudomonas aeruginosa st233 strain in brazil [103] . this increased transmission of high-risk multidrug-resistant pathogens from humans to animals was related to the closer relationships between humans and companion animals. some authors doubt the generalized pet-effect on human mental and physical health because of conflicting results that are prevalent in this area of science and the lack of publication of negative results [13, [104] [105] [106] . the majority of research evidence was also considered inconclusive due to methodological limitations such as reliance on self-reports, small sample sizes that may not be representative of the general population, homogeneous populations, varying research designs, narrow range of outcome variables that were examined, and the use of cross-sectional designs that do not consider long-term health outcomes [107] [108] [109] . other studies found for example that pet ownership was associated with a higher incidence of heart attacks and readmissions in heart attack patients instead of a lower incidence [110] or that pet owners had higher diastolic blood pressure than those without pets [111] . müllersdorf (2010) showed that pet owners had better general health but suffered more from mental problems such as anxiety, insomnia, and depression, than those who did not own pets [112] . other studies failed to support earlier findings that pet ownership is associated with a reduced use of general practitioner services [113] or psychological or physical benefits on health for community dwelling older people [114] . negative effects of pet ownership include dog and cat bites or scratches, the spreading of disease (zoonoses), and fall injuries, caused by falling or tripping over dogs and cats [115] . allergic reactions may be a consequence of animal contact and affect 15%-30% of individuals (often genetically) predisposed [116] . allergies relating to more uncommon pets such as fish, birds, and amphibians seem to be increasing in prevalence [117] . other studies prove that pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged 6-10 years. therefore, advice to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given [118] . there are also less-positive effects that pets can have on health. more excessive forms of anthropomorphism became clear in our study for the presence of zoonotic parasites in healthy dogs and cats. fifty percent of owners allow pets to lick their faces. sixty percent of the pets visit the bedroom; 45-60% (dogs-cats) are allowed on the bed, and 18-30% (dogs-cats) sleep with their owner in bed. six percent of pets always sleep in the bedroom. of the cats, 45% are allowed to jump onto the kitchen sink [119] . this means that in addition to the detected zoonotic parasites (the hazard), there was a significant potential exposure to these pathogens. in addition to parasites, other pathogens such as bacteria, viruses, and fungi can also be transmitted by animals by direct contact through biting, licking, scratching, sneezing or coughing, handling pets or their body fluids or secretions and by indirect contact through contaminated bedding, food, water, or bites from an arthropod vector [120] . not every individual will develop symptoms after being infected with a zoonosis. this is the result of various factors such as the causative pet species, housing, the degree of contact and contamination, the ability of a micro-organism to cause disease in humans and animals, but especially due to the degree of immunity of the recipient. in order to assess the risk of disease transmission from pets it is important that the nature and frequency of contacts between pets and their owners or other people are evaluated [120] . we traditionally know that young children (age < 5 years), the elderly (age ≥ 65 years), patients with an impaired immunity, and pregnant women that carry a fragile fetus are at more than average risk of becoming ill after an infection. moreover, they may have more severe disease, have symptoms for a longer duration, or develop more severe complications compared to other patients. young children (notably those aged 3-5 years) and some people with developmental disabilities often have suboptimal hygiene practices or higher risk contact with animals which further increases risk [121] . in children, hand-to-mouth behavior is part of their natural development and they mouth their fingers and other objects. in a meta-analysis, the average indoor hand-to-mouth frequency ranged from 6.7 to 28.0 contacts/hour and the average outdoor frequency ranged from 2.9 to 14.5 contacts/hour. the lowest value was attributed to the 6-to 11-year-olds and the highest to the 3-to < 6-month-olds [122] . fifteen percent of dog owners and 8% of cat owners always wash their hands after contact with the animals [119] . in addition, due to improved healthcare in recent decades, the group of immunocompromised patients has increased sharply. this includes, for example, patients with diabetes, post-splenectomy, after placement of implants and patients being treated with chemotherapy or immunosuppressants. the risk groups are also referred to as yopis (young, old, pregnant, and immune suppressed). patient surveys and epidemiological studies suggest that the occurrence of pet-associated zoonotic disease is low overall [121] . many of these pathogens are not reportable and presumably underdiagnosed or not recognized by family doctors due to the general, mostly flu-like, symptoms. therefore, any reported frequency of such infections is likely underestimated. to get a better picture of zoonotic risks, a risk analysis is required where a risk score can be calculated using exposure, contagiousness of the infection, and its consequences in the human. in addition, the disease burden of an infection for the population can be calculated and expressed in disability adjusted life years (dalys). this quantifies the health loss based on two components: the life years lost due to premature death and secondly the proportional loss of quality of life as a result of the disease. there is a trend towards closer physical contact between owners and their pets or their environment which poses an increased risk of transmission of zoonotic pathogens. these trends (a general direction in which something is developing or changing) will be explained. our publication [119] showed that a high percentage of pets were allowed in the bedroom and in bed, with 6% always sleeping in bed with their owner. is that a problem? already from a hygienic point of view, it is not advisable to sleep with animals or take them into bed. they do not pay attention to where they are walking outside and do not wipe their feet after arriving home. dogs like to roll in carcasses and both dogs and cats regularly lick the anus and thereafter the fur. in a pilot study with 28 healthy dogs and 22 healthy cats that slept with their owners, we tested 68% of the dogs (19) and 32% of the cats (7) positive for enterobacteriaceae on the fur or footpads. fleas and flea larvae were found on 14% of pets [123] . as a result of our publication, chomel investigated in 2011 whether transmission of infections by sleeping with pets and licking the face could be found in literature. he reported that also in the usa, france and the uk, a relatively large number of pets slept in bed with their owner (14%-33% of dogs and 45%-60% of cats) [124] . similar results of sleeping with pets were also reported from canada (26% of pets slept with children), the czech republic (45%), and qatar (63.3%) [125] [126] [127] . chomel found bacterial infections such as yersinia pestis (plague), bartonella henselae (cat scratch disease), methicillin-resistant staphylococcus aureus, and sometimes fatal bite wound infections such as capnocytophaga canimorsus and pasteurella multocida. furthermore, parasite infections such as cheyletiella spp. were reported. feline cowpox is a rare viral infection, but it can be transferred to the human after direct contact. both animals and humans reveal local exanthema on arms and legs or on the face. in most cases the disease is self-limiting, but immunosuppressed patients can develop a lethal systemic disease resembling smallpox [128] . it can be concluded that, although uncommon with healthy pets, the risk of transmission of zoonotic agents by close contact between pets and their owners through bed sharing is real and has even been documented for life threatening infections such as plague [129, 130] . although pets do not transmit arthropod-borne diseases to people (e.g., lyme borreliosis, ehrlichiosis, anaplasmosis), they do bring zoonotic disease vectors such as ticks and fleas, in close proximity to people, e.g., when they are sleeping with their animals [121] . while fleas are considered a vector of bartonella henselae (the causative agent of cat scratch disease) tickborne diseases are reported as increasing as ticks expand their ranges [131, 132] . with an estimated 65,000 cases a year, lyme borreliosis is responsible for the largest disease burden of any vector-borne disease in the european union [133] . another increased risk associated with close contact with fur is when it is contaminated with zoonotic parasite eggs. especially with echinococcus multilocularis (fox tapeworm) or e. granulosus (hydatid worm, or dog tapeworm). these eggs are immediately infective and may cause serious health problems in the human, many years post infection [134, 135] . despite a low prevalence of infectious (embryonated) eggs of toxocara spp. on dog's fur, the potential zoonotic risk should not be disregarded [136] . the same risk is applicable for the persistence of sporulated toxoplasma gondii oocysts in dogs' fur [137] . in relation to this, it is noteworthy that many publications report striking increases of ringworm, a common zoonotic fungal skin infection in mainly children caused by microsporum spp., trichophyton spp. or arthroderma spp., where the presence of pets is always mentioned [94] . however, nowhere has it been suggested that close contact with infected pets in bed increases the infection risk [138, 139] . rodents or rabbits are mainly infected with t. mentagrophytes, while m. canis is primarily found in dogs and cats. infection occurs by direct or indirect contact with infected hair, scales, or materials. infected animals may be asymptomatic carriers without clinical signs. examples are 16% m. canis carriage in a study of european cats [140] , 27% in suspected brazilian cats [141] , and the isolation of t. mentagrophytes dermatophytes from 4% of clinically healthy rabbits and 17% of guinea pigs in dutch pet shops [142] . licking the face of humans by mainly dogs is an expression of their naturally submissive, positive social behavior. the owner is recognized by the dog as the dominant superior in the ranking. in a pack of dogs, submissive dogs lick their dominant counterparts at the corners of the mouth from a typical submissive attitude [143, 144] . owners apparently allow this as a token of affection from their pet. such behavior is more common in young animals and has been considered as attention-seeking or care-soliciting gestures. it indicates to the owner the strength of the social bond between dogs and people [120] . licking or nudging of veterans by service dogs may help take their mind off any negative thoughts, emotions, or memories that they might be experiencing [145] . on the internet, many images can be found of mainly dogs, but also cats and even rats licking their owner's face. various studies show that around 40%-50% of owners allow this [119, 120] . the question is whether this is harmful due to the potential transmission of infections. the review by chomel (2011) shows in the literature that infections, especially pasteurella spp. and capnocytophaga canimorsus, were reported to have transmitted to humans by dogs, cats, kittens, and rabbits [124] . pasteurella multocida meningitis has been reported in 36 infants where 87% had been exposed directly or indirectly to the oropharyngeal secretions of household dogs or cats through licking or sniffing [146] . zoonotic transmission via this route is also assumed for various other pathogens such as gastric helicobacter spp. [147] , periodontal pathogens [148] , and bartonella henselae the etiological agent in cat scratch disease. the b. henselae bacteria may cause ocular complications, including parinaud oculoglandular syndrome, a severe eye infection. the route of infection is unknown, although direct conjunctival inoculation, most likely with infected flea feces, seems to be most plausible [149] . knowing, however, that b. henselae is present in up to 40% of cat saliva [150] , it is more plausible that salivary fluid could be rubbed directly into the eye from the skin after been licked by a cat. there are several anecdotal reports of infections in mainly young children that were transmitted by being licked. one recent example is an 8-month-old baby that presented with fever and preseptal cellulitis with purulent discharge. the causative agent was surprisingly corynebacterium bovis, a bacterium that is normally found in bovine mastitis. it became clear that the dog was frequently allowed to lick the baby's face and was fed on raw meat [151] . there is an ineradicable belief among a large part of the public that the licking of human wounds by dogs can disinfect them and that the saliva thereby has healing properties [152] . in addition, it is regularly reported that a dog's tongue is believed to be sterile. this is of course not the case and the oral flora of a dog comprises hundreds of species (including pathogenic) bacteria, fungi, and viruses [153, 154] . various wound healing saliva components have indeed been demonstrated in human and animal studies [155, 156] . the bactericidal effects of male and female dog saliva facilitate the hygienic function of maternal licking of the mammary and anogenital areas by protecting newborns from fatal coliform enteritis caused by e. coli and neonatal septicemia caused by streptococcus canis. however, the saliva is only slightly, and non-significantly, bactericidal against wound bacteria such as coagulase positive staphylococci and pseudomonas aeruginosa [157] . capnocytophaga canimorsus and pasteurella multocida are common commensals in the oral cavity of dogs, cats, and other species [158, 159] . transmission has been reported after the licking of mucous membranes or open wounds [160] [161] [162] . in patients at high risk, severe wound infections, sepsis, disseminated intravascular coagulation, or death can occur. patients with immunodeficiency, splenectomy, or alcohol dependence are at a particularly increased risk of infection with c. canimorsus [159] . even immunocompetent persons who have been licked by a dog can develop fatal sepsis [163] . a further negative effect of companion animal ownership is, of course, accidents inflicted on humans by these animals. these accidents can involve tripping over a cat or being dragged by an enthusiastic dog, but in literature, most evidence points towards biting and scratching incidents. dog biting and cat scratching incidents can cause physical health problems both at the time of infliction but also afterwards by triggering trauma-related secondary infections. dog biting incident reports are numerous, and numbers vary from country to country. in the uk, 18.7 dog bites per 1000 population per year were reported [164] , while a commission in the netherlands reported in 2008, 150,000 bite accidents per year in a population of 16 million (9 events per 1000 inhabitants) [165] . children are especially vulnerable to dog bites. the majority of dog bites occurred in children 5 years of age or younger (68.0%) and almost all (89.8%) of the dogs were known to the children [166] . recently, a systematic review has been published that analyzed more than 26,000 bites from the literature of the past 30 years about the risk of bites relative to specific breeds of dogs, combining bite incidence with bite severity [167] . the analysis by breed revealed that pit bulls were responsible for the highest percentage of reported bites across all studies (22.5%), followed by mixed breed (21.2%), and german shepherds (17.8%). dog bite incidents can result in medical treatment, hospitalization and even death. in the netherlands it was calculated that from the 150,000 dog bite victims per year, around 50,000 seek medical attention and 230 are hospitalized [165] . between 3% and 30% of dog bites become infected and complications become more severe when infection occurs. more than 100 species of bacteria have been isolated from bacterial infections of dog bites, suggesting that most oral flora of dogs have the potential to be pathogenic [168] . the top 3 pathogens found are pasteurella, staphylococcus, and streptococcus. in literature, specific attention is given to wound infections caused by capnocytophaga canimorsus, because this bacterium is seen as the relatively deadliest pathogen. it was suggested that only 2% of dog bite wounds contained capnocytophaga spp. [154] while others reported infection percentages of 4.2% [169] . wound infection with this bacterium can lead to severe complications like septicaemia, meningitis, osteomyelitis, peritonitis, endocarditis, pneumonia, purulent arthritis, and disseminated intravascular coagulation. c. canimorsus septicaemia has been associated with 30% mortality. the true number of c. canimorsus infections is probably largely underestimated due to the fastidious growth of the organism. however, infected dog bites in predisposed persons should be taken seriously especially after splenectomy [170] . cat bite incidents occur less frequently. in only 5%-10% of reported bite incidents in australia, cats are to blame. the long incisor teeth inflict less severe superficial wounds but because of the penetrating effect, joint and tendon infections more easily occur. in a review it is reported that 28%-80% of cat bites become infected mostly by pasteurella multocida [169] . the bacterium bartonella henselae can also be transmitted by cats through biting incidents but the transmission of this bacterium is much more related to a cat scratch accident. even less frequently reported animal biting incidents are those inflicted by rodents (2% of cases in australia). these bites have an infection rate of approximately 10%. this could result in rat bite fever in humans, an infection with streptobacillus monoliformis or spirillum minus, characterized by the triad of fever, rash, and arthritis [169] . cat scratch disease (csd) was first described in a french boy in 1931 and is a common, often self-limited, disease that usually presents as tender lymphadenopathy caused by bartonella henselae [171] . the cat is considered the primary reservoir for this bacterium, with infected fleas and ticks serving as vectors and humans and dogs as accidental hosts. vector transmission of this bacterium occurs via two primary routes: inoculation of bartonella contaminated arthropod feces via animal scratches, most often cat scratches, or by self-inflicted contamination of wounds induced by the host scratching arthropod bites [172] . immunocompromised human hosts (kidney transplant patients or patients with hiv) are especially susceptible to infection. in these individuals, the disease may be present as a more disseminated form with hepatosplenomegaly, meningoencephalitis, or angiomatosis [173] . an increasing number of pocket pets and exotic pets are kept by humans. numbers of ornamental birds in europe are estimated as 50 million, fish tanks 15.5 million (300 million ornamental fish), small mammals 27 million, and reptiles 8 million [15] . these animal species can also be a source of many zoonotic diseases, especially in young children and immunocompromised individuals. most cases of these conditions are not serious, and deaths are very rare but some of these diseases can be life threatening, such as rabies, rat bite fever infections, and plague [174] . however, there is also a trend to keep more unusual exotic animals, legally or illegally. these are "wild" animals that are kept in the home such as bats, foxes, skunks, raccoons, meerkats, prairie dogs, kinkajous, sloths, monkeys, apes, prosimians (mammals), parrots, mynah birds, finches (birds), crocodiles, turtles, tortoises, lizards, snakes (reptiles), frogs, toads, newts, salamanders (amphibians), fish, eels, rays (fish), crabs, crayfish, snails, insects, spiders, and millipedes (invertebrates) [175] . even fruit bats are kept as pets [176] and it is known that bats harbor a higher proportion of zoonoses than all other mammalian orders, including rabies-like viruses that are highly pathogenic for people. [177] . another example of a zoonosis is monkeypox following the importation of prairie dogs in the usa [178] . most reptiles and amphibians such as turtles, lizards, and frogs carry salmonella bacteria in their gut, in most cases without visible signs of infection. the infection may cause symptoms of sickness, diarrhea and fever in humans [179] . zoonotic transmission of salmonella infections causes an estimated 11% of salmonellosis annually in the united states. in cases involving pet turtles, almost half (45%) of infections occurred in children younger than 5 years [180] . salmonella infections are often transferred by feeder rodents [181] and outbreaks highlight the importance of improving public awareness and education in countries who receive imported reptiles [182] . it is advised to exclude reptiles, amphibians, rodents, exotic species, baby poultry, and raw animal-based pet food items from the households of patients at high risk. in lower risk households, an understanding of the risk of salmonellosis and other pet-associated zoonoses and preventive hygiene measures is needed. the pet trade in general, with its high turnover and diversity of available species, creates a reservoir for pathogens originating from all over the globe. reptiles account for approximately 10% of live animal shipments imported to the united states [182] . importation of live reptiles and amphibians for commercial purposes is for a great part unregulated at eu level except cites and customs regulations [176] . in a risk assessment study, the five pathogens with the highest public health risk caused by the import of exotic animals were salmonella spp., crimean-congo hemorrhagic fever virus, west nile virus, yersinia pestis, and arenaviruses. the risk via legally imported animals was considered low, but substantial for illegally imported animals due to the unknown health status of the animals [183] . avoiding exposure to exotic pet pathogens in the home is difficult and best achieved by not keeping them in the first place. otherwise, it is advised to always wash the hands immediately and thoroughly after contact with exotic pets and after handling raw (including frozen or defrosted) mice, rats and chicks. children should be supervised so that they do not put their mouths close to or kiss exotic animals. reptiles and other animals should be kept out of rooms in which food is prepared and eaten. the number of abandoned and homeless dogs and cats in europe is estimated to be over 100 million. countries with more than 1 million abandoned animals are italy, romania, russia, and ukraine [184] . there is an increasing trend to rescue and import dogs from countries with stray animal problems, in europe often from southern or eastern europe and in the us from puerto rico, the dominican republic, mexico, the middle east, turkey, china, and korea [185] . many charities and independent groups are involved to rescue dogs and seek adoption in more animal-friendly countries [186] . new owners primarily choose to adopt from abroad based on a desire for a particular dog they had seen advertised and on concern for its situation, while some were motivated by previously having been refused dogs from local rescues [187] . in the usa 85% of all household dogs were neutered and today there are no longer enough dogs being born in the usa annually to replace the approximately 8 million dogs that die each year. developing countries have hundreds of millions of street dogs available for export, for example egypt has an estimated 15 million, india, 30 million, and afghanistan, 100 million [188] . in 2006, more than a decade ago, the centers for disease control (cdc) estimated annual dog imports at around 287,000. today the number of imported dogs is estimated to be more than a million a year [189] . imported dogs are reintroducing diseases and parasites that were previously eliminated in the usa [190] . in the usa new and lethal strains of distemper and canine influenza as a result of imported rescue dogs were reported as well as canine brucellosis, rabies, and vector-borne diseases like ehrlichiosis, heartworm, babesiosis, and leishmaniasis [188, 191] . in many southern european countries, there is also a risk of exposure to diseases not encountered in the northern, importing, countries. animals could be infected with anaplasma phagocytophilum, babesia canis, brucella canis, borrelia burgdorferi, dirofilaria immitis (heartworm), dirofilaria repens (subcutaneous worm), echinococcus multilocularis (fox tapeworm), echinococcus granulosus (hydatid or dog tapeworm), ehrlichia canis, hepatozoon canis, leishmania infantum, linguatula serrata (tongue worm), onchocerca lupi, rabies, rickettsia conorii, strongyloides stercoralis, and thelazia callipeda. except b. canis, e. canis, and h. canis, the infections are zoonotic and regularly reported [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] [202] . most of these are transmitted by ticks, sand flies, or mosquitoes that are non-endemic in the receiving countries, but a reservoir of infections has been created and the risk is that vectors will become present as result of climate change. [203] . animals that are infected with rabies or echinococcus spp. may infect people directly. the importation of dogs from endemic, predominantly mediterranean, regions to northern europe, as well as travelling with dogs to these regions carries a significant risk of acquiring an infection. pet owners are therefore advised not to travel with dogs and to seek the advice of their veterinarian prior to importing a dog from an endemic area or travelling to such areas [193] . for this reason, esccap developed maps on their website featuring european countries and regions with advice on endemic parasites, diseases and recommended treatments when travelling with dogs [204] . a three-year european union funded project entitled callisto (companion animal multisectorial interprofessional and interdisciplinary strategic think tank on zoonoses), has investigated zoonotic infectious diseases transmitted between companion animals and humans and food producing animals [176] . the committee advised that special attention should be given to stray cats and dogs. stray dogs, in particular, may pose serious health and welfare problems for humans and animals [81] , including the transmission of zoonotic diseases such as rabies. consideration should be given to controlling companion animal movement between areas of the eu endemic for particular zoonoses and areas that are not currently endemic for that disease. reliable figures are not available, probably because of the fact that many voluntary organizations are responsible for saving and transporting these rescue animals. such data are essential in order to be able to quantify the actual risks of zoonotic diseases attributable to companion animals and to develop sustainable interventions to prevent transmission to humans and livestock [176] . as long as there are no official guidelines, to prevent the spread of (zoonotic) diseases to new countries, the time, expense, disease risk, and the implications of adopting a dog from abroad should all be carefully considered before importation. as an alternative, the conditions for native dogs could be improved by supporting local charities to organize neutering campaigns and rehoming programs, to build local animal shelters and to improve attitudes towards dogs and their living conditions [205] . cats and dogs harbor the enteric nematodes toxocara canis and toxocara cati, and cats are the final host for the protozoal parasite toxoplasma gondii. these parasites can be transmitted to humans because they have an oral-fecal transmission cycle. humans can be infected by ingestion of infective toxocara spp. eggs or toxoplasma oocysts from contaminated soil (gardens, sandpits, and playgrounds) [206, 207] . a recent meta-analysis of data from published records indicates that public places are often heavily contaminated with a pooled global prevalence of toxocara eggs of 21% [208] . both parasites are considered by cdc as part of five neglected parasitic infections. these infections are considered neglected because relatively little attention has been devoted to their surveillance, prevention, and/or treatment. the diseases that they cause have been targeted as priorities for public health action based on the number of people infected, the severity of the illnesses and the ability to prevent and treat them [209] . tens of millions of people worldwide are estimated to be exposed to, or infected with, toxocara spp. and recent findings suggest that the effect of toxocarosis on human health is increasing in some countries. almost one fifth (19%; 1.4 billion individuals) of the world's human population is seropositive to toxocara. the highest seroprevalence rates were found in africa (mean: 37.7%) and the lowest in the eastern mediterranean region (mean: 8.2%) [210] . toxocara larvae migrate into the body of the human to several organ systems with a preference for the central nervous system (brain, eyes). human toxocarosis can manifest itself as syndromes known as visceral larva migrans, ocular larva migrans, neurotoxocarosis, and covert or common toxocarosis [211] . asthma is one of the most common chronic respiratory diseases worldwide, with a negative impact on the quality of life and socio-economic status of patients. two decades ago, the first evidence was published that suggested that toxocara infection is a neglected risk factor for childhood asthma [212] . the finding that children infected with toxocara spp. are more likely to have asthma compared to non-infected children was recently confirmed in a systematic review and meta-analysis [213] . cognitive or developmental delays in children or young adults who become infected is of particular concern. toxocarosis appears to be associated with decreased cognitive function [214, 215] . the annual toxoplasma oocyst burden measured in community surveys has been reported as up to 434 oocysts per square foot (4670 per square meter) and is greater in areas with loose soil, that cats like to use to cover their feces in gardens, children's play areas, and especially sandboxes, also called sandpits and sand piles [207] . because a single oocyst can possibly cause infection, this oocyst burden represents a major potential public health problem. an estimated one third of the world's population harbor anti-toxoplasma antibodies. due to keeping pigs indoors, more education and awareness, the prevalence of the disease in the usa and europe declined by 50% over the last decades [216] . during an acute invasion of toxoplasma parasites there is mild to major tissue damage without clinical symptoms (latent toxoplasmosis). the most important form is congenital toxoplasmosis when a woman receives her first exposure to toxoplasma during pregnancy. in early pregnancy, this can lead to abortion or to malformations that are not compatible with life shortly after birth. congenital infections may also be characterized by mental retardation and ocular defects. acquired infection after birth may result in clinical symptoms such as lymphadenitis, fever, and malaise and probably leads to a clinically symptomatic disease state more frequently than the congenital condition, with an estimated incidence of 30% of all ocular toxoplasmosis cases [216] . playing in a sandbox is also found to be a predominant risk factor for s. typhimurium salmonellosis in children aged 4-12 years [217] . this can be the result of fecal contamination of the sand by dogs and cats that have been fed raw meat (see section 5.1.1.). young children are especially at risk as they put their hands or other objects in their mouths every 2-3 min [208] . it has also been reported that children ingest a median of 40 mg of soil per day and that one child consumed 5-8 g of soil per day on average [218] . it is therefore advisable not to let children play in public places or on playgrounds with loose sand, but only in sandboxes that can be covered. furthermore, washing hands after playing outside is important and fingernails must be trimmed to prevent sand being left behind. in this context, a strange trend can be observed as young children play in mud baths on 29 june "as a way to connect and celebrate the natural joys of playing in the mud". this international mud day originated in 2008 and was initiated by an australian pedagogue who had observed this phenomenon during a visit to nepal [219] . if the origin of the soil needed for producing the mud is unknown, there is of course an infection risk for the above discussed parasites. to prevent the transmission of zoonotic diseases from pets, risk analysis is of great value. this starts with an assessment of the potential zoonoses in an area, depending on the endemicity (the hazard, h). hazard characterization also includes prevalence in animals (the reservoir), virulence for man, transmission routes, and survival of the agent in the environment. the second step is exposure assessment (e). who is exposed to the potential hazard and for how long or how often? how much of the potential pathogen is needed to become a health risk? this inevitably is directly related to human behavior in relation to their pets. the third step is to assess the impact of getting infected (i). how serious is the disease, what is the chance of complications, and what economic consequences may be expected (e.g., labor hours lost)? each of the parameters can be ranked in classes from negligible to the most serious possibility. ranking is based on literature data, own observations (measuring), or experts' opinions. the final risk assessment can be achieved by multiplying the outcome of hazard characterization, exposure assessment and impact (h × e × i = a number). the outcome can be compared with other zoonotic agents and a ranking of significance can be made [220] . there is one important parameter to reduce the risk of contracting a zoonotic infection that can directly be influenced, which is exposure. recommendations are particularly targeted to households with very young children, the elderly, pregnant women, or immunocompromised patients. they are based on reducing exposure to hazards and involve four categories of advice (table 1) . table 1 . recommendations to prevent the transmission of zoonotic pathogens from pets [121, 221] . • washing the hands thoroughly -after animal contact, at least before eating and drinking and before preparing food or drinks -after handling raw pet food -after handling pet habitats or equipment -after cleaning up feces -after removing soiled clothes or shoes reflecting on the changed human-companion animal bond, it can be concluded that pets undoubtedly have a positive effect on human health. conversely, the human-pet bond seen nowadays is facing many challenges, putting pet welfare under more pressure due to issues such as anthropomorphism, which mainly results in obesity, breeding on extreme appearance rather than health, behavioral problems connected to unfulfilled species specific mental and physical needs, and the provision of inadequate food because owners mistakenly think they feed more naturally. with regard to the negative effects of pets this article attempts to give an impression of increasing trends in the human-companion relationship that can be observed in society, which appears to increase the risk of transmission of infection between pets and humans. it is mainly a consequence of the increasing contacts between humans and pets and with pathogens secreted by animals in the shared environment. more than 6 out of every 10 known infectious diseases in people can be spread from animals, and 3 out of every 4 new or emerging infectious diseases in people come from animals [222] . the recent pandemic of the covid19 coronavirus (sars-cov-2), that may be originated from bats, is a good example of a recent emerging zoonotic infectious disease. a few cats and dogs have tested positive but are not considered as a source of infection for people. the proportion of zoonotic human disease that is attributable to pets is largely unknown. reports about the frequency of such infections are likely underestimated [120] ; however, the risk of infection is relatively small for many zoonoses and the severity of the disease is often limited. a person's age and health status may affect their immune system, and thereby increase his or her chances of getting a disease from animals. pregnant women should avoid contact with pet rodents, reptiles, cat feces, and raw meat to prevent infection of the unborn child, abortion, or birth defects. if symptoms occur in immunocompetent, non-pregnant persons between 5 and 64 years of age, these are mainly of a general nature such as diarrhea or flu-like symptoms. physicians do not regularly ask about the presence of pets or pet contact, nor do they discuss the risks of zoonotic diseases with patients, regardless of the patient's immune status, which means that many cases of zoonotic diseases go undiagnosed. the general public and people at high risk of pet-associated disease are not aware of the risks associated with high risk pet practices or recommendations to reduce them. since unfamiliarity with hazards reinforces fear, communication plays an important role in this. veterinarians play a key role in education regarding risk reduction by giving advice about responsible pet ownership and the required preventive hygiene. healthcare providers such as family doctors, school doctors, and pediatricians can also provide information about safe pet ownership. physicians should ask as part of the medical history about eventual contact with pets, particularly with patients at high risk [120] . the "one health" initiative aims to reduce this professional gap between vets and physicians [2] . when giving recommendations to prevent zoonotic transmission, one of the often-made remarks is that people nowadays are already too hygienic. it is assumed that there is a protective influence of postnatal infection and that it might be lost in the presence of modern hygiene. this belief is based on the hygiene hypothesis that was formulated in 1989 [223] . it was observed that hay fever in young adults was inversely related to the number of siblings in their family. this hypothesis focused exclusively on allergic conditions as a result of the modern way of life and the assumption that modern hygiene was reducing contact with bacteria. another view is that some chronic inflammatory disorders have increased over the last decades as a result of decreased frequency of infections due to pathogenic organisms [224] . another related theory is the "old friends" mechanism that is based on the positive influence of gut parasites, non-pathogenic environmental bacteria (saprophytes, pseudocommensals), and gut commensals or microbiota. however, decreased exposure to these microorganisms is not the only reason for the increasing frequency of allergies and chronic inflammatory disorders in industrialized countries. nowadays there is more information about the immunological roles of skin, oral mucosa, and gut microbiota as well as helminths and the influence these have on the immune system [225] . gut flora may be modified due to diet, obesity, hygiene, antibiotics, but also to psychological stress, vitamin d deficiency, and pollution [225] [226] [227] . pollution also has a significant effect on the development of several respiratory problems and diseases. not only due to outdoor pollution such as fine dust, harmful solids, liquids, or gases [228] , but also due to indoor molds as result of insufficient ventilation in energy efficient homes [229] . finally, there is a clear increase in the allergen production of house dust mites and pollen leading to more exposure and sensitization in susceptible individuals [230] . all together it must be clear that there is much more known about other causes of increasing allergies worldwide than simply excessive cleanliness as suggested in the hygiene hypothesis. regarding field infections with helminths such as trichuris trichiura in early life, these are associated with a reduced prevalence of allergies later in life and infants of helminth-infected mothers have a reduced prevalence of eczema. hookworm infections in developing countries are associated with a reduced prevalence of asthma [231] . the rate of eczema in such countries was found to be about five times higher in infants whose mothers had never had helminths compared with persistent helminth-infected mothers [232, 233] . helminths are nowadays even used under controlled conditions to stimulate immunity. examples are trichuris suis therapy for crohn's disease and necator americanus larvae to treat crohn's disease and other autoimmune disorders [234] . there are no clinically apparent childhood infections found to be associated with protection from allergic disorders [235] . it can even result in an opposite effect in the case of toxocara infection by children after soil contact. a recent meta-analysis showed that children infected with toxocara spp. are more likely to have asthma compared to non-infected children [213] . this parasite and asthma both have elevated immunoglobulin e (ige) levels and eosinophilia in common. that means that precautions should be taken in children to prevent soil contact not only to prevent toxocara infection, but also to prevent acquired ocular toxoplasmosis. there is no need, therefore, to stimulate the contraction of pathogenic bacteria or helminths to achieve a healthy gut microbiota and to reduce allergic conditions. recommendations based on the hygiene hypothesis should preferably be based on results from controlled studies to prevent unintentional negative effects. it is both humans and companion animals who experience negative effects of a changed human-companion animal bond. the education given by vets to their clients should therefore also focus on preventing these negative health and behavioral effects. for instance, by giving science-based advice on feeding practices. in general, regulating authorities should encourage the development of enforcement criteria for breeding dogs and cats to reduce health and welfare risks. pets undoubtedly have a positive effect on human health and well-being, while owners are increasingly aware of pet health, welfare, and well-being. anthropomorphism, also resulting in behavioral problems and breeding on appearance rather than health, and trends such as keeping exotic animals and importing rescue dogs may result in an increased risk of contracting zoonotic infections. recommendations regarding responsible pet ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conform the biology of the animal, are key in preventing such negative aspects of the human-animal bond. there is no need to stimulate unhygienic practices following the hygiene hypothesis. education can be performed by vets and physicians as 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friends' hypothesis metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment current understanding of the human microbiome environmental and health impacts of air pollution: a review. front. public health 2020, 8, 14 abridged version of the awmf guideline for the medical clinical diagnostics of indoor mould exposure house dust mite allergy under changing environments interactions between helminth parasites and allergy the impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection an update on the use of helminths to treat crohn's and other autoimmune diseases infections presenting for clinical care in early life and later risk of hay fever in two uk birth cohorts this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-023353-2yoz1t6a authors: nan title: abstracts from the 2010 evdi annual meeting date: 2010-12-28 journal: vet radiol ultrasound doi: 10.1111/j.1740-8261.2010.01774.x sha: doc_id: 23353 cord_uid: 2yoz1t6a nan oral malignant melanoma is the most common canine oral tumour being characterized by local invasion and a high rate of regional and distant metastases. the overall prognosis is guarded, mainly because of the poor response to adjuvant systemic treatment like chemotherapy. melanoma is sensitive to radiation, which may reduce/eliminate tumour volume in macroscopic tumours or prolong local control in microscopic settings. neither surgery nor rt can control the high risk of distant metastases though. interesting approaches for immunogenic tumours such as melanoma are the use of tumour vaccines. the objective of this study was to evaluate toxicity associated with concurrent administration of an intradermally administered tumour vaccine and radiation. materials and methods: nine dogs with histologically confirmed oral malignant melanoma were treated with rt in combination with a human recombinant tyrosinase dna vaccine. patients were staged according to the who tnm-staging-scheme for oral tumours1. in 5 dogs a ct scan was performed to assess local disease, 4 dogs were evaluated clinically for local disease extension. for assessment of metastatic disease, regional lymph node aspiration, thoracic radiographs and abdominal ultrasound were performed. all dogs received 4 fractions of 8 gray each at 7-day intervals to a total dose of 32 gray (6mv linear accelerator) to the primary tumour and the regional lymph nodes (mandibular and medial retropharyngeal). the vaccine was given 4 times at 14-day intervals with the first vaccine being administered at the first fraction of rt. the acute radiation toxicity was evaluated according to the vrtog scoring scheme2 at the last rt and 2 to 4 weeks after the last rt. the anatomic tumour locations were as follows: mandible (n ¼ 4), maxilla (n ¼ 3) and buccal mucosa (n ¼ 2). metastases were initially identified in 8 of 9 dogs: ipsilateral mandibular lymph node (n ¼ 5), ipsi-and contralateral mandibular lymph node (n ¼ 1), ipsilateral mandibular lymph node and lungs (n ¼ 1), liver (n ¼ 1). local acute rt toxicity (mucositis, alopecia/dermatitis, conjunctivitis) never exceeded grade 2 toxicity and did not have an appreciable negative impact on quality of life. the melanoma vaccine was well tolerated by all dogs, minimal side effects were observed in few dogs: fever (n ¼ 2), lethargy (n ¼ 2) and local swelling (n ¼ 2). locally and loco-regionally all tumours could be controlled with a mean follow-up of 195 days. three dogs developed lung metastases after 41 to 68 days. three dogs died of tumour unrelated reasons. three dogs are still alive (for 109 to 351 days). discussion/conclusion: we could not observe an increase in severity of acute local radiation toxicity administering rt and vaccine concurrently, as it has been suspected initially. this is an important fact, since early timing of the vaccination might be essential for its activity. more patients will be collected to evaluate these preliminary results. owen ln: tnm classification of tumors in domestic animals, world health organisation geneva, 1980 ladue t, klein mk: toxicity criteria of the veterinary radiation therapy oncology group. vrus 42(5) [475] [476] 2001 introduction/purpose: pulmonary angiography with multi-detector computed tomography (mdct) is the standard of care in humans for non-invasive diagnosis of acute and chronic pulmonary embolism, pulmonary hypertension and other diseases or anatomical variants of the pulmonary arteries. however, there is paucity of data in the animal literature despite the expected diagnostic performance of mdct for pulmonary angiography. therefore, aim of this feasibility study was to evaluate the anatomy and visualization of the normal canine pulmonary vasculature as the basis for further application of mdct in diagnosing canine pulmonary arterial disease. materials and methods: experiments were previously approved by the responsible animal care and use committee (v1430). in four one-year old beagles, pulmonary mdct angiography (mdcta) was timed based on an individualized timing bolus and calculated delay after bolus injection, scanning the entire thorax in caudal to cranial direction using helical scan mode at 0.625 mm collimation, 0.625 helical reconstruction interval utilizing all 64 detectors. images were retrospectively reconstructed at 1.25 mm and 2.5 mm collimation for each dog. the pulmonary arteries were evaluated for anatomy (relationship to major vessels, cardiac and bronchial structures visible), visibility and visible generations of branching using all three reconstruction thicknesses. results: all lobar pulmonary arteries and adjacent anatomical structures were visualized at all reconstruction thicknesses. subjective blurring of small sublobar arteries occurs using 1.5 mm and 2.5 mm reconstruction thickness, particular with multiplanar reconstructions. fewer generations of sublobar arterial branches were detected on 1.5 mm and 2.5 mm as compared to 0.625 mm reconstructions. computed tomography of the brain in small animals can be limited by contrast resolution. the ability of the observer to differentiate grey and white matter is an important measure of image quality, but this can be reduced on images with low contrast. various factors affect the contrast resolution, including exposure, slice thickness and patient size. while slice thickness can be adjusted on retro-reconstruction, the exposure settings are fixed for the study. the purpose of this study was to determine the effect of kvp and ma on objective and subjective measures of image quality, and to identify the technique that results in the best image quality. materials and methods: six fresh canine cadavers of medium size were imaged using a 16-slice ct set in axial mode with 2.5 mm slice thickness and soft tissue algorithm. twelve different protocols were used to image the brain in each dog: kvp was set at 140, 120 or 100 and for each kvp the ma was set at 360, 280, 200 and 120. all other parameters were kept constant. a slice at the level of the internal capsule was selected for analysis. using image analysis software identical regions of interest were placed in the white matter and grey matter, and the mean hounsfield units and standard deviation calculated for each slice. image quality was evaluated objectively by measuring a contrast to noise ratio (cnr), calculated as cnr ¼ (mean gm roiàmean wm roi)/ f(sd gm roi) 2 þ (sd wm roi) 2 g 1/2 . images were evaluated subjectively by a radiologist blinded to the technique who graded the image quality on a 5-point scale (subjective score; ss). a two-factor anova with repeated measures was used to determine the effect of ma and kvp on cnr and ss. the spearman test was used to correlate cnr and ss. significance was set at po0.05. the exposure factors had a significant effect on objective and subjective measures of image quality. increasing ma significantly increased cnr (po0.001) and ss (po0.001). increasing kvp also significantly increased cnr (p ¼ 0.0176) and ss (p ¼ 0.0055). a technique of 360 ma 140 kvp resulted in the greatest cnr, whereas 360 ma 120 kvp resulted in the highest subjective score. a technique of 360 ma 140 kvp resulted in a higher cnr, but a lower ss than 280 ma 140 kvp. ss was positively correlated with cnr (r ¼ 0.61; po0.0001). discussion/conclusion: ct acquisition with a high ma and kvp technique is recommended for canine brain imaging. it results in improved contrast resolution, primarily due to a reduction in image noise. while this was expected for ma, the reduction in noise with increasing kvp also offset the corresponding reduction in contrast. when tube heating limits the use of a 360 ma technique, acquisition at 280 ma provides subjectively good contrast resolution. introduction/purpose: classroom performance systems (cps) [audience response systems (ars); ''clickers''] provide a means of encouraging student interaction in a traditional didactic lecture setting. cps tm is the leading response system in education, being used by over 5 million students worldwide. cps tm response pads (''clickers'') are handheld devices that allow students to respond to questions asked during a lecture enabling students and instructors to instantly assess their comprehension of lecture material. materials and methods: student performance in and student evaluations of the 3rd year (5th semester) didactic radiology course were evaluated from [2005] [2006] [2007] [2008] [2009] . additionally, student interactions were evaluated subjectively by instructors who used cpstm. results: from 2005 -2008 , all lectures and laboratory sessions were held in conventional fashion. in 2009, cpstm was utilized by 3/5 instructors. there was no significant difference in overall student performance and student performance on individual instructor's exam questions. on a scale from 1 (worst) to 5 (best) the radiology course was evaluated higher by students when cpstm was used than not, however, this difference was not significant (p ¼ 0.075). subjectively, students appeared more interested and involved when cpstm was used. discussion/conclusion: cps may be beneficial in radiology education. benefits include interactive involvement of students in a lecture setting and immediate feedback for students and instructors. disadvantages include time constraints, technical problems and dependence on willing student participation. introduction/purpose: non-invasive imaging techniques have been developed for phenotyping genetically engineered mice created to model human cardiovascular diseases like atherosclerosis. b-mode and doppler ultrasound may be useful because it is a fast and real-time imaging technique which gives structural, functional and hemodynamic information and it makes possible to perform temporal longitudinal studies. atherosclerotic lesions do not occur at random sites. the spatial heterogeneity of their distribution is in part dependent on hemodynamic factors. in murine models of atherogenesis lesions tend to develop at the ascending aorta, near the ostia of the three major branches, and on the lesser curvature of the aortic arch. the purpose of this study is to describe normal hemodynamics in mouse aorta and its major branches in the thorax by ultrasonography. materials and methods: 27 inbred c57bl/6 adult mice were studied (10 males and 17 females; 17-25 g body weight). anesthetized mice were scanned in supine position with an ultrasound biomicroscope (vevo 770, visualsonics, toronto, on, canada) provided with a 30 mhz transducer. aorta was studied at five different regions (ascending aorta, near the ostia of the three major branches in the arch, and in the proximal portion of the descending aorta). brachiocephalic trunk (bt), left common carotid artery (lcca), and left subclavian artery (lsca) were imaged near its junction with the aortic arch. systolic area was measured in b-mode, and peak systolic velocity (psv), minimum diastolic velocity (mdv), and time average maximum velocity (tamx) were measured with pulsed doppler ultrasound. pulsatility (pi) and resistive indices (ri), and mean blood flow (fv) were calculated. mean values and standard deviations were determined averaging a total of three doppler waveforms in every mouse. flow velocity profile and flow pattern were recorded in each region. the statistical analysis was performed with the statistical analysis system (sas institute, cary, usa) by applying the glm and ls means procedures. a p-value less than 0,05 was considered statistically significant. all anatomical terms are in accordance with the nomina anatomica veterinaria. results: aorta in the thorax has a plug flow profile and a typical high resistance flow pattern. bt, lcca and lsca have an intermediate flow velocity profile and a high resistance flow pattern. there were not differences between doppler data obtained in males and females. the ostium of the brachiocephalic trunk, because of its topographical disposition and flow direction changing, was the region were doppler has been most difficult to record. statistical differences were found between systolic area, psv, mdv, tamx, and fv of the aorta at the five different regions described above. no significant differences were found between systolic area, psv, and fv of the aorta major branches, although differences were observed between the mdv and tamx in these branches. values of pi and ri don't differ along aorta and its major branches in the thorax. this study provides normal doppler wave pattern and vascular values of the aorta and its major branches in the thorax of adult c57bl/6 mice by ultrasonography. these values may be used as normal reference to compare with mutant mice with cardiovascular alterations. pascaline pey 1 , massimo vignoli 2 , hendrik haers 1 , luc duchateau 3 , federica rossi 2 , jimmy saunders 1 . 1 department of medical imaging of domestic animals and orthopedy of small animals, ghent university, belgium. 2 clinica veterinaria dell'orologio, via gramsci 1/4, 40037 sasso marconi (bo), italy. 3 department of physiology and biometry, ghent university, belgium introduction/purpose: the microbubble-based contrast agents for ultrasonography have become more used in recent years in dogs1, and have been reported to be useful in differentiating adenomas from nonadenomatous lesions in human patients with adrenal masses2. the purposes of this study were to evaluate the feasibility and to define the perfusion parameters of contrast-enhanced ultrasonography of the adrenal gland in the normal beagle dog. materials and methods: six healthy female beagles were injected with an intravenous bolus of sonovue s (0.3 ml/10 kg, mechanical index ¼ 0.10). subjective and objective analyses were evaluated. objective measurements were performed using an off-line image analysis system (imagejr). results: subjective evaluation: the aorta was first rapidly opacified after injection of the contrast medium, followed by the renal artery and the medulla. subsequently, a homogeneous centrifugal enhancement from the adrenal medulla to the adrenal cortex was observed. at this point, a homogeneous decrease in opacification of both cortex and medulla started, associated with an enhancement of the phrenicoabdominal vessels. then a persistent enhancement in the splenic parenchyma, in the renal vein, the caudal vena cava and phrenicoabdominal veins was noticed. at the end of the parenchymal phase a second contrast enhancement was observed, corresponding to the refilling time. discussion/conclusion: the shape of the time-intensity curve reflecting the adrenal perfusion was similar in all dogs. however, the absolute values of the measurements and of the parameters of perfusion displayed considerable variations. ratios of the peak values of the cortex and the medulla to the peak values of the renal artery showed significant difference allowing differentiation between the cortex and the medulla for both adrenal glands. contrast-enhanced ultrasonography of the adrenal glands is feasible in dogs and the optimal time for adrenal imaging is between 5 to 90 seconds after injection. introduction/purpose: testicular anomalies are common in dogs. the aim of this study was to describe contrastenhanced ultrasonographic features of focal and diffuse testicular lesions, compared with normal tissue. materials and methods: seventeen dogs with scrotal anomalies were considered. all dogs underwent b-mode ultrasound, anesthesia, contrast-enhanced ultrasound (ceus), bilateral orchiectomy and histological examination. ceus was performed with cnti mode (esaote s , italy) and a secondgeneration contrast medium (sonovue s -bracco, italy) injected at a dosage of 0.03 ml/kg, iv. parenchymal perfusion was recorded for 2 minutes. time-intensity curves were reconstructed by a commercial software (qontrast s -bracco, italy), using the gamma-variate bolus-corrected model. the peak intensity (p), time to peak (ttp), regional blood flow (rbf) and mean transit time (mtt) were calculated. data relative to neoplasms were compared to those of normal/non neoplastic tissue by fisher's exact test. a po0.05 was considered significant. results: nine normal testes, 18 tumors (11 interstitiomas, 3 sertoliomas and 4 seminomas), 9 degenerated testes and a chronic orchitis were histologically diagnosed. eight dogs had bilateral involvement, while 9 monolateral. two seminomas (intratubular type) were not visualized. perfusion parameters of each group are summarized in the following table. peak intensity time to introduction/purpose: studies with contrast enhanced ultrasonography (ceus) on focal splenic lesions have shown discrepancies in accuracy for differentiating benign from malignant lesions.1-3 a speculative explanation for false positives may be the absence of a dual blood supply to the spleen compared to the liver. we therefore hypothesized that the early wash in/early wash out (ewew), and the hypoechogenicity during all phases are unreliable in differentiating malignant from benign splenic lesions. materials and methods: a retrospective study reviewing ceus studies of 17 splenic lesions (7 malignant, 10 benign), with concurrent cytology, core biopsy and/or excision biopsy was conducted. sonovue& at a dose of 0.03 ml/kg was used in all dogs. tortuosity of feeding vessels, presence of ewew, and hypoechogenicity during all phases were assessed visually and used to characterize the lesions. there were 4/10 benign, and 4/7 malignant lesions with ewew. therefore, sensitivity, specificity, and accuracy for ewew in differentiating malignant from benign lesions are 57%, 60%, and 59% respectively. there were 3/10 benign, and 3/7 malignant lesions with persistent hypoechogenicity throughout all phases. therefore, sensitivity, specificity, and accuracy are 43%, 70%, and 59%. there were 0/10 benign, and 5/7 malignant lesions with tortuous and persistently visible feeding vessels. sensitivity, specificity, and accuracy are 71%, 100%, and 88%. finally, there were 7/10 benign and 7/7 malignant lesions with a combination of at least one of the above parameters. sensitivity, specificity, and accuracy are 100%, 30%, and 59%. discussion/conclusion: these preliminary results support our hypothesis that the interpretation of splenic lesions cannot be performed accurately on the basis of ewew, or persistent hypoechogenicity throughout all phases. instead, we recommend assessing the presence of tortuous vessels in the periphery of the lesion during the early phase, and the persistence of these hyperechoic tortuous vessels in the late phase. further studies with larger numbers are necessary to better assess these findings. introduction/purpose: ceus has been used to detect focal perfusional changes in the spleen in humans and dogs. no earlier studies exist of contrast ultrasound of the spleen in cats. the purpose of this study was to evaluate perfusion of the cat spleen, and to compare the perfusion patterns in awake and anesthetized cats. the spleen of 18 healthy cats was imaged with ceus (acuson sequoia 512, siemens or esaote mylab70, biosound esaote inc.). two groups of cats were imaged: group i comprised of ten young, anesthetized cats and group ii of eight young to middle aged cats that were initially imaged awake (iia) and later anesthetized (iib). mechanical index (mi) was set at 0.32 (group i) or 0.04 (group ii) in the focal zone area. parameters were standardized including depth gain, power, number, and location of focal zones in relation to the spleen. all cats received multiple bolus injections (0.1 ml) of contrast medium (definity s o) per imaging. a 1-to 2-min digital clip was recorded for each contrast injection and analyzed off-line. the timing and grade of heterogeneity of the spleen were estimated in a standardized manner, blinded and in a randomized order. the grade of heterogeneity (1) (2) (3) (4) was estimated when the spleen was the most heterogeneous. the grade of homogeneity (i-iii) was estimated when the spleen was the most homogeneous. student's t-test was used to compare the time to first appearance of contrast in the spleen (at), and the time to the spleen becoming heterogeneous or homogeneous. frequencies of each heterogeneity and homogeneity grade within separate groups were calculated with statistical software (statistix version 9, analytical software). statistical significance for analysis was set at po0.05. results: enhancement of the spleen was bi-phasic in all cat groups. at was significantly faster in awake than anesthetized cats. a marked heterogeneous perfusion pattern (grade 4) was significantly more prevalent in anesthetized (50%) than awake (12.5%) cats. these results indicate focal perfusion defects, more pronounced with general anesthesia, of the spleen with ceus should be evaluated with caution and only after disappearance of the initial heterogeneity. introduction/purpose: diabetic nephropathy in small animals is a controversial issue. it is known that some spontaneously diabetic dogs develop significant proteinuria and hypertension. in cats ''diabetic nephropathy'' is a term describing fatal, chronic renal insufficiency with low urine specific gravity and proteinuria often occurring in feline diabetes. measurement of intrarenal resistivity index (ri) values is described in human medicine as an useful method in early diabetic nephropathy diagnostics. up to the present time the results of such tests in dogs (novellas and others)three diabetic ones and four suffering from diabetes caused by hyperadrenocorticism-did not reveal any ri value deviations in animals suffering from diabetes not caused by hyperadrenocorticism. the aim of this study was to compare the ri values in diabetic dogs and cats with the healthy ones and evaluate the possible relationship between ri, age and duration of diabetes. one of the study groups consisted of 10 dogs of both sexes and different breeds. the mean age was 10,9 ae 0,3, the duration of diabetes was from 1 month to 6 years. another study group were cats -five european shorthairs of both sexes (mean age 7,8 ae 3,56, the duration of diabetes from 1month to 2,5 years). the data confirming diabetes, using blood and urine analysis were strongly documented. patients with indication for ultrasound examination, but without any signs of serious or systemic diseases (eg. the animals with diseases of musculoskeletal system, routinely monitored geriatric patients) were assigned to control groups. control groups consisted of 15 dogs and 15 cats of both sexes and different breeds and ages (mean age in canine group 6, 9 ae 4, 5, 1 ae 3, 07 years) . animals with the suspicion of urinary tract infection or with history of previous kidney conditions were excluded from this study. renal ri values were higher in diabetic dogs and cats than in healthy animals. in the group of diabetic dogs in four animals ri exceed the accepted limit 0,71. mean ri for the group of the dogs with diabetes was 0,66 ae 0,07, in control group 0,62 ae 0,05 (p ¼ 0,08). as many as four diabetic cats had significantly elevated ri and in two of them the diabetic nephropathy was suspected. mean ri for the group of diabetic cats was 0,71 ae 0,07 and for the healthy cats 0,59 ae 0,04 (po0,001). in all animals the positive correlation between the ri and age was observed (healthy dogs r ¼ 0,38, healthy cats r ¼ 0,51, diabetic dogs r ¼ 0,39, diabetic cats r ¼ 0,71). correlation between ri and duration of diabetes was more relevant in cats (r ¼ 0,56) than in dogs (r ¼ 0,06). discussion/conclusion: our study proves that renal vascular resistance value is elevated in some of small animals with diabetes. it is not possible to conclude much about usefulness of doppler ultrasound in monitoring the kidney condition in animals with diabetes, from study performed on such a small group of patients. our results may serve as an introduction to the research on bigger group of animals, which would be enriched in detailed laboratory and histopathological data providing new information about diabetic nephropathy in dogs and cats. brkljacic b., renal vascular resistance in diabetic nephropathy: duplex doppler us evaluation. radiology 1994; 192: 549-554 introduction/purpose: ultrasound (us)-guided percutaneous renal biopsy has become invaluable as diagnostic method in kidney diseases that is associated with minor or major complications, reportedly being more extensive using large-bore 14-gauge needles. 1 contrast harmonic us (chus) increases the intensity of the blood pool echo signals in parenchymal organs and enhances existing lesions. this study investigated the value of chus as a screening method of post-biopsy renal complications, and evaluated the traumatic importance of 14-gauge biopsy needles. renal biopsy was performed in 11 healthy beagle dogs at 3 occasions (0, 4 and 6 months) for a total of 33 biopsies using 14-gauge needles (vet-core tm , surgivet, usa). a chus examination was performed 30 minutes after biopsy using a mylab30vet us machine (esaote, genoa, italy) with a multifrequency linear transducer of 5-7.5 mhz. approximately 0.3 ml/10 kg of contrast medium (sonovue, bracco), was injected twice iv. the chus study was repeated each week, until complete dissolving of the lesions. the images were recorded and reviewed using subjective and semi-quantitative methods for description of the lesions including their number, shape, size, sharpness, echogenicity, and evolution over time. results: chus performed 30 minutes after biopsy revealed hypoechoic lesions in all kidneys, visible as 2 tracts (n ¼ 17), 1 tract (n ¼ 13), triangular or nodular area (n ¼ 2), or area þ tract (n ¼ 1). one beagle had perirenal bleeding. ten lesions were small to mild and 23 lesions were moderate to large. ten tracts had a thickened nodular base, subcapsular at the renal cortex. at week 2, all lesions were still present but slightly (n ¼ 8) or significantly (n ¼ 25) decreased in size and sharpness, with increased echogenicity. the perirenal bleeding disappeared. one beagle showed ureter and renal pelvic dilation and anechoic round lesions were present compatible with hematoma. at week 3, 27 kidneys were normal, 5 showed vague lesions and one was still abnormal showing the same features as at week 2. at week 4, all kidneys were normal. discussion/conclusion: chus enables assessment of the evolution of post-biopsy renal lesions and can be an adequate screening method. moreover, 14-gauge needles were not found to be quite as traumatic as generally believed. introduction/purpose: variable ultrasonographic measurements of normal and abnormal canine adrenal glands have been reported.1-3 moderate correlation was present between ultrasonographic and gross measurements of thickness (dorsoventral measure) for both glands, but no correlation was found for width (mediolateral measure) or length.1 however, the repeatability of such measurements has not been determined. the purpose of the study was to assess the intra-and interobserver variability of sonographic measurements of adrenal glands. materials and methods: six healthy beagle dogs were used. three observers, two ecvdi diplomates (j.h.s. and v.b.) and one ecvdi resident (p.p.) scanned both adrenal glands of each dog three separate times. the observers were asked to measure the maximal length (craniocaudal direction), height (dorsoventral direction) and width (mediolateral direction) of each gland. length and height of both cranial and caudal poles were measured in longitudinal plane. height and width of both cranial and caudal poles were measured on transverse images. a random effects model was fit to the data with dog, observer and measurement within observer as random effects. measurements of the length of both glands had the highest intra-and interobserver variability. height of the caudal pole measured on longitudinal images had the lowest intra-and interobserver variability. measurements that also presented low intra-and interobserver variability were: height and width of the caudal pole on transverse images and height of the cranial pole but on longitudinal images only. discussion/conclusion: length measurements appeared unreliable due to difficulties in obtaining the complete length of the gland on one image and in defining clearly the cranial border of the right gland. measurements of height and width of the cranial pole of both glands on transverse images were unsatisfactory due to its inconsistent shape and position. hence, height of the caudal pole measured on longitudinal images should be preferentially used, as it is the less variable and the most accurate sonographic estimation of gross adrenal size. grooters am, biller ds, merryman j. ultrasonographic parameters of normal canine adrenal glands: comparison to necropsy findings. vet radiol ultrasound 1995; 36 (2) introduction/purpose: dogs and cats presented with signs of upper airway obstruction frequently have to undergo bronchoscopic examinations for a final diagnosis. although usually followed by surgery to relieve the obstruction the procedure carries a high risk due to the need for general anesthesia. the goal of this study was to assess computed tomography and virtual bronchoscopy in awake dogs and cats with signs of upper airway obstruction. materials and methods: five dogs and 2 cats presented with signs of upper airway obstruction were included. final diagnosis was made by bronchoscopy, physical examination of upper airway under general anesthesia, fluoroscopy or histopathology. the patients underwent head and neck ct examination with a 16 detector-row helical ct unit without sedation or general anesthesia. all patients received oxygen during the entire ct examination. five patients presented with respiratory distress (dogs 1, 2 and 4 and all cats) and one with chronic cough (dogs 3 and 5). other clinical signs included cough (dog 2) and stertor (cat 1). the imaging findings and final diagnosis were: dog 1: diagnosed on ct as focal severe tracheal collapse and final diagnosis of tracheal collapse on bronchoscopy. dog 2: diagnosed on ct as tracheal stenosis; final diagnosis on necropsy of severe submucosal granulation tissue and stricture due to scar tissue. dog 3: diagnosed on ct as everted sacules and elongated soft-palate; final diagnosis on bronchoscopy of everted sacules, elongated softpalate and grade 2-3 laryngeal collapse. dog 4: diagnosed on ct as laryngeal paralysis and final diagnosis of laryngeal paralysis on physical examination. dog 5: diagnosed on ct as tracheal collapse and final diagnosis of tracheal collapse on fluoroscopy. cat 1: diagnosed on ct as mass, probable neoplasia; final diagnosis on histopathology of pyogranulomatous laryngitis. cat 2: diagnosed on ct as regional wall thckening (inflammation or neoplasia); final diagnosis of severe suppurative laryngitis. discussion/conclusion: ct of awake patients using the vetmousetrap was successful in accurately demonstrating upper airway disease. ct vetmousetrap imaging was a fast and non-invasive procedure that allowed simultaneous oxygen administration with minimal stress to respiratory compromised patients. introduction/purpose: the purpose of the study was to design, construct and test a device for clinical computed tomographic imaging of awake dyspneic cats. this abstract reports on device design and clinical testing on normal eupneic cats. materials and methods: ten normal cats were placed inside the device for measurement of carbon dioxide (co2), fraction of inspired oxygen (fio2), temperature at rest. twenty two awake normal cats were imaged with two different kvps (80 and 120) and 2 different pitches (0.562 and 1.75). the signal intensity of the pulmonary parenchyma (si-lung), signal intensity of background (si-backgr.), contrast, noise, signal-to-noise ratio (snr) and contrast-to-noise ratio (cnr) were calculated. images were evaluated for sharpness of liver margins, motion artifact, aliasing and windmill artifacts. significance was set at p ¼ 0.001. the final device design was a transparent acrylic tube with a wall thickness of 5 mm, outer diameter of 21 cm, 40 cm in length, with oxygen and catheter line access, and no metallic components. no artifacts related to the device were detected and the cats could be visually monitored throughout the imaging and quickly removed. oxygen and catheter line access was uncomplicated and effective. carbon dioxide levels were mildly elevated by 5 minutes. the mean fio2 at 25 minutes was 68%. temperature levels inside the device increased over time with the highest mean of 26.81c at 30 minutes. overall, 75 out of 80 (94%) examinations were judged to have absent or minimal motion artifact. higher kvp protocols had higher snr and cnr. higher pitch protocols had lower noise, higher snr and cnr, and less motion artifact. low pitch protocols were deemed clinically better because of significantly lower windmill and aliasing helical artifacts. discussion/conclusion: the vetmousetrap was an effective device for imaging awake cats with minimal elevation in carbon dioxide and significant elevation in ambient oxygen. the only clinically relevant artifacts noted were windmill and aliasing in the large pitch (1.75) protocols. motion was not a clinically significant issue. section of diagnostic imaging, department of companion animal clinical studies, 3 section of epidemiology, department of production animal studies, faculty of veterinary science, university of pretoria, onderstepoort 0110, south africa introduction/purpose: canine chylothorax is a problematic condition to manage due to poor response rates with both medical and surgical management. surgical ligation of the thoracic duct (td) has been the most frequently reported treatment; however success rates with td ligation alone have been reported to be between 53% and 59%. failure to ligate all of the td branches has been described as the most common cause of surgical failure. lymphography has conventionally been conducted by catheterization of a mesenteric lymphatic, but has recently been described using the less invasive technique of percutaneous injection into a popliteal lymph node. computed tomography (ct) has been shown to be superior to radiographs in detecting td branches and providing td topographical information. this study assessed the ct comparability of the two lymphography techniques and determined the equivalency of helical and sequential ct modalities for td lymphography. materials and methods: seven beagles underwent both mesenteric lymphography (ml) and ultrasound guided percutaneous popliteal lymphography (ppl). iohexol 300 mg/ml was administered at 1 ml/kg for both techniques. for the ppl, contrast medium was administered at 100 ml/hr where as for ml it was given as a bolus over a one minute period. helical ct was initiated immediately after completion of contrast medium injection, followed by sequential ct of the same area. for both administration techniques and ct modalities, images were taken at the mid-vertebral bodies of t9-l1. images were assessed for the total number of td branches and their position relative to the aorta, as well as the cross-sectional area and mean hounsfield units (hu) for the largest td branch. the effects of administration technique, ct modality, vertebral site and animal on the observed td number were assessed using zero-truncated poisson regression, while their effects on cross-sectional area and mean hu, were analyzed using multiple linear regression. the number of td branches detected did not differ significantly between the two contrast administration techniques (p ¼ 0.256) or ct modalities (p ¼ 0.417), although the count ratio (cr) indicated a trend for the ppl administration technique (cr ¼ 0.830) and the helical ct modality (cr ¼ 0.876) to detect slightly fewer td branches. ml and helical ct both resulted in significantly greater largest td branch cross-sectional area and mean hu (po0.001). this study suggests that ppl is an acceptable alternative to ml for identifying td branches when using either helical or sequential ct. introduction/purpose: thoracic computed tomography (ct) is superior to conventional radiography by eliminating superimposition and enhancing resolution, thus improving detection of pathology. acquisition timing during computed tomography angiography (cta) is critical to optimize contrast medium enhancement. due to the large differences in veterinary patients optimal timing of contrast enhancement cannot be standardized. this study serves to determine if there is any statistically significant difference in the two cta techniques, namely bolus tracking (bt) and time delay (td) utilizing a test bolus in terms of enhancement, scan time and ct dose index (ctdi). materials and methods: 6 healthy beagles (36-48 months), underwent dual slice thoracic cta using the bt and td techniques. the time interval between studies was 6 weeks and the order was randomized. images were interpreted at the level of the cranial aspect of the 8th thoracic vertebra (t8), mid and caudal t8, where the aorta (ao), pulmonary artery (pa) and caudal vena cava (cvc) were visualized simultaneously. images were viewed in a mediastinal window. in this within-subject study design the bt and td techniques were compared with respect to the hounsfield units in the ao, pa and cvc during the arterial and venous phases, respectively. findings were compared using the students paired t-test and in each case the result was confirmed by the non-parametric wilcoxon's method pairs signal-ranks test. the shapiro-wilkes test for normality was done. additionally, the 95% confidence interval for the difference of the means between the bt and td techniques was rewritten to a 95% confidence interval for the bt as a ratio of the td studies, where the latter was considered the gold standard for an individual. (gsd) . although various studies dealing with radiographic appearance of the lumbosacral region in gsd exist, radiographic anomaly of the lumbosacral transition has not been confirmed by an adequate population size so far. the aim of this study was to analyse the anatomical variance and frequency of early onset pathologies of the lumbosacral transition between gsd and other large breed dogs. furthermore, a genetic analysis of radiographic signs related to lumbosacral stenosis was performed for the gsd. a total number of 1.267 lateral lumbosacral radiographs of clinically sound large breed dogs -733 of which gsd (mean age 15 months) and 534 dogs of other breeds (mean age 31 months) -were studied using established methods (1, 2) . nine specific findings and 17 anatomic dimensions were assessed with emphasis on malalignment and vertebral canal height at the lumbosacral level as well as lumbosacral osteochondrosis and transitional vertebrae. the results of radiographic examinations of 572 gsd (95% born in [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] with complete pedigree data were included into heritability estimation. there were significant differences between the gsd and the other breeds in most of the assessed morphologic parameters. the prevalence of lumbosacral osteochondrosis and 4 other specific findings was significantly higher in the group of the gsd. the ratio of ventrodorsal lumbosacral subluxation was 2:1 between gsd and other breeds and was significantly higher for the gsd. dorsoventral dimension of the lumbosacral vertebral canal was significantly lower for the gsd in any location, whereas the ratio of caudal lumbar vertebral canal height to cranial sacral vertebral canal height was significantly higher in this group. genetic analysis of the collected parameters in the group of the gsd resulted in moderate to high heritability estimates for specific findings (h 2 0,22-0,64) and predominantly high heritability estimates (h 2 0,31-0,73) for anatomic dimensions. discussion/conclusion: compared with other breeds there is a primary aberrant anatomic conformation of the lumbosacral transition and a higher frequency of promoting factors such as sacral osteochondrosis, malalignment and inherently narrow vertebral canal, that predispose gsd's to lumbosacral stenosis. genetic determination of these predisposing factors suggests that a selective breeding scheme based on radiographic parameters could be promising. introduction/purpose: previous studies reported the german shepherd dog (gsd) being overrepresented in the occurrence of cauda equina compression syndrome (ces) as a consequence of degenerative lumbosacral stenosis (dlss). a congenital predisposition for the early degeneration of the lumbosacral intervertebral disc has been suspected. male dogs show a higher incidence of ces and multiple anatomic variations such as lumbosacral transitional vertebra or osteochondrosis of the sacrum are reported as predisposing factors. the purpose of the present prospective study was to evaluate degeneration of the lumbosacral intervertebral disc in the gsd compared to other breeds. the lumbosacral spines of 126 gsds and 54 other large breed dogs were examined using magnetic resonance imaging (mri). the signal intensities of the entire disc and the nucleus pulposus were determined. the dimensions of the disc and the lumbosacral angle were measured, and step formation as well as conformation of the lumbosacral junction was recorded. in addition the degree of degeneration of each intervertebral disc was graded using three different classification systems. results of gsds without clinical signs of ces were compared to healthy dogs of other breeds and to dogs showing signs of ces. statistical regression analysis was performed to identify possible associations between the morphology of the intervertebral disc and the training level of the dogs. results: a significant difference in the severity of degeneration of the lumbosacral intervertebral disc in gsds in comparison to other breeds was found. degeneration of the lumbosacral intervertebral disc was not associated with the degree of degeneration of other lumbar discs. morphologic differences concerning gender and breed could be deminstrated. discussion/conclusion: we concluded that male dogs and gsd dogs have a higher risk to develop degenerative changes of the lumbosacral intervertebral disc. introduction/purpose: when interpreting mr images in veterinary diagnostic imaging a thorough understanding of brain anatomy and species characteristics are of particular importance. although feline and canine brains have been examined for decades most other mammals are only occasionally scanned. materials and methods: scans of anatomical of various mammalian species specimen were analyzed and retrospectively compared. results: metatheria (marsupials): the corpus callosum (and septum pellucidum) is only present in eutherian mammals, kangaroos and other marsupial brains are remarkable for their lack of this and have a well developed rostral commissure that connect the hemispheres. in the opossum the cortical surface only shows two major furrows, one is the lateral rhinal fissure, separating a large ventrolateral rhinencephalon from the neocortical part and the other is the orbital fissure at the rostrodorsal end of the hemisphere. in higher marsupials, as the kangaroo a gyrencephalic brain is present. the olfactory bulbs are huge and solid, without an olfactory recess. lagomorphs: in hares the surface of the pear shaped brain is smooth with only the lateral rhinal fissure and a dorsal longitudinal fissure, the marginal sulcus. neocortical and older cortex divisions almost equal in size and the large ammons horn parallels the neocortical surface over a large extent. in contrast to higher mammals, the paleocortex is less developed, thinning rather in its dimension when running caudad. in the cerebellum, the vermis is straight and the most pronounced structure is the ventral flocculonodular lobe that extends far laterally. ungulates: the scans nicely reflect the important characteristics of artiodactyl brain, e.g. the dominant position of the visual and olfactory systems. this is seen in the large diameter and size of the eye, optic nerve/chiasm, and rostral colliculus respectively, as well as in the impressive dimension of the olfactory bulb and tracts as well as of the piriform lobe. the cortex of ungulates telencephalic hemisphere is densely folded and the major sulci show a high variability in ramification. most pronounced in the horse. the corpus callosum is well developed in artiodactyls. in the diencephalons a large pituitary gland and pineal gland are striking. discussion/conclusion: for the identification of structural deviations from normal anatomy in species other than cats and dogs a thorough understanding of brain anatomy is necessary. introduction/purpose: gliomatosis cerebri is a neoplastic condition of the central nervous system characterized by widespread diffuse infiltration of the brain and spinal cord by neoplastic glial cells with preservation of the brain architecture. the condition is rare in all species and has been confirmed histologically in dogs. a case report of a single dog described mri findings as hyperintense and ill-defined in t2-w and flair sequences within one hemisphere of the forebrain and extension to the brainstem and cerebellum. the purpose of the present case series is the description of the mr features in five dogs and their correlation to histopathology. the present case series includes 5 dogs, one yorkshire terrier and four bearded collies, presented because of neurological disorders including circling (3/5), central blindness (2/5) or decreased menace response. the disease was localized to the forebrain. mri was performed at two different institutions: 3 dogs were examined using a 0,3t mri unit and two dogs using a 1,5 t unit. minimal sequences consisted of t2-w sequences, t1 sequences pre-and postcontrast in different planes and flair (3/5). results: mri findings included diffuse hyperintense areas in t2 w sequences in all dogs. the ill defined lesions were hypointese (4/5) or isointense (1/5) in t1 w images. no contrast uptake was present in any case. the lesions were bilateral with one side always more severely affected and continued along the white matter extending partially into the gray matter with contact to the brain surface. in all cases the thalamus and cerebellum were affected. mild to moderate mass effect and midline shift was present (5/5) and there were signs of increased intracranial pressure with flattening of the sulci, transtentorial herniation (3/5), herniation through the foramen magnum in two of these dogs, and periventricular edema on flair images (3/5). other signs included syringohydromyelia (4/5) in the cervical spine. additional hyperintense areas interpreted as infiltration within the spinal cord were visible in two cases. histopathologically all cases revealed a glial cell population with homogenous morphology infiltrating diffusely the white and gray matter of both hemispheres, the thalamus, brainstem and cerebellum. in two out of three cases an infiltration of neoplastic cells in the spinal cord was found. discussion/conclusion: mri features of all five dogs were very similar in distribution; signal behavior, lack of contrast uptake, had signs of mass effect and increased intracranial pressure. descriptions in men are similar except mass effect, which might be explained by the later presentation of dogs and progression of disease. distribution and extent of the lesion corresponded well to histopathological findings. on mri interstitial edema could not be differentiated from cellular infiltration, however, hypointense signal in t1-w sequences in more severely affected areas could be a sign for brain edema (high water lesion), which was confirmed by histology. because four of the five presented dogs were bearded collies we hypothesize that gliomatosis cerebri in this breed may have a hereditary background. c. haarstrick, c. niesterok, g. oechtering, m. alef, i. kiefer. department for small animal medicine, university of leipzig introduction/purpose: in small animals the soft palate can only be examined by oral inspection, such as pharyngoscopy enabling only examination of the organ surface. investigation of the internal pattern is possible by ct or mri, but the expensive equipment and required anaesthesia form limitations of performing these examinations. the examination of the palate gains importance due to the increasing popularity of brachycephalic dog breeds. because of their anatomical conditions and problems during recovery from anesthesia, less risky procedures, such as ultrasonography, are needed. the aim of this study is to characterize the normal sonographic anatomy of the soft palate and to define predetermined landmarks for ultrasonographic measurements of the soft palate diameters in dogs. the study was performed on seven canine heads. the dogs were euthanized because of various diseases not related to the head. ultrasonography was performed via a submental approach. depending on the shape of the ultrasonic probe also intraoral ultrasonography was performed. the studies were performed in three or four probe positions in sagittal and transversal direction. thickness of the soft palate was measured at two defined locations. subsequently the detected anatomic structures were reexamined by submental approach in a water bath. afterwards the single anatomic layers were dissected starting with the submental layers working up towards the soft palate. after every single preparation a sonographic examination was carried out. finally the isolated soft palate was analyzed in the water bath by ultrasound. the produced images were assessed regarding recognizable structures and analyzed for image quality by a subjective score system. the transition between the hard palate and the soft palate as well as the middle section of the soft palate was distinguished from the surrounding tissue by submental approach in all cases. evaluation of the echogenicity of the soft palate parenchyma was difficult. also visualization of the caudal part of the velum depended strongly on testing conditions. the differentiation between cutaneous and subcutaneous tissue, sublingual musculature and tongue is easily achievable. measurement of the thickness of the palate was done at two defined locations. the first was defined one centimeter caudal to the transition between the hard and the soft palate. the second more difficult location was determined at the caudal soft palate adjacent to the presumed palatopharyngeal arch which is identified as hyperechogenic line caudodorsal to the soft palate. discussion/conclusion: in this study the sonographic appearance of the canine soft palate using cadavers was described. the submental approach enables the differentiation between intraoral structures. evaluation of the echogenicity and length or thickness of the soft palate is limited, due to influence of surface pressure by the investigator for instance. the normal echographic anatomy of the esophago-gastric junction and the gastro-duodenal junction has not been studied in the normal cat. the aim of the present prospective study is to describe the normal aspect of the pylorus, proximal duodenum and the cardia of the stomach. a prospective study was performed in 15 fasted adult cats (11 purebred, 2 crossbreed and 2 domestic shorthair cats) free of gastro-intestinal disease and deworming. cats were scanned in dorsal recumbency, without sedation. a linear probe (12 or 18 mhz) was used for better resolution during the study. measurements were obtained for caudal esophageal wall thickness just cranial to the cardia (from inner mucosa to external serosa in longitudinal section: ew), cardia wall thickness (cw, in longitudinal section), pyloric wall thickness (pw, in longitudinal section), muscularis of the pyloric thickness (mp, in longitudinal section), length of the thicker part of the cranial duodenum submucosa (dl), gastric lymph node thickness (glnt). freezed images and videoclips were recorded for retrospective review. several cats free of digestive symptoms were excluded of the study because of: incomplete emptying of the stomach, abnormal gastric thickening, abnormal digestive lymphadenopathy, incomplete alignment of the gastroduodenal junction, agitated cats. images obtained during the prospective study were reviewed by two ecvdi diplomates (lc and dr). the most caudal part of the esophagus and the gastro-esophageal junction were identified in 10/15 cats (66.7%). submucosa of the most cranial part of the duodenum was thicker in 13/15 cats (86.7%). in all cats, the muscularis part of the cardia and the pylorus was thicker. the former was strip shaped and the latter had a short triangular shape. on the 15 cats studied, only 3 measurements could be systematically performed: pw, mp, glnt. measurements (1st number correspond to mean, minimal and maximal values are in brackets, confidence intervals of 95%) were as follows: ew: 3.1 mm (2.3-3.8 mm); cw: 4.7 mm (3.6-5.1 mm); pw: 4.2 mm (3.1-5.2 mm); mp: 2,5 mm (2-3.6 mm); dl: 7.4 mm (3-13.7 mm); glnt: 4.1 mm (3.4-5.1 mm). discussion/conclusion: linear probe seems to be an efficient tool to identify the pylorus in cats but limited for the cardia and abdominal esophagus especially in fatty cats. the description of the normal echographic anatomy of the ß borders ý of the stomach could help further identification of cardia and pyloric disease in cats. olivier taeymans, dvm, phd, dipl ecvdi, dominique penninck, dvm, phd, dacvr, dipl ecvdi. from the department of clinical sciences, foster small animal hospital, cummings school of veterinary medicine, tufts university, north grafton, 01536 ma, usa introduction/purpose: few reports exists on normal 1,2 and pathological ultrasonographic appearances of the structures at the ileocecocolic junction (icj) in the cat. during routine abdominal ultrasound of feline patients, we observed that it is not uncommon to find changes in this area particularly in cecal wall thickness, cecal contents, mesenteric root fat, and regional lymph nodes. we hypothesize that these changes are not incidental, and may be representative of gastrointestinal (gi) disease in cats. materials and methods: the hospital information system of tcsvm was searched for ultrasound records of feline patients containing a description of abnormalities at the level of the cecum, ileocecocolic area, ileocolic junction, ileocecal (cecal) lymph nodes, or colic lymph nodes between january 2002 and june 2008. patients having gastrointestinal neoplasia were removed based on available patient outcome. this resulted in 29 cats, 14 males and 15 females. mean age was 87 months. results: there were 18/29 cats with enlarged cecal lymph nodes. focal hyperechoic mesenteric fat was noted in 18/29 cats. mild fluid accumulation at the ileocecocolic junction was seen in 7/29 cats. fluid content was noted in 7/29, and foreign cecal material in one cat. six cats showed a rounded shape of the cecum. the cecal wall was thickened in 19/29 cats, 6/19 showing wall alterations. the ileal wall was mildly thickened in 6 cats, and 4 of these had altered wall layers. 18/29 cats had no ultrasonographic evidence of gi changes in other portions of the gi tract, and 17 of these 18 cats were symptomatic for gi disease. 8 cats had fine needle aspirate or biopsies. histopathology confirmed mild colitis/enteritis in one case, and mild reactive lymph nodes in another case. cytology showed reactive lymph nodes in two cases. our results show that icj changes was the only gi finding in 62% of our population, and that all except one of these cats were symptomatic for gastrointestinal disease. these results support that ultrasonographic changes noted at the level of the icj may have clinical relevance, and may represent typhlitis. introduction/purpose: comparison of the kidney long axis dimension to the second lumbar vertebra (l2) on ventrodorsal projections has proved to be an accurate method for assessment of kidney size on radiographs. in the adult dog, the kidney length has been reported to be 2.5-3.5 times the length of l2. 1 the aim of the study was to test the hypothesis that the suggested maximum normal kidney size obtained from radiographs is too high and also to evaluate whether breed type (brachycephalic, doliocephalic, mesocephalic), age, gender, weight and body condition of the dog have an impact on kidney size. abdominal radiographs of 100 dogs with no evidence of concurrent disease that might have an effect on renal size and that had no clinical or ultrasonographic evidence of renal disease were included in the study. all animals had normal renal blood profile and electrolytes. the animals were radiographed in right, left lateral and ventrodorsal recumbency although not all animals had all projections performed. the number and type of projections depended on necessity, and the reason for the clinical investigation. radiographic length of the kidneys was measured on digital images and compared to the length of the second lumbar vertebra. the ratios of kidney length to length of l2 (kidney/l2) of each dog in all available views were calculated from measurements obtained from radiographs. the length of kidneys of 3.5 times the length of l2 was above the 97.5th percentile of the distribution for all the ratios except the left kidney/l2 measurements on vd views. significant differences (p ¼ 0.008) in kidney/l2 ration between the groups were present, especially between the brachy-and dolicocephalic groups. the brachycephalic group appeared to have the highest kidney/l2 ratio. the dolicocephalic group never reached a kidney/l2 ratio of 3.5. there was no significant difference between genders. higher kidney/l2 ratios were observed in intact animals compared to neutered animals. the weight did not have a significant impact on kidney/ l2 ratios although there was very close to a significant difference between weight categories (o10, 10-30, 430 kg) with the largest difference between the categoryo10 and the other two weight groups. discussion/conclusion: mild renomegaly should be suspected if the kidney/l2 ratio is equal to or larger than 3.5 in dogs other than brachycephalic breeds. the kidney/l2 ratio of 3.5 in our study was found only within the brachycephalic dog group. all other groups had slightly lower ratios. it is suspected that this may be due to shorter vertebrae rather than longer kidney length in brachycephalic dogs but further studies are needed. introduction/purpose: granulomatous meningoencephalitis (gme) is an acute progressive and often fatal inflammatory disease of the central nervous system (cns) mainly affecting small and toy breed dogs, often young and middle-aged. a definitive diagnosis of gme can only be achieved after postmortem and histological examinations. the purpose of this study was to describe the ultrasonographic findings, using color doppler mapping and spectral analysis in dogs with postmortem histologically confirmed gme. retrospective study of transcranial doppler sonography (tcds) examinations performed between august 2005 and september 2006. during this period, 116 dogs with clinical signs of cns disease were submitted to tcds. but only 11 dogs were selected with gme diagnosis confirmed at postmortem examinations. b-mode images were taken to evaluate brain parenchyma anatomy and echotexture. color doppler mapping was performed to identify the circle of willis on both hemispheres. after identifying the vessels, pulsed doppler was accomplished on each one, obtaining resistive index (ri). seven female (64%) and 4 male dogs (36%) presented gme diagnosis confirmed at postmortem and histological examinations. the mean age was 6 years (range: 3-15 years). decreased parenchymal echogenicity was observed in 9 dogs (82%); 8 dogs presented ventriculomegaly. focal lesions were detected in 6 dogs (54.5%). color doppler study revealed conspicuous vessels in the circle of willis of 10 dogs (91%). pulsed doppler detected the six major cerebral arteries in 7 dogs. in 2 dogs only one artery was not depicted and in one dog, two cerebral arteries were not outlined. pulsed doppler showed normal and high ri values in the outlined arteries. discussion/conclusion: tcds findings were correlated with pathologic examination diagnosis. there was a positive correlation between ultrasound and pathology findings concerning the focal lesions. color doppler study showed conspicuous intracranial vessels using minimum color gain, possibly due to the increased diameter. vascular occlusion and stenosis caused by space-occupying masses or inflammatory infiltrates may be the possible cause of undetectable vessels. csf pressure is normal or high in gme patients. the dogs showed normal or high ri values. focal granulomas and vascular changes in different locations and various degrees of presentation can modify microvascular impedance. sonographic findings in this study corroborate previous gme pathology reports. although it is known that an in vivo diagnosis is difficult, tcds can be a useful instrument to evaluate cns when gme is considered. introduction/purpose: in recent years, with the new development of high resolution, electronic broad band transducers, ultrasound (us) is being considered as an optimal image technique to evaluate normal anatomy and abnormalities of the peripheral nerves. to the author's knowledge, information regarding the appearance and ultrasonographic approaches of peripheral nerves in the cat has not been documented. the aim of the present study was to determine the ultrasonographic appearance and the approaches to assess the sciatic nerve (scn) in cats. materials and methods: anatomical nerve study: eight limbs from four feline cadavers were used for anatomical dissections. another two fresh feline cadavers were frozen and cut in cross sections to determine the anatomic landmarks. ultrasound nerve study ''in vitro'': eight pelvic limbs from four fresh feline cadavers were employed. all limbs were scanned using a 4-13 mhz linear transducer (mylab 70, esaote s ). the examinations were performed in three regions: glutea, femoris and poplitea. the accuracy of the ultrasound localization was demonstrated by injecting ink around the targeted nerves which were, then, immediately dissected. the obtained ultrasonographic images were compared with the anatomical cross sections to facilitate their interpretation. ultrasound nerve study ''in vivo'': five healthy adult experimental cats were sedated to perform the ultrasonographic examination of the scn using the technique described for the ''in vitro'' study. the ultrasonographic images of the scn showed a good correlation with the anatomical nerve study. the scn was easily identified on glutea, femoris and poplitea regions. on the longitudinal view, the ultrasonographic appearance of the scn was a tubular hypoechoic structure with internal echoes, outlined by hyperechoic lines. on the transversal view, it was observed as an ovoid hypoechoic structure surrounded by a hyperechoic rim. there were no differences between the ultrasonographic appearance of the scn in fresh cadavers or healthy cats. discussion/conclusion: this study shows the usefulness of ultrasound to evaluate the scn in cats, from its origin until its division into the peroneus communis and tibialis nerves. the ultrasonographic appearance of the scn was as a hypoechoic structure with internal echoes, outlined by hyperecogenic lines. benigni, l., corr s. a., lamb c. r. ultrasonographic assessment of the canine sciatic nerve. vet. rad. ultrasound. 2007. 48 (5) : 428-433. echeverry d., gil f., laredo f., ayala m. d., belda e., soler m., agut a. ultrasound-guided block of the sciatic and femoral nerves in dogs: a descriptive study. vet j. 2009 (in press). introduction/purpose: ultrasound examination performed by and experience sonographer is becoming the elective exam when suspecting of a gastrointestinal obstruction, replacing contrast radiographic studies for confirmation of these. to the author knowledge only hyperechoic interface foreign bodies with acoustic shadowing have been described in the literature. this report describes the appearance of hypoechoic foreign bodies, specifically rubber pacifiers. three dogs with gastro-intestinal clinical signs where referred for an ultrasound. in all of them, a throughout exam of the gastro-intestinal tract as of the remaining abdominal cavity was made (sonosite titan with microconvex transducer from 5-8 mhz). a gelatin model (collagen based) was made containing 3 rubber pacifiers (1 latex and 2 silicone). an ultrasound scan was performed to access the ultrasonographic appearance of the pacifiers without the interference of gas or ingesta from the gastrointestinal tract. results: three foreign bodies were detected ultrasonographically. one foreign body was detected in the stomach, 2 were detected in the small bowel (jejunal loops). only two animals had ultrasonographic signs of mechanic ileus, the ones that were detected in the small bowel, the one visualized in the stomach was only partially obstructive. all of the foreign bodies shared the following ultrasonographic appearance: hypo to anechoic band with a hyperechoic capsular interface. their shape varies according to the plane sectioned as well as the part of the pacifier scanned. the teats had an oval shape, were composed of and hypo to anechoic band with strong hyperechoic contours, they were open on one side, allowing for the content of gastrointestinal tract to enter it. all of the foreign bodies detected were surgically removed. the pacifiers visualized in the gelatin model had low or null acoustic impedance, and therefore were anechoic with a hyperechoic contour. discussion/conclusion: rubber pacifiers, latex and silicone based, are present in many households with children and seem to be a commonly ingested foreign body by our pets. their specific ultrasound appearance makes their diagnosis easy by ultrasound, since its ultrasonographic features are patognomonic. rubber pacifiers size and position as well as the size of the animal, will determine if they are completely or partially obstructive. nonetheless even in partially obstructive cases, their surgical removal is advised. introduction/purpose: recently, a fatal syndrome characterized by mesenteric lymphadenopathy and hypergammaglobulinaemia has been recognized in domestic ferrets. histological lesions are similar to those of the non effusive form of feline infectious peritonitis (fip), and with immunohistochemical techniques coronavirus antigen in several affected organs have been detected. a recent mutation or shift in the ferret enteric coronavirus (fecv) is thought to be the cause of this syndrome (known as ferret systemic coronavirus infection). this report describes radiographic and ultrasonographic findings observed in the abdomen of seven ferrets with confirmed diagnosis of systemic coronavirus infection. materials and methods: 7 domestic ferrets (2 female and 5 male; 8-18 months) were presented at the veterinary teaching hospital with a history of lethargy, hyporexia or anorexia, and weight loss. diarrhea was recorded in 3 and vomiting in 1 of the ferrets. abdominal masses were palpated in 4 animals. pale mucous membranes, abdominal enlargement, and abdominal pain were observed in 2 of the ferrets. faecal examination, complete blood count (cbc), serum biochemical profile, abdominal radiographs (right lateral and ventrodorsal views) and abdominal ultrasound (linear high frequency, 11-13 mhz, transducer) studies were performed in all the ferrets. hyperglobulinaemia with monoclonal gammopathy (7/7), anaemia (5/7) and severe neutrophilic leucocytosis (4/7) were detected. ferret systemic coronavirus infection was diagnosed based on histological findings (pyogranulomatous inflammation of the viscera and peritoneum) and on immunohistochemical confirmation of coronavirus antigen presence in affected tissues. radiographically, signs of emaciation (loss of lumbar musculature) and decreased serosal detail were recorded in 5 animals. presence of mid abdominal soft tissue mass was observed in 4 ferrets. other radiographic findings include moderate splenomegaly and mild to moderate dilation of the intestinal tract with gas (2/7), pendulous ventral abdominal wall and left kidney enlargement (1/7). sonographically, a mild amount of echogenic effusion and thickening of the peritoneum, omentum, and mesentery with a hyperechoic and hyperatenuated aspect was the most common finding (6 ferrets). heterogeneous, ill-defined soft tissue masses, surrounded by hyperechoic fat and mesenteric lymphadenopathy were imaged in 5 animals. mild to moderate splenomegaly was observed in 3 patients and inguinal inflammation accompanied by signs of vascular thrombosis was recorded in 1 ferret. although definitive diagnosis of systemic coronavirus infection is based on histological and immunohistochemical results, a tentative diagnosis can be made on the basis of the animal's history, clinical signs, and clinicopathological, radiographic, and ultrasonographic findings. imaging signs of peritonitis and the presence of soft tissue masses are highly suspicious of systemic coronavirus infection. aleutian disease, lymphoma, and chronic inflammatory bowel disease should be included in the differential diagnosis. susanne stieger-vanegas, christopher cebra, jason wiest. department of clinical sciences, college of veterinary medicine, oregon state university, corvallis, oregon, usa introduction/purpose: in llamas and alpaca colic symptoms can be obscure. additionally, the cause of colic symptoms can be difficult to localize due to the limited availability of diagnostic techniques evaluating the abdomen. colic symptoms caused by bacterial enteritis or enteritis caused by eimeria macusaniensis can be very difficult to differentiate from colic symptoms caused by intestinal strangulation, entrapments, or intraluminal obstruction. the lack of the ability to differentiate between medical and surgical causes of colic symptoms affects treatment and therefore the survival rate and outcome of new world camelids with colic symptoms. computed tomography has been performed in llamas 18 hours after barium was given with an orogastric tube.1 however, 18 hours can be a long delay time for medical or surgical treatment in a patient with colic symptoms. the purpose of our study was to evaluate a new gastrointestinal imaging protocol using iodinated contrast media diluted in water. the objective of this study was to evaluate the feasibility of this new ct protocol to evaluate the gastro-intestinal tract in clinically normal new world camelids. three hours prior to the computed tomography study, three alpacas and two llamas without clinical signs indicating gastro-intestinal disease received iodinated contrast media diluted in water via an orogastric tube. following sedation, the animals were positioned in sternal recumbency on the ct table and axial, sagittal and coronal images were obtained. to enhance the definition of the organs and gastrointestinal wall intravenous iodinated contrast was applied and images were obtained with a 60 sec delay. in all animals the first, second and third compartment of the stomach (c1, c2, c3), small intestine, spiral colon and ascending colon were identified. in all animals the cranial and caudal glandular saccules of the stomach were observed. the ampulla of the duodenum was well seen and could be separated from the remainder of the duodenum. the walls of the intestine could be identified. in all patients the contrast reached at least the cranial aspect of the spiral colon. introduction/purpose: although radiographs are essential in the evaluation of thoracic diseases, the findings may not be specific or limited. in dogs and cats, the use of ultrasonography in the diagnosis of thoracic extracardiac diseases demonstrates an important role, providing images in real time and providing additional information regarding the location, size, extension and nature of the injuries. this study aims to compare the radiographic and b-mode and doppler ultrasonographic findings of the thorax, excluding the heart. materials and methods: twelve dogs and one cat suspected of thoracic disease undergoing thoracic radiography with formation of potential acustic window (caused by fluid, lung consolidation, mass lesions and/or pulmonary nodules) where submitted to ultrasonography of the thorax between march 2009 and march 2010 in the veterinary department at the federal university of viçosa, minas gerais, brazil. four cases had the post mortem examination. in 12 cases the radiographs showed effective acoustic window. in 10 cases the thoracic ultrasonography added important information such as: location of the lesion in the pulmonary parenchyma because of the sliding sign (9), best estimative of the amount of free pleural fluid (3), visualization of structure not detected during radiographic exam [cranial mediastinal lymph nodes(1) and atelectatic lung (2)]. in 10 cases the lesion was suitable to doppler evaluation: mass lesions and/or pulmonary nodules showed peripheral vascularization (9) and it was possible to separate pulmonary vessels from fluid filled bronchi. in 3 cases ultrasound did not add to the diagnosis. discussion/conclusion: in this study the b-mode and doppler ultrasonography played an important role in better characterizing of pulmonary, pleural and mediastinal conditions. besides it is an easy, fast, inexpensive, safe method. when available it should be considered to be done to help to reduce the list of differential diagnoses. christian niesterok, eberhardt ludewig, gerhard oechtering, michaele alef, ingmar kiefer. departement of small animal clinic university of leipzig introduction/purpose: both ct and radiography are commonly used for detection of metastases or primary lung tumors as well as for staging of neoplastic disease. on the other hand, they play a minor role in detection of benign round-shaped nodes, being differential diagnoses. the aim of this study was to estimate the value (sensitivity/specificity) of plane radiographs as a first line diagnosticum compared with computed tomography (msct philips brilliance mx 8000) as gold standard. in our study, we were looking for animals which were diagnosed in ct with a nodular lung pattern. in a second step, we then compared those results with those findings from the radiographs. altogether there were 55 dogs and 24 cats that met our inclusion criteria, undergoing both thoracic radiography and ct between june 2005 and dec. 2009. ct diagnostic was performed with a 6 line-spiral-ct ( philips brilliance), with a slice thickness of 2 mm, collimation of 1.5, pitch of 0.9 and a reconstructing matrix of 512 â 512. finally, statistic analysis was performed on order to determine the radiographic sensitivity. overall in 56 of 79 animals (71%), nodular changes already were detected during the radiographs and confirmed later on by the results of the computed tomography. in detail 39 of 55 dogs (71%) and 17 of 24 (71%) cats showed nodular round herds undergoing plane radiographs. the remaining percentage, which we missed in the radiographs mostly consisted of either very small changes or -even more often -were the result of highly changed lung lobes (like pneumothorax etc.), that lead to an overall lung opacity, providing only poor contrast. therefore, in these cases, soft tissue interpretation was difficult or even impossible. discussion/conclusion: radiography still plays an important role as a first line diagnosticum in detecting and differentiating nodular lung lesions. however, one needs to be aware that quite a high percentage of nodular lung changes can be missed with radiographs showing an overall sensitivity of 71% with no differences between dogs and cats. furthermore our study showed, that plane radiographs are of poor diagnostic values wherever concurrent problems (e.g. pneumothorax, pleural effusion etc) exist, that lead to an increased lung opacity. introduction/purpose: oesophageal pathology is not always easy to detect on survey thoracic radiographs and the exact nature of the pathology may be difficult to determine. endoscopy and ct are reliable diagnostic methods to address these deficiencies but are not always available in general practice. it is hypothesized that pneumo-oesophagraphy may be a cost-effective/affordable and readily performed technique to provide additional information on oesophageal pathology. thirty dogs with endoscopically confirmed spirocerca lupi nodules were selected from a larger prospective spirocercosis trial. each dog had rlr and dv survey thoracic views. dogs then had endoscopy under general anaesthesia immediately followed by rlr, llr, dv and vd pneumo-oesophagraphy thoracic views (pneumo-views) made whilst still anaesthetised. endoscopic nodule number, location and characteristics were recorded and were used as the gold standard. the oesophagus was inflated with a ambu bag s through a cuffed endotracheal tube placed in the oesophagus. radiographs were evaluated for degree of oesophageal distension (graded 0-4), nodule/mass visibility (yes/no), nodule surface characteristics and their mural attachment location. data were analysed in excell using paired t-test and chi-square tests and significance was set at po0.05. results: dogs had a mean age of 52 months (range 9-125) and weight of 19.6 kg (range 3.6-41.4 kg) and a variety of breeds were represented. lateral pneumo-views had more oesophageal distension than the dv/vd pneumo-views. dv survey and rlr pneumo-views detected significantly more nodules than the rlr survey and pneumo-dv views respectively. if all four pneumo-views were used 90% of nodules found on endoscopy, could be detected and if this was combined with the 2 survey views this became 93%. there was no significant difference in apparent length of oesophagus affected between the views or the modalities used. in six of nine malignant nodules an irregular mucosal surface, (indicative of neoplasia) could be seen on at least one of the pneumo-views. the mural attachment location could readily be seen on pneumo-views. the associated gas filled stomach gave the best visualization of the cardia on the rlr view. discussion/conclusion: this study indicates that pneumo-oesophagraphy is a useful and simple technique to use in a clinical environment to detect oesophageal pathology and its characteristics. introduction/purpose: pulmonary edema is the most common complication of congestive heart failure in dogs. interstitial pulmonary edema, which usually precedes more severe alveolar edema, may not be radiographically detectable. in human medicine, transthoracic ultrasonography is used to detect narrow repetition artifacts, or comet-tail artifacts, that indicate the presence of edema in the lung. the hypothesis of this study is that comet-tail artifacts can be identified using ultrasono-graphy in dogs with cardiogenic pulmonary edema, and that these artifacts occur in greater numbers and are more widely distributed in dogs with congestive heart failure than in normal dogs. transthoracic ultrasonography was performed in a normal group of clinically healthy dogs and dogs with heart failure with radiographic and echocardiographic signs of congestive heart failure. physical examination, two-view thoracic radiographs, two-dimensional and doppler transthoracic echocardiogram and heartworm antigen test were performed on all dogs. during ultrasonography, dogs were gently restrained in a standing or seated position. the thorax was divided into 4 quadrants on each side, and video clips and still images of each quadrant were recorded. distribution, characteristics and number of comet-tails were determined using qlab software (philips healthcare, bothell, wa). statistical analysis was performed using sas software version 9.1.3 (sas institute, cary, nc). results: 31 dogs were recruited in the normal group and 6 dogs were included in the heart failure group. transthoracic ultrasonography was easily and quickly performed on all dogs, with an average examination time of ten minutes or less. comet-tails were detected in 32% of the normal dogs, with a mean of 2 per dog. no statistical association was found between the variables age, sex, side and distribution using a phi correlation coefficient. subjectivley, in dogs with cardiogenic pulmonary edema, comet-tails occurred in greater numbers and were more widely distributed. in severe cases, comet-tail artifacts were so numerous that they became confluent and obscured the borders of the pleura. the presence of artifacts was consistent with the distribution of edema on radiographs. discussion/conclusion: artifacts are rare and isolated in normal dogs, but numerous and widely distributed in dogs with congestive heart failure, as has been described in human patients. further studies are needed to determine if the number and distribution of comet-tail artifacts in dogs with pulmonary edema correlates with the severity of clinical and radiographic signs. thoracic ultrasonography is a fast, easy, and effective method to characterize pulmonary edema in dogs with mitral valve disease. it may provide additional information in combination with traditional diagnostic tests for the assessment of dogs with heart failure, the most important being the detection of early signs of heart failure and monitoring of the response to treatment. introduction/purpose: to evaluate multiobserver diagnostic performance and reader agreement for meniscal tear detection using low-field mri (lfmri) in dogs with suspected cranial cruciate ligament (crcl) disease, with arthroscopy as the reference standard. materials and methods: fifty consecutive stifles with clinical and radiographic signs suspicious for crcl insufficiency underwent mri using a 0.5 tesla philips gyroscan t5 nt magnet with a human quadrature knee coil. the protocol contained sagittal und dorsal pd-and t2-, sagittal and transversal t1and sagittal fat suppressed t2-weighted sequences with images of 3 mm slice thickness and a total scan time of 32.5 min. after mri, diagnostic arthroscopy was performed by 1 surgeon (p.b.) unaware of the mri findings. mri images were read independently by 15 observers, unaware of historical and clinical data, asked to identify presence of a meniscal tear, which would necessitate invasive verification of meniscal integrity probably requiring meniscal surgery. diagnostic performance of each individual observer was analyzed. the impact of the individual observer's experience in stifle mri on the observer's diagnostic performance was estimated by correlating the level of experience (5 self reported levels) with all parameters of test performance. the overall diagnostic performance for the lateral and medial menisci expressed as median and associated interquartile range (iqr) was: sensitivity 69.2 (61.5-73.1), specificity 91.9 (85.6-94.6), ppv 72.7 (62.4-81.8), npv 87.7 (86.0-90.3), accuracy 85. 6 (80.8-87.6 ) and diagnostic odds ratio 24.5 (14.7-45.0). the level of experience in stifle mri did not influence the diagnostic performance of the individual observer (p40.05). discussion/conclusion: in a previous study with a single observer, using 42 data sets out of the 50 data sets from the present study, sensitivity and specificity of lfmri for detection of meniscal tears were 0.56 and 0.97, respectively, with a ppv and npv of 0.88 and 0.84, which might have been related to the low oberserver experience. these results with 15 observers of various levels of self reported experience in stifle mri show that the level of experience did not impact on the performance of the individual observer, with a median diagnostic performance similar to the first study. introduction/purpose: the temporomandibular joint (tmj) is formed from the temporal fossa of the temporal bone and the condylar process of the mandible. investigation of tmj diseases such as osteoarthritis and dysplasia requires a clear diagnostic image. while advanced imaging such as computed tomography allows clear imaging of the tmj, superimposition of the tmjs on conventional lateral radiographs creates difficulties in visualization and interpretation. oblique projections are generally used to separate the tmjs on a radiograph, but these are technically challenging to achieve. the aim of this study was to compare 5 different oblique radiographic views with the head in lateral recumbency, assessing the clarity of visualization of the normal tmj anatomy. the views under investigation were the laterorostral-laterocaudal oblique at a 101 and 201 rotation of the head (''nose-up'' view), lateral view with a rostro-caudal x-ray beam angulation of 101 and 201, and a parallax view with the beam centered over c2 and collimated to include the tmj region, using the divergence of the x-ray beam to project the tmjs separately on the radiograph. the views were performed on both tmjs of thirty canine cadavers with no breed specification although brachycephalic dogs were excluded. the radiographs were graded independently by experienced (board certified radiologist) and inexperienced (undergraduate veterinary student) observers. grading was performed on the temporal fossa, condylar process, joint space, retroarticular process and the overall tmj, and was based on a four point scale, with 0 indicating the structure could not be visualised and 3 indicating clear visibility and definition of the structure in question. mean grades for each component and for the overall joint were compared for each observer and each projection. mean grades were significantly (po0.005) higher for the ''nose-up'' projections than the angled beam or parallax projections, and both observers showed significantly higher (po0.05) mean grades for the 20o ''nose-up'' angulation than the 10o ''nose-up'' angulation. cohen's kappa was used to assess interobserver agreement, and was also highest for the ''nose-up'' projections. discussion/conclusion: these results suggest that a latero 20o rostral-laterocaudal oblique gives the best representation of the anatomy of the tmj of the dog of the projections assessed, and should be considered when investigating clinical cases of tmj disease. introduction/purpose: foot pain is a common cause of lameness in horses. when horses with foot pain have no radiographic abnormalities, soft tissue assessment remains a diagnostic challenge without magnetic resonance imaging (mri). the purpose of this study was to retrospectively review ultrasonographic (us) findings in the front feet in a series of clinical cases with lameness abolished by palmar digital nerves block but without radiographic bone abnormalities. medical records of horses that underwent a complete us examination of the digit were collected. history, clinical findings, radiographic and us results were retrospectively reviewed. the records of 39 horses satisfied the inclusion criteria. thirty of the 39 horses had lesions affecting the deep digital flexor tendon (ddft), 27 horses had abnormalities in the distal interphalangeal joint of which 6 had a visible abnormality in one collateral ligament. distension of the podotrochlear bursa was seen in 22 horses and abnormalities in the ligaments of the navicular bone were detected in 2 horses. irregularity of the navicular bone flexor surface was found in 8 horses. in only 3 of the 39 horses the ddft was the only structure affected. the other 36 horses had us abnormalities in more than one anatomical structure. discussion/conclusion: us diagnosis of soft tissue damage in the foot was possible in cases where radiographic diagnosis was unsuccessful. us offers an easily available diagnostic tool for an evaluation of the digital soft tissues and may decrease the need for mri when this modality is difficult to apply. introduction/purpose: trochanteric bursitis is said to be common cause of lameness in horses and is usually diagnosed based on clinical signs and findings during physical examination. the purpose of this study was to describe the anatomical features of the trochanteric bursa and to develop a technique for centesis of the bursa. the trochanteric bursae of 2 cadavers were injected with latex to demonstrate their boundaries. four techniques for centesis of the trochanteric bursa were evaluated by attempting to inject dye into the bursae of 20 horses which were scheduled to be euthanized for unrelated reasons. following euthanasia, the region of the bursa was dissected to determine the location of the dye. injection was successful in 8/10 bursae when the foot was situated in a hickman block without using ultrasonographic guidance, in 6/10 bursae when the foot was situated in a hickman block using ultrasonographic guidance and a needle guide, in 3/10 bursae when the needle was inserted without ultrasonographic guidance and without placing the foot in the hickman block, and in 9/10 bursae when the foot was situated in a hickman block and the needle inserted using ultrasonographic guidance without using the guide. discussion/conclusion: placing the foot in a hickman block facilitates centesis of the trochanteric bursa. warren-smith cmr, s. danika, n. saunders, l. sambrook, t. bouts, j. hutchinson, r. weller. royal veterinary college university of london, zoological society, london (sambrook l. and bouts t.) introduction/purpose: foot problems are considered the most important ailment seen in captive elephants and can involve the integument and/or the musculoskeletal system, their aetiology can be infectious, traumatic or degenerative. radiography is currently the most commonly used modality in the clinical investigation of musculoskeletal problems in elephants. in this study we described the radiographic and computed tomographic (ct) changes observed in a range of musculoskeletal pathology in captive elephant feet. we also established the intra and inter observer agreement regarding the topographic location and type of these disorders on both radiography and ct. radiographs and ct scans of fifteen cadaver foot specimens from one african and fourteen asian elephants (euthanased for various reasons unrelated to this study) were acquired. in addition 24 radiographic sets were obtained from six live asian elephants from a single zoological collection. a total of 22 front feet and 17 hind feet were evaluated. radiographic and ct assessment was performed by two observers according to an evaluation grading scheme developed based on the reported radiographic and computed tomographic findings in elephant foot disorders. each observer performed the reading of all images twice. pathological changes were observed in 50% (observer 1) and 90% (observer 2) of cases with pathology equally divided between front and hind feet (contrary to reported studies where forefeet have been more commonly involved).changes were seen in all five digits but most commonly in digits iii and iv. pathological changes included osteitis (39.2%), enthesiopathies (17.5%), infectious arthritis (14%), osteoarthritis (7%) and soft tissue infections and defects (21%). intraobserver agreement ranged from ''good'' to ''moderate'' for topographic location, from good to substantial for radiographic diagnosis and was good for severity of radiographic lesions (k: 0.656 to 0.029). however interobserver agreement was only ''moderate'' for diagnosis (k ¼ 0.595). both intra and interobserver agreement for ct agreement was ''poor'' at all levels including topography and diagnosis (k ¼ 0.000 to 0.198. discussion/conclusion: radiography offers an effective way of identifying and differentiating pathological changes both in vivo and post mortem in the elephant's foot, allowing diagnosis and treatment plans to be generated. however ct appears not to offer any advantage over radiography at this time. significant differences in inter observer agreement were seen likely reflecting different levels of expertise in reading radiographs of elephant feet. further large scale studies are warranted to evaluate the different pathologies seen and their respective causes. introduction/purpose: foot related problems are the most common cause of euthanasia in captive elephants and at the moment radiography is the only imaging modality that is available for their assessment. the aim of this study was to (1) develop standard protocols for front and hind legs that are practical to use and allow the thorough examination of all digits and (2) to describe the radiographic anatomy of the elephant feet on those projections. materials and methods: fifteen elephant cadaver feet of different ages and sizes were radiographed using a portable, digital radiography system in a variety of projections for which a custom-made block was designed. in addition computed tomographic (ct) scans were acquired before the cadavers were finally dissected. anatomical features and the pathological changes which were observed on ct scans and gross inspection were identified on the different radiographic projections. the projections that allowed the best visualisation of the most common changes were subsequently trialed on eight live animals using a custom-made block. 3d reconstructions were created from the ct images and registered onto the radiographs to demonstrate the radiographic anatomy of the various skeletal elements of the elephant foot. the dorsoproximal-palmarodistal and the plantarodistal-dorsoproximal projections were found to be best for the examination of the most common sites of pathology. a custom made block allowed the accurate positioning of the foot without endangering the personnel involved. ideally each digit should be radiographed individually and the angle adjusted accordingly, however it was found that the time needed to do this exceeded the patience of any live elephant (and their keepers). hence the number of radiographs was reduced to two per foot, resulting in a total acquisition time of less than 30 minutes for all four feet, which was deemed to be acceptable, but may still be too long in less well-handled individuals. the radiographic anatomy of the elephant foot itself is not overly complex however pathological changes are very common and often result in severe changes in the shape of whole bones. discussion/conclusion: the combination of a dr system and our custom-designed block facilitates radiographing elephant feet considerably. the proposed standardised protocol will not only aid in monitoring the development of foot pathology in individual elephants but will also allow the comparison between elephants in different collections. the detailed description of the normal radiographic anatomy provides a reference against which suspected pathological changes can be compared. introduction/purpose: infiltration anaesthesia of the alveolar branch of the mandibular division of the v th cranial nerve at the mandibular foramen offers superior pain relieve for invasive dental procedures of the mandibular cheek teeth (1) . precision of infiltration is enhanced, if needle placement is guided by computed tomography (2) . this pilot study explores the feasibility of the technique in horse cadavers as well as the possibility of a catheter placement. materials and methods: isolated heads of nine euthanized warmblood horses were scanned using a single slice 3rd generation ct scanner as well as a multislice scanner. all heads were placed in dorsal recumbency within the ct (according the positioning during a head ''real'' scan). the mandibular foramen was located on transverse images and marked with a radiopaque marker or a hypodermic needle using the laser light. the nerve block-simulation was performed with a spinal needle (quinke type point, 18 g, 152 mm). the needle was introduced through the skin medially to the mandibular notch and advanced between 13-15 cm perpendicular to the table surface in the direction of the mandibular foramen. subsequent needle adjustment was guided by ct scans until correct position of the needle's tip at the mandibular foramen was confirmed. diluted contrast medium lopamidol (scanlux r 370 mg j/ml) was injected in eight horses and imaged using a control ct scan. methylene blue was injected in one cadaver and verified by anatomic dissection. the assumed complete coverage of the mandibular nerve could be demonstrated by injected methylene blue during dissection. final positioning of the needle required only a few additional minutes following the first scout ct-scan of the head. one to three corrections in needle placement with subsequent ct scans were necessary to reach the final location. the placement of a catheter (perifix r epidural catheder, 1000 mm, 20 g) through the needle is possible and stays in place after the removal of the needle. a proper fixation on a living horse has to be evaluated. discussion/conclusion: ct guided infiltration anaesthesia of the mandibular nerve in horses is feasible and can be performed immediately after diagnostic ct scans. precision of local anaesthetic deposition at a location that is not readily accessible can be enhanced using this technique. further pain management could be improved by the positioning of a catheter next to the mandibular foramen. metal is a known source of image artefacts in mri by creating image distortion and large areas of signal void. radiography has not been validated for metallic screening, nor has the relationship between particle size and resulting mri artefact size been established. purpose of this study was to gather sensitivity and specificity data for radiographic screening procedures and to create a scoring system for metal particles for their resulting artefact size. materials and methods: 14 cadaver horse feet were radiographed (dprpadio, lm views) and a standard foot protocol mri (t1w gre, t2 ã w gre, stir fse) was performed. stainless steel ball bearings with known diameter (1, 2 and 3 mm) were placed into the most dorsomedial and palmarolateral nail hole of the foot, and dprpadio and lm radiographs were again obtained. the mri foot protocol was repeated with the metal particles in situ. radiographic and mri studies were evaluated by one board certified radiologist. radiographs were scored for presence, diameter and location of metal particles. on mri studies the maximum signal void size was measured in all sequences. radiographic identification and accurate measurement of all metal particles was possible using both projections. on mri metal artefacts were seen as an area of signal void, often with a high intensity rim and distortion in adjacent tissues. sequences in transverse and sagittal planes that contained the entire particle volume within the slice thickness showed the biggest signal void. the size of the mri artefact was approximately 10 â larger than the original metal particle in fse and 15 â larger in ge sequences. t1w gradient echo sequences showed void signal areas 5-10 mm larger than fse sequences. discussion/conclusion: radiography is a sensitive and specific screening tool for mri-artefact-inducing metal particles of diagnostically relevant size. two orthogonal radiographic views are necessary to correctly localize metallic debris. if a metal particle cannot be removed, the diagnostic quality of a foot mri exam can be predicted by plotting the predicted artefact size on the anatomical area of clinical interest. ge sequences should be avoided due to their higher artefact sensitivity. the magic angle effect is a magnetic resonance phenomenon responsible for increased signal intensity in tendons imaged at 551 relative to the magnetic field using short echo time pulse sequences. the magic angle effect has previously been considered as a deleterious artifact responsible for false positive diagnosis of tendinopathy. recently it has been suggested that intentionally imaging tendons at 551 relative to the magnetic field may enhance the detection of some lesions. the purpose of our study was to assess the benefits of magic angle imaging for the characterization of lesions in a diode laser-induced tendinopathy model. materials and methods: 6 isolated equine limbs were imaged in the study. a total of 16 lesions were created using a surgical laser. in 2 limbs, 2 lesions per tendon were induced post-mortem both in the deep digital flexor tendon (ddft) and in the superficial flexor tendon (sdft). in the other 4 limbs, 2 sdft lesions per limb were induced in vivo 3 weeks prior to euthanasia. the limbs were imaged immediately after euthanasia with a 1.5t mr scanner using a surface general-purpose flexed coil. the limbs were imaged parallel to the magnetic field (01) using sagittal t1-w and transverse t1-w, t2-w and stir sequences. imaging with t1-w sagittal and transverse sequences was then repeated with the limbs positioned at 551 relative to the magnetic field. the conventional (01) and magic angle (551) images were reviewed independently for presence and pattern of lesions. all lesions were examined histologically. all lesions could be identified on the magic angle images. the post-mortem induced lesions were not apparent on any of the sequences with the limbs positioned at 01. on magic angle images, the post-mortem induced lesions appeared as a hyperintense core surrounded by a hypointense rim, whereas the ante-mortem induced lesions appeared as mildly hypointense relative to the normal tendon. on 01 images, the ante-mortem induced lesions were hyperintense relative to normal tendon on all sequences. discussion/conclusion: magic angle imaging allows detection of lesions that are not detected using conventional imaging. the differences observed between ante-mortem and post-mortem induced lesions may be explained by the presence or absence of an inflammatory process. with the postmortem induced lesions, in the absence of an inflammatory response, changes in the tendon result from a modification in the organization of the collagen fibers, which can be detected with magic angle imaging. these results suggest that magic angle imaging could be clinically relevant to detect reorganization of collagen fibers in chronic tendinopathy in the absence of an inflammatory response. introduction/purpose: experimentally induced tendinopathies represent an accurate means of evaluating the efficacy of new treatments using blinded studies. in these investigations, imaging modalities are frequently used to assess tendon healing. the purpose of this study was to compare ultrasonographic (us) and magnetic resonance imaging (mri) evaluations to assess the healing of induced superficial digital flexor tendon (sdft) lesions. materials and methods: bilateral metacarpal sdft lesions were induced surgically in 10 standardbred adult horses (week 0). us evaluations were performed before induction and at week (w): 3, 7, 11, 15. at each time, the maximal tendon cross section area was measured and the echogenicity and architecture were graded on a 0-4 scale (0 ¼ sound tendon). low field (0.2 t) mri was conducted under general anesthesia at w11. ultra-high field (7t) mri was acquired post-mortem at w15. for both examinations, the lesion was graded subjectively using the same 0-4 scale. quantitative data were also measured using a homemade matlab image analysis software for the 0.2t data and imagej routines for the 7t ones. these included: tendon and lesion volumes, t1-weighted signal values, t1 and t2 (only for 7t) relaxation times of the lesions. correlations were calculated using pearson coefficients and statistical differences were evaluated using paired student t-test. in both cases, statistical significance was set at po0.05. results: us parameters changed significantly over time: the mean tendon grade decreasing from 2.8 ae 0.2 at w3 to 0.9 ae 0.2 at w15. no significant differences were found in the mri grades of the lesion between the 0.2t (2.6 ae 0.5, w11) and 7t (2.7 ae 0.7, w15) examinations. these grades were significantly higher compared to us ones. significant correlations were found between 0.2 and 7t values for the tendon volume (r ¼ 0.92), lesion volume (r ¼ 0.78), t1-w lesion signal (r ¼ 0.55). in contrast, the lesion volume, expressed in percentage of the tendon volume, was significantly greater in 7t (62 ae 15%) compared to 0.2 t (47 ae 11%). a positive non significant correlation (r ¼ 0.31) was found between the t1 lesion relaxation times at 0.2 t (362 ae 16 ms) and 7t (1749 ae 86 ms). compared to 0.2t, 7t mri allowed to calculate the t2 lesion relaxation time (17 ae 1 ms) and to assess, based on high resolution images, the fascicular architecture of the injured tendons through the visualisation of the connective septa dividing the collagen fiber bundles. this study provides reference data that can be used for future assessment of new treatment efficacy. it confirms the interest of us for evaluating the healing process, particularly in the first stages. despite a significant us improvement, lesions remained severe on mri which may indicate the interest of mri to evaluate the late healing stages. high correlations found between 0.2 and 7t data validate the interest of low field mri, even if 7t mri permits a more accurate evaluation, particularly through the assessment of the tendon fascicular architecture. introduction/purpose: lung diseases occur commonly in captive snakes, especially when snakes are housed under inadequate conditions and/or when errors of management are made. the assessment of the snake's lungs by computed tomography (ct) has already been reported but not compared with radiographic examination. the purpose of this study is to describe and compare the radiographic and ct findings of normal snake lungs and lungs with bacterial pneumonia. materials and methods: six healthy snakes (4 common boa constrictor (boa constrictor), a brown water python (liasis fuscus) and an african rock python (python sebae)), and four snakes (a carpet python (morelia spilota) and three indian python (python molurus)) suspected of respiratory diseases based on clinical examination, transtracheal wash and culture, were used for this study. radiographic examination including a lateral view of the pulmonary area was performed without sedation. ct was performed using a high resolution ct (somatom 16, siemens, germany, erlangen) and obtaining transverse reformatted images of 1 mm thickness. also for the ct examination, the snakes were not sedated and held with plastic drainpipe and straps. results: radiographically, the normal lung appeared as a radiolucent, cylindrical shadow with a ''honeycomb'' weft. on ct images, the healthy respiratory alveolar parenchyma was a circular, hypoattenuating, homogeneous area with a ''honeycomb'' weft around a central lumen. the plexus of blood vessels is seen as a circular hyper attenuating line. in boidae (boas) the left lung appeared to have less volume then the right lung. this difference was less evident in pythonidae (pythons). radiographic findings of the diseased lungs included one nodule of soft tissue opacity in 1 patient, several small nodules in 2 patients, and 2 thick lines of soft tissue opacity perpendicular to the lung's axis in 1 patient. on ct images the following findings were made: the respiratory parenchyma was in all patients more heterogeneous with several focal hyper attenuated areas in 2 patients, or with more extended hyper attenuating areas in 2 patients. these lesions were more obvious in the cranial part of the right lung. the inner margin of the parenchyma appeared focally hyper attenuated in 3 cases. when comparing the lateral radiographs to the ct images the extent of the pulmonary lesions could be assessed in detail more on the ct images. discussion/conclusion: even though the radiographic findings of bacterial pneumonia in snakes were less obvious than on ct images. pulmonary changes could be diagnosed in most cases in this series. ct images could show early changes and may be useful for monitoring the disease process during treatment. introduction/purpose: the production efficiency in hatching senegal sole (solea senegalensis) is highly influenced by the development of bone deformities during the rearing process. the presence of vertebral deformities may be caused by a variety of factors such us genetic, nutritional, environmental conditions in the tank, or possible infections. the present study is a radiological description of the meristic characters of solea senegalensis and the skeletal bone malformations present in juvenile specimens coming from farms in northern spain. for this study, 30 specimens of senegal sole (solea senegalensis) with 481 days after hatching (140-220 mm long) having macroscopic signs of spinal malformation were selected. after a morphometric study, the specimens were radiographed (dorsoventral projection) using mammography films. from the images obtained, the number of vertebrae and their associated spines in the different body regions were observed. metric measurements of vertebrae of each region were taken and the vertebral anomalies and of their associated elements were counted and described. finally, the severity of the injuries was assessed. results: skeletal conformation: the number of vertebrae ranged from 42 to 45, including 6-8 abdominal, 31-34 caudal and 3 in the caudal complex. 60% of specimens showed variations in the number of rays present on the caudal fin. variations in other regions were less frequent. skeletal abnormalities: 96.7% of individuals had skeletal abnormalities, and they were severe in 30% of them. the most frequent abnormalities detected were abnormal vertebral bodies, either isolated or as a part of more serious lesions including deviations from the axis (60%) or spinal fusions (23,3%). 70% of specimens showed abnormalities in their neural or hemal elements. the most commonly affected region was the caudal complex with 90% of the animals affected mainly involving the rays of the caudal fin (86.7%). the discrimination of juvenile specimens based on their appearance is an effective method of selection. it is suspected that slight initial bone changes might be accentuated in the adult stage and its presence may be an early indicator of suboptimal rearing conditions. therefore, new studies are necessary to determine possible causes, and thus to obtain better performance in captive breeding. introduction/purpose: diagnostic imaging techniques are being utilised more frequently for the diagnosis of disease in wild animals. however, there is a lack of literature describing the normal appearance of the gross and imaging anatomy the purpose of this study was to describe the normal anatomy of the grey seal using radiography, ultrasound, ct and mri. a 26 days old male grey seal was admitted for treatment of a humeral fracture. radiographs of the humerus and the chest where taken with the patient under general anesthesia. given the difficulty in obtaining standard views of the humerus and to completely characterize the fracture ct of the humerus was performed and the chest was also scanned. surgery was attempted to reduce the fracture, but it was found that the fracture site was infected and the seal was euthanized. postmortem ct scans of the whole body, with bone, lung and abdominal windows depending on the area was performed to obtain images of normal anatomy. abdominal ultrasound with a 5-8 mhz probe and mri of the brain including t2 weighted sagittal, dorsal and transverse sequences and t1 transverse sequences using a 1.5 tesla scanner were also performed. ct images provided images of the skeletal structures and thoracic cavity. abdominal contrast however was poor and the different organs were difficult to individualize-this was likely caused by the young age of the patient. ultrasound of the abdomen allowed the identification of the different organs. these had similar appearance to those in the dog or cat with the exception of the kidneys which showed a heterogeneous echostructure with a hyperechoic parenchyma and multiple small irregular shaped-anechoic areas. this structure is thought to represent the kidney composition of many closely adherent renculi surrounded by pericapsular plexus and interlobular veins in the seal. mri of the brain allowed identification of the brain and other head structures and organs. the seal underwent post mortem examination and the gross anatomy was compared with imaging findings. discussion/conclusion: having normal references for anatomy is important especially when imaging species unfamiliar to the radiologist. ct provided good quality images for the skeleton and the thorax of the seal. ultrasound and mri allowed identification of abdominal organs and brain, respectively. description of normal imaging anatomy of more individuals and different ages would be useful to aid in recognizing disease. introduction/purpose: the most commonly presented problem in reptiles are abscesses. subcutaneous abscesses, superficial, or abscesses localized near the oral cavity are well noticeable and readily palpable on clinical examination, thus easy to diagnose. internal abscesses remain a diagnostic challenge owing to their different locations, which in turn, determine clinical manifestations observed. a male 5-year-old boa constrictor, 800 g bodyweight was presented with deep cutaneous inflammatory lesions due to burning, caused by a heating cable 5 months ago. since the burning event, the snake refused any food. the clinical evaluation showed emaciation with concomitant severe dehydration and decreased responsiveness to external stimuli. the boa was assigned to strict quarantine in a terrarium for observation and diagnostic procedure. the microbiological study of the skin lesions revealed the presence of pseudomonas aeruginosa. the blood examinations indicated elevated count of total white blood cells twbc (18 10 3 /ml) and heterophils (78%). due to poor body condition, the snake underwent ultrasonographic evaluation. the study was performed with 7.5 mhz linear transducer. a nodular lesion of 2 â 2,5 cm dimensions, localized caudally from the thinned-wall, anechogenic gallbladder was visualized. the tumor-like lesion showed heterogeneous echogenicity. centrally located an irregular anechogenic area of 1,3 â 0,9 cm was visible. the ultrasonographic appearance suggested abscess or neoplasia with the presence of large focus of tumor lysis. the other abdominal organs did not show any changes in the ultrasound image. other diagnostic procedures and surgical intervention were not possible to perform due to poor animal condition. despite therapy the snake died after 6 days. the necropsy confirmed the presence of a well formed encapsulated abscess located in the duodenal wall. the lesion caused marked narrowing of the intestinal lumen. the microbiological study of the abscess contents showed the presence of the same bacteria that were detected on the skin at the earlier bacterial culture. discussion/conclusion: infectious factors producing internal abscesses may be identical with those developing epidermal lesions. most probably they spread via the hematogenic route 1 introduction/purpose: in order to establish the venous pattern of the body cavity in the common buzzard (buteo buteo), a venographic study is carried out by means of a postmorten technique. materials and methods: six common buzzards (buteo buteo), coming lifeless from a wild animal recovery center, are kept freeze at à201c until the moment of the venographic study. for the contrast medium application, the right and left jugular veins are located. a lead oxide jelly solution with a 150% concentration of pb3o4 is injected at 501c. the filling process is controlled under radioscopy. after the radiographic study, the anatomical dissection of the body cavity is performed. pictures of the anatomical dissection, radiological projections and the tomographic images with their corresponding schemes are shown. this postmortem technique is a simple technique, and provides fairly good vascular images, allowing appropriate study of the vessels in the body cavity. the venographic studies and the anatomical dissection permit the accurate identification of the main veins and its branches and allows to establish the venous pattern for this species. introduction/purpose: ectromelias are congenital deformities characterized by total or partial absence of one or more limbs. total absence is named amelia while partial absence is anamed transversal or longitudinal hemimelia (''congenital amputation''). these malformations are reported sporadically in children and in several mammalian species. herein we describe the radiographical and ct pattern of hemimelia and amelia in a group of mediterranean italian buffaloes. materials and methods: 14 mediterranean italian buffaloes, 8 males and 6 females, from one day to six months old, affected by limb malformations of the thoracic or pelvic limbs or both, from different farms in campania, were studied. radiographs were performed on the euthanized animals or on the isolated limbs, using the two orthogonal views. in two buffaloes affected by amelia of the pelvic limbs, ct study of the pelvis was also made. results: 7 buffaloes had unilateral transversal hemimelia of the left pelvic limb; 3 buffaloes had bilateral transversal hemimelia of the pelvic limbs; 1 buffalo had bilateral amelia of pelvic limbs; 1 buffalo had transversal hemimelia of both the pelvic limbs and of the left thoracic limb; 1 buffalo had bilateral pelvic amelia and longitudinal hemimelia of the right thoracic limb; 1 buffalo was affected by transversal hemimelia of all the limbs. in all the buffaloes the malformed limbs presented more or less developed outlines of claws. in 2 animals affected by transversal hemimelia of both the pelvic limbs the bones amputated were exposed. the transversal hemimelia showed different degree of amputation. in the case of longitudinal hemimelia the third digit was absent. the two buffaloes affected by amelia of pelvic limbs showed small calcified oval-shaped nuclei near the left acetabular rim. the ct study demonstrated a cartilaginous bud of the proximal femur. discussion/conclusion: radiographs were valuable to characterize all the bones malformations distal to the carpus or the knee. ct demonstrated to be more accurate in cases of amelia since it demonstrated that clinical lack of a limb does not always imply the complete absence of the underlying bones. the more frequent radiographical pattern of the transversal hemimelia was represented by a ''wedge-shape'' of the bone affected. our findings show that in italian buffalos pelvic limbs are more frequently affected than the thoracic ones and this is in contrast with the reports in human medicine. since the true prevalence of these malformations may be underestimated and since a possible toxic teratogenic toxic etiology (e.g. dioxins) is possible, our report could also have an epidemiological significance. introduction/purpose: the equine tarsal joint is an anatomically complex region and has already been thoroughly described with radiography and ultrasonography.1-3 however in clinical conditions these techniques are sometimes inconclusive and computed tomography (ct) is recommended. this study was established to describe a detailed ct reference of the normal equine tarsal joint. materials and methods: ct was performed on 3 normal equine limbs from patients euthanized for reasons unrelated to this study. in all limbs ct was also achieved after intra-articular injection of the tarsocrural, centrodistal and tarsometatarsal joints with 60 ml, 3 ml and 3 ml contrast (30 mg iodine/ml) respectively. 1,3 mm and 0.6 mm slices were made (120 kv and 352 mas), dorsal and sagittal planes were reformatted. the ct images were compared with corresponding anatomical slices. the tibia, talus, calcaneus, central, fused first and second, third and fourth tarsal bones could be clearly visualized. the long digital extensor (with tarsal extensor retinaculum), superficial digital flexor, deep digital flexor (with tarsal flexor retinaculum), gastrocnemius, peroneus tertius and tibialis cranialis tendons and long plantar ligament could be clearly identified. the lateral digital extensor, medial digital flexor, splitted peroneus tertius and tibialis cranialis tendons and collateral ligaments could be localized but not always clearly identified. some of the numerous small tarsal ligaments could be identified: the plantar, lateral and interosseus talocalcaneal ligaments, interosseus talocentral-centrodistal-and tarsometatarsal ligaments, proximal and distal plantar ligaments and talometatarsal ligament. the articular cartilage at the non weightbearing surfaces of the distal tibia and proximal talus could be clearly assesed on the postcontrast images, the articular cartilage at the weight-bearing surfaces of the distal tibia and proximal talus was more difficult to examine and the articular cartilage at the small tarsal joints was not visible. discussion/conclusion: ct of the tarsal joint can be of great value when radiography and ultrasound are inconclusive and in pre-operative planning of complex fractures. with ct having a superior anatomic resolution and detailed bone and soft tissue visualization this technique can increase our knowledge of the pathology in the equine tarsus. introduction/purpose: the aim of this study was to provide a detailed anatomical description and reference values of the structures of the thorax in clinically normal goats by means of ct for use by radiologists, clinicians, researchers and veterinary students. twenty-six clinically healthy, anesthetized saanen goats (median age 4.0 years; sd 1.1 years) were positioned in sternal recumbency. transverse images were obtained with a 40-slice ct scanner (120 kv, 350 ma, 1 s tube rotation and a slice collimation of 5 mm reconstructed to 1.5 mm). subsequently, 12 animals were euthanized, frozen and sliced into 10-15 mm transverse sections, of which the caudal aspect was photographed. the study was approved by the animal ethics council of the canton of zurich. corresponding ct and anatomic images were compared. absolute and relative measurements of thoracic structures were obtained. corresponding thoracic structures on ct and anatomic sections were well identified. speciesspecific features, like the synostosed sternum, bronchus trachealis and large caudal mediastinal lymph node were consistently identified. contrary to the dog, in all goats the lung lobes could be differentiated, except the right middle lung lobe. the thymus was inconsistently seen (12/26). minimal to moderate interstitial pneumonia or bronchopneumonia (24/26) and pleural thickening (5/26) were diagnosed in the caudal and dorsal lung fields. mean values of thoracic structures included: angle between trachea and spine (16.51); ratio of tracheal to thoracic height (0.13); vertebral heart score (8.0); ratio of thoracic aorta to caudal vena cava (1.0); ratio of maximal height of cranial vena cava to the length of the thoracic vertebrae at the level of the tracheal bifurcation (0.8); mean cross-sectional area of the tracheal lumen just cranial to the thoracic bifurcation (2.1 cm 2 ). further, the square area of the principal bronchi, the caudal lobar bronchi and the concomitant pulmonary arteries and veins was measured. discussion/conclusion: with the exception of small vessels, nerves and certain small lymph nodes, all the structures of the thorax that were identified on the anatomic sections could be identified on the corresponding ct scans. subclinical lung disease in the majority of goats, most likely attributable to parasitic infection (protostrongyloides), was documented in ct and confirmed with fecal examination and macroscopic pathology. introduction/purpose: contrast enhancement in computed tomography (ct) plays an important role in the detection of several diseases. in human medicine, use of a saline solution injected intravenously immediately after the contrast material bolus, also known saline chaser, has been reported to allow a significant reduction of the contrast material volume in helical ct. however, there was no study about the reduction of the amount of contrast material using a saline chaser in veterinary medicine. the purpose of this study was to assess the degree of dose reduction of the intravenously injected contrast material without decreasing vascular enhancement using a saline chaser in multidetector-row ct of cattle. dynamic ct was performed in the head of six normal holstein. in a crossover study design, group a was administered only contrast material (600 mgi/kg) whereas group b and group c were given contrast material at a reduction of 30% and 40%, respectively, followed by 50 ml of saline solution. in all groups, a power injector was used with an injection rate of 4 ml/s and a pressure of 4.7-5.7 kg/cm 2 . attenuation values were obtained from the right and left maxillary arteries and dorsal sagittal sinus. there was no significant difference in the maximum enhancement value (mev) of the maxillary arteries and dorsal sagittal sinus between groups a and b. mev of the maxillary arteries and dorsal sagittal sinus in groups a and b was significantly greater than that in group c (po0.05). discussion/conclusion: results indicate that use of a saline chaser allowed 30% reduction in the dose of contrast material of without decreasing vascular enhancement. introduction/purpose: subchondral bone damage has been diagnosed using magnetic resonance (mr) in the equine distal limb as areas of reduced signal intensity in t1 weighted sequences and increased signal intensity in stir sequences 1 . a recent study showed no correlation between degree of lameness and radiographic changes in the tarsus 2 . the purpose of the present study is to describe mr findings in horses with acute onset of severe lameness localized in the tarsal region. horses were selected for inclusion that had acute onset of severe lameness localized to the hock by local analgesia and the results of radiographic and ultrasonographic examination did not conclusively explain the degree of lameness. mr images were acquired by use of a 0.2t imaging system, with the horse under general anesthesia; 3d t1 gradient echo, t2 fast spin echo and stir sequences were acquired on sagittal, dorsal and transverse planes. we included 3 horses (used for show jumping, age range 6-12 years) showing 3/5 unilateral pelvic limb lameness localized to the hock. radiographic examination showed enthesiophyte formation on the dorsal aspect of the central and third tarsal bones and dorso-proximal aspect of the third metatarsal bone in case 1; case 2 showed mild degenerative joint disease of the distal intertarsal joint and no radiographic changes were observed in case 3. mr images showed marked irregular thickening of the subchondral bone plate and abnormalities of the adjacent spongiosa of the third and central tarsal bones in all cases. increased signal on stir sequences involved the dorso-medial aspect (case 2 and 3) or axial portion (case 1) of the third and central tarsal bones. in addition, case 3 showed a linear area of hyperintense signal in each sequence on the dorso-medial aspect of the central tarsal bone, interpreted as an incomplete slab fracture. therapeutic plan included distal intertarsal joint medication (case 1) and extracorporeal shockwaves (case 2). case 1 and 2 were rested for 1 and 8 months respectively before return to the previous competition level. case 3 is still in the rehabilitation process. discussion/conclusion: increase signal intensity in stir sequence could be related to different pathologic situation such as hemorrhage, edema, bone necrosis or fibrosis 1 . in the present report, horses showed acute onset of lameness suggestive of bone trauma, microfracture of the subchondral bone and fluid accumulation within the bone as the more likely cause of the abnormal signal observed. subchondral bone trauma can involve the small tarsal bones and mr is crucial for lesion identification and therapeutic plan when radiographic and/or ultrasonographic findings are equivocal. introduction/purpose: the purpose of study was to register the reason for extracting the incisors in upper and/or under jaw and to examine the consequences of extraction of all incisor teeth by taking radiographs regularly for 1-2 years post operatively. materials and methods: four horses, 14 to 32 years old, were presented at the university clinic for large animals. the horses had problems with eating including weight loss, related to dental disease. in all four cases treatment included extraction of all incisors in the mandible and/or maxilla. the horses were monitored using radiography prior to tooth extraction, and at regular intervals over a period of one to two years post-operatively. images vd and dv for the mandible and maxilla respectively were taken using intraoral position of the cassettes and developed in a digital method (cr-system). the underlying pathology detected in these horses included both infectious and non-infectious diagnoses. specific clinical diagnoses included tooth resorption, periodontitis, dental-fractures and malocclusion. all horses showed signs of oral cavity related pain and gingival inflammation. all horses commenced eating within 24 hours post surgery. after eight to 10 weeks the alveolar sac was filled with regular trabecular bone. areas of sclerotic bone on the preoperative images remained slightly visible for some months after surgery. in cases where there was severe bone resorption associated with the initial dental disease, there was no significant re-growth of bone, so that the outer bone margins in these cases remained irregular in outline. all horses improved after surgery, gained weight and returned to normal use as pleasure riding horses. discussion/conclusion: no problems were evident with grazing; eating apples and carrots however presented problems unless these items were broken into small pieces. in general these horses became slower at mastication post surgery and this should be kept in mind if they are kept in groups with normal horses where feeding is from a common source. all horses showed a dramatic improvement after removal of the incisors. there were no side effect or long term special requirements for these horses other than ensuring that they are fed individually if housed in large groups. the tongue was occasionally visible in some of the horses. the experience reported in the abstract suggests that removal of all incisors is a viable option in cases where these teeth have significant irreversible and major disease. l. evrard ã , s. rabba ã , g. bolen ã , d. verwilghen ãã , v. busoni ã . university of liège -university veterinary clinic. ã diagnostic imaging section. ãã surgery section introduction/purpose: when horses with foot pain have no radiographic abnormalities, the soft tissues have to be assessed. however, some radiographic changes in the foot (e.g. enlarged synovial fossae, distal sesamoid bone fragments) are commonly seen in horses without lameness and their significance may be difficult to correlate to the clinical presentation in some clinical cases. the aim of this study was to review us findings in horses with lameness abolished by palmar digital nerve block but with inconclusive radiographic examination. medical records of horses that underwent a complete radiographic and us examination of the digit were collected and reviewed. horses with a clinical history of infection or direct trauma and horses without radiographic abnormalities were excluded. results: twenty-three horses fulfilled the selection criteria. on radiographs, 16/23 horses had enlarged synovial fossae, 8/23 had distal sesamoid bone fragments, 1 had a suspected irregularity of the flexor surface. at us distal interphalangeal joint (dipj) synovitis was seen in 16 horses, of which 6 had a visible abnormality in one of the dipj collateral ligaments. lesions of the deep digital flexor tendon (ddft) were seen in 9 horses and distension of the podotrochlear bursa in 14. the ligaments of the navicular bone were abnormal at us in 3 horses. at us the flexor surface of the navicular bone was irregular in 11 horses. us abnormalities in more than one anatomical structure were seen in 20/23 horses. discussion/conclusion: in horses with foot pain but inconclusive radiographic examination, us may reveal useful complementary information about soft tissue involvement and integrity of the navicular bone flexor surface. k peremans, s vermeire, a dobbeleir, i gielen, e vandermeulen, j saunders, k dik, h van bree. medical imaging department, faculty of veterinary medicine, university ghent, belgium introduction/purpose: canine orthopedic problems in the forelimb frequently originate from the elbow. the origin of pain can be caused by developmental, traumatic or degenerative processes requiring different treatment strategies. bone scintigraphy is included in the diagnostic frame work when clinical/ radiographic examination is inconclusive or non-localizing. determination of the exact localization of pathology within the elbow is hampered by resolution limits of conventional gamma camera systems. the use of conventional micro-spect systems is precluded by limited gantry opening. the aim of the study was to investigate the value of hispect (multi-pinholes mounted on a conventional gamma camera) in the evaluation of elbow pathology in the dog. materials and methods: 5 dogs with elbow disease were included (7 elbows). radiographic examination was negative (n ¼ 1), inconclusive (n ¼ 3) and not performed in 1 case. two dogs had undergone arthroscopic interventions for an elbow problem, but lameness had switched to the other leg. all animals underwent planar bone scintigraphy. micro-spect was performed using a conventional triple head gamma camera (triad, trionix), adapted with 3 multi-pinhole collimators (6 holes, 3 mm diameter, resolution 2.5 mm) (bioscan). data were acquired in step-and-shoot mode (10 steps, 361 angular step, 120 sec per step). images were reconstructed using a dedicated ordered subset-expectation maximisation (osem) algorithm (scivis). structural imaging and/or arthroscopy were performed to confirm pathology. the lame leg was correctly identified based on intensity of uptake on planar scintigraphy but no localization within the elbow could be determined. on the hispect images, pathology could be confined to anatomical regions. four elbows showed high uptake in focal regions. pathology was confirmed with structural imaging or arthroscopy and included: insertion desmitis of the flexor carpi ulnaris (n ¼ 1), detachment of the medial coronoid process (n ¼ 2) and a cystic lesion in the anconeal process (n ¼ 1). in one elbow, several areas of increased uptake were noted in the humerus and radius representing severe subchondral erosion on arthroscopy. in the previously operated elbows residual mild increased uptake in one elbow was focally at the level of a removed fragment of the coronoid process (scar tissue on arthroscopy) and more generalized in the other (erosion of the joint cartilage on arthroscopy). discussion/conclusion: with the hispect system increased uptake was discernible in localized elbow areas correlating with pathology found on structural imaging modalities and/or arthroscopy. hispect improves anatomical localization of pathology and may allow a more accurate choice of subsequent structural imaging modalities and therapy. introduction/purpose: idiopathic pulmonary fibrosis (ipf) is a chronic, progressive parenchymal lung disease described mainly in west highland white terriers (whwts). high resolution computed tomography (hrct) is used for visualizing pulmonary parenchyma and diagnosing ipf in man. the study objective was to investigate hrct findings in whwts with ipf and in healthy control whwts. materials and methods: hrct was performed under general anesthesia to 7 whwts with ipf (mean age 11.6 years) and 11 control whwts (mean age 8.5 years). a ct-scanner with axial high-resolution algorithm, 1.0 mm slice thickness and 7.5 mm table movement was used. the findings were evaluated and classified according to previously published criteria 1. quantitative ct-values were measured from the nondependent parts of the lung. the radiologist was blinded to the dog's disease status. the radiologist was able to distinguish all sick whwts from healthy controls. the hrct findings of ipf dogs were ground glass opacity (n ¼ 7), traction bronchiectasis (n ¼ 5), parenchymal bands (n ¼ 5), consolidation (n ¼ 5), peribronchovascular interstitial thickening (n ¼ 3), honeycombing (n ¼ 2), subpleural lines (n ¼ 2), and subpleural interstitial thickening (n ¼ 1). control dogs had only 1-2 focal lesions in the dependent part of the lung. the ct-values were significantly higher in ipf (mean à731 ae sd 51 hu) compared with controls (à821 ae 36 hu), po0.001. no correlation with pao2 was detected. discussion/conclusion: hrct was accurate in differentiating dogs with ipf from healthy controls. ipf causes a diffuse increase in radiodensity and lesions which are not visible in healthy lungs. unlike in man, ground glass opacity is common but honeycombing only rarely seen in canine ipf. introduction/purpose: feline injection-site sarcoma (fiss) is a locally invasive and rapidly growing tumor. computed tomography (ct) is widely used in the preoperative evaluation for clinical staging. however, ct can also demonstrate the volume of the mass. the aim of the present study was to retrospectively analyze the ct features of 200 cases of fiss and to assess whether there is a statistical relation between tumor volume and local invasiveness of fiss. materials and methods: ct images and medical records of 200 cases of histologically confirmed fiss were reviewed. diameter and volume of the mass were measured on ct (tomographic diameter, td, and tomographic volume, tv) and compared to the diameter and volume recorded during previous clinical examination (clinical diameter, cd, and clinical volume, cv). statistical analyses were performed on sas 9.1 software to evaluate the influence of tumor variables (site, first presentation or recurrence, aspect of the mass, number of involved muscles, involvement of underlying bones, presence of skip metastasis, of lymphadenomegaly, of radiographic or histological evidence of lung metastasis), and animal variables (breed, gender, age and body weight of the cat) on cd, cv, td and tv. values of po0.05 were considered significant. results: no significant difference was observed between clinical and tomographic tumor measurements. a statistical association between the mean diameter (mean of the cd and td) and each tumor variable was observed. a statistical association was also observed for mean volume (mean of the cv and tv) and each tumor variable. a greater diameter and volume was associated with an interscapular localization, first presentation, heterogeneous aspect, greater number of involved muscles, involvement of underlying bones, and lymphadenomegaly. smaller diameter and volume were associated with radiographic or histologic evidence of pulmonary metastasis. no statistical association was observed between any animal variable and mean diameter/mean volume. discussion/conclusion: the volume of fiss is related to the local invasiveness. larger tumor mass is more invasive locally, involving a greater number of muscles and underlying bone structures. a larger volume is also associated with an increase of lymph node volume, suggesting further diagnostic steps, but not necessarily proving metastatic disease. in this study smaller tumor volume was associated with a higher occurrence of pulmonary metastatic disease. however, 35% of the animals with lung metastasis were patients with recurrent tumor growth. furthermore, histological confirmation was not always available. introduction/purpose: elbow dysplasia(ed) is common in labrador retrievers and is frequently found in radiographic screening. grade 1 (mild osteoarthritis) is the most common screening result of affected individuals in finland. regardless of extensive screening based mainly on secondary radiographic signs of the disease, progress in decreasing morbidity has been modest. the aim of this prospective study was to compare ct findings and radiographic findings of mildly affected dogs to find out if finnish protocol using one mediolateral flexed radiograph with emphasis on osteoarthritis is sufficient to reach a diagnosis of ed in this breed, or if signs of the primary lesion should carry more weight. thirteen clinically healthy labrador retrievers with screening results 0/1 or 1/0 were studied. their age ranged from 16 to 28 months (mean 21 months). age range at the time of the original screening was 12-19 months (mean 14 months). mediolateral 451 flexed and oblique craniocaudal radiographs were made and a ct study was conducted on both elbow joints. mediolateral radiographs were first graded according to finnish kennel club's regulations, where main emphasis is on osteophyte formation at the dorsal border of the anconeal process. secondly, the mediolateral and craniocaudal radiographs and also the original mediolateral radiographs were evaluated by taking into account also blurring of cranial edge of medial coronoid process (mcp) and ulnar trochlear notch sclerosis. ct was used as golden standard for diagnosing ed. on ct nine dogs had fmcp, three dogs were affected bilaterally. one dog had bilateral osteochondrosis of the trochlea humeri. ununited medial epicondyle was diagnosed in one joint, which was excluded from the study. on radiographs 12 of 14 joints with ed and two of 11 joints free of ed had blurring of the cranial edge of the mcp. sclerosis of ulnar trochlear notch was seen in seven joints with ed and in three joints free of ed. in the radiographs taken for the original screening five joints with ed had blurring of the cranial edge of the mcp and seven joints had subtrochlear sclerosis. in the joints free of ed one joint had both signs. the most reliable radiographic sign in the present study was osteophytes distal to the medial epicondyle seen in the craniocaudal view. they were visible in all dysplastic joints but in none of the healthy joints. in the present study, evaluation based on secondary radiographic signs was correct in 21/25 joints (sensitivity 79% and specificity 91%), and in 16 of 25 joints in the original screening (sensitivity 64% and specificity 64%). discussion/conclusion: accuracy of screening for ed in labrador retrievers using a single flexed mediolateral projection based either on secondary radiographic signs or radiographic signs of the primary lesion was only moderate in the original screening. however, accuracy based on either of these signs was noticeably higher in the present study. screening for ed at the age of 24 months instead of 12 months and including the craniocaudal view in the radiographic protocol could be considered in this breed. skrzypczak p., atamaniuk w., kiezbowicz z., biez ' yński j., nicpoń j. department and clinic of veterinary surgery, faculty of veterinary medicine, wroczaw university of environmental and life sciences, poland introduction/purpose: vascular ring anomalies in 95% of affected dogs are represented with persistence of the fourth right aortic arch. double aortic arch is a very rare anomaly of the vascular ring that results in esophagal compression [1] . the use of computed tomography angiography (cta) has not been described in a dog with such an anomaly. in humans, double aortic arch is the least common but most symptomatic form of vascular ring anomalies. a ten-week-old czechoslovakian wolfdog (ceskoslovensky vlcak), male, 18 kg, was admitted to the department and clinic of surgery with a suspected vascular ring anomaly. the dog regurgitated regularly shortly after feeding. there were no changes upon clinical examination. the dog was smaller than the littermates. during laboratory examination, there was mild hypoalbuminemia and neutropenia. rx, esophagoscopy and subsequent cta were carried out. for this purpose, contrast material (omnipaque s 240 mg/ml) was injected into the saphenous vein at a rate of 2.5 ml/s for 30 s. transverse images were obtained using helical ct (ge s , light speed vct) scanner (5 mm thick slides, kvp 120, mas 4200 matrix 512 â 512, pitch1.2). images were acquired 8 seconds after the start of injection. following data acquisition, 3d image reconstruction was performed using vascular software. radiography and esophagography examination revealed dilatation of the esophagus with no signs of displacement at the level of the aortic arch. this was confirmed with the contrast study. the initial diagnosis was the presence of a persistent right fourth aortic arch, although leftward displacement of the trachea was not observed. computed tomography angiography was performed to further classify the anomaly. 3d ct reconstructions of the patient depicted the presence of second hypoplastic aortic arch. after surgery, the ct angiography procedure was repeated. the initial diagnosis of the presence of a vascular ring was confirmed for this case. ct angiography was used to better characterize a suspected vascular ring anomaly [2] . it fills the gap between standard diagnostic modalities, such as conventional radiography or echocardiography, and angiography, which is more complicated and hazardous. non-selective ct angiography is a reasonable choice in the diagnostic process for vascular ring anomalies. moreover, 3d reconstruction provides an excellent illustration of the problem for the owner or for students who are not involved in diagnostic imaging. introduction/purpose: ultra high field mri (uhf mri) has recently become available in a few research units. beside the experimental use of spectroscopic and functional mri applications, it is also used to obtain high spatial resolution images of the cerebral system of humans. the aim of this poster is to present the mri cerebral images of a clinical case with neurologic disease obtained post-mortem at 7 tesla mri and to compare them to macroscopical and histopathological examination. a 9 year old yorkshire terrier was administered to the critical care unit of the veterinary school of liège. the dog was in shock and had difficulties to maintain his head in equilibrium after he had fallen from stairs. the shock animal was finally euthanized in comatose state. a ct-and mri-scan were performed post-mortem. ct revealed fracture of the skull with cerebellar and cerebral trauma. the mri scan was performed using a philips 7 t mri with a 1-channel cylindrical rf-transmitting coil and a 16-channel receiver coil. a t2-weighted gre -sequence (fov of 70 â 70 mm, tr: 60 ms, te: 25 ms, fa: 15, slice thickness: 0.3 mm, scan time: 58 minutes) and a t1-weighted gre sequence (fov of 70 â 70 mm, tr: 7,7 ms, te: 3,5 ms, fa: 8, tfe-factor: 219, tfe dur. shot: 1754,4 ms, tfe dur. acq: 1685,3 ms, slice thickness: 0.55 mm, scan time: 9 minutes) were performed. after scanning, the head was fixed in a 10%formaline solution, the brain was removed from the skull and a microscopic study using hestaining of histological sections was performed. on mri, there was significant haemorrhage visualised in the left cerebral and cerebellar hemispheres, which were confirmed by macroscopic and microscopic examination. mr image quality was influenced by susceptibility artefacts of blood and air. high resolution t2-weighted images of the left and right cerebellar hemisphere allowed delineation of anatomical areas corresponding to zones of high neurons concentrations on histology. these areas correspond to the areas identified in human neuroanatomy as nucleus dentatus, fastigii, emboliformis and globosus. in humans these structures are responsible for the equilibrium, the muscular tonus and the coordination of movements. haemorrhage and traumatic damage of these areas may explain the clinical signs of this dog. discussion/conclusion: uhf mri is a promising tool for identification, illustration and description of small anatomical structures in dogs. post-mortem application of this technique can be a potent help to neuro-anatomists and neuropathologists for describing anatomical details and addressing microscopic examination in the light of abnormalities detected. secondly, if scan times and cost can be reduced in the future, using such systems in a clinical way can make diagnosis and prognosis more precise and appropriate due to the possibility to visualise neurological structures that can not be visualised at 1.5 or 3 t, because image quality and spatial resolution increase with magnetic field strength. a 5-year-old 36 kg intact male german shepherd dog was referred to the veterinary teaching hospital with a 15 days history of right hind limb lameness without previous history of trauma. on physical examination, the right thigh muscles were atrophied and signs of pain and swelling were noted on the right stifle. the rest of the examination and blood analysis were unremarkable. radiographs of the right stifle were taken. multiple foci of lysis with amorphous periosteal reaction were observed within the patella. there was an increase of periarticular soft tissue opacity. differential diagnoses included primary neoplasia of the patella and osteomyelitis (bacterial or fungal). an echo-guided aspiration of the joint fluid was performed. cytological results suggested a mixed inflammatory and neoplastic lesion, but infectious etiology could not be ruled out with cytology. samples from a biopsy of the patella were submitted for histopathology and microbiological culture. numerous poorly-stained yeast-like organisms were isolated and identified as cryptococcus neoformans. discussion/conclusion: survey radiographs are the primary tools for investigating osteomyelitis. radiographs may reveal foci of lysis, periosteal reaction, sequestra and moderate soft tissue swelling. primary or secondary neoplasias are considered the main differential diagnoses of osteomyelitis. patellar osteosarcomas have been rarely described in dogs, and radiographic findings are very similar to those found in fungal osteomyelitis. ultrasound examination can detect fluid collections and surface abnormalities of bone, whereas ct scan can reveal small areas of osteolysis in cortical bone, small foci of gas and minute foreign bodies. mri has great sensitivity, specificity and accuracy for the detection of osteomyelitis. although imaging studies help to distinguish osteomyelitis from other differentials, histopathology and microbiological culture continue to be the gold standard for definitive diagnosis. in conclusion, cryptococcosis should be considered as a differential diagnosis of osteolytic patellar lesions. introduction/purpose: the purpose of this study was to evaluate and compare the radiographic efficacy and safety of a non-ionic dimeric and isotonic iodinated x-ray contrast medium iodixanol [visipaque320 mg i/ ml (amersham health cork, ireland)] and a non-ionic monomer contrast medium iohexol [omnipaque 300 mg i/ml (nycomed, spain)] in feline cervical myelography. materials and methods: five adult healthy cats were studied after injection of iodixanol (0.5 ml/kg) and iohexol (0.5 ml/ kg) into the cerebellomedullary cistern through the atlantooccipital space. ventrodorsal and left to right lateral radiographs of cervical, thoracic and lumbar vertebrae were obtained before and 10, 20, 40, 60 minutes after injection. after completion of the study, the radiographs obtained were evaluated and compared independently by a radiologist who was unaware of the types of contrast agents. myelographic quality was assessed by numerical scoring method [0 (no contrast) to 4 (excellent myelogram)]. statistical analysis (nonparametetric test) was performed by spss package. diagnostically adequate radiographic examinations were obtained with both agents. adequate opacity in thoracic and lumbar vertebrae was obtained after 10 and 20 minutes post injection for both contrast agents. after 20 minutes contrast agents had reached the end of lumbar vertebrae column. there was no statistical difference between both contrast medias. evaluation of each of the radiographs showed good to excellent opacification. no adverse effect occurred up to one week after end of the study. discussion/conclusion: in conclusion, iodixanol and iohexol proved to be safe and effective contrast materials for myelographic studies in cats. introduction/purpose: interpretation of thoracic radiographies is difficult for beginners. image and computer graphic processing with quantitative analyses of image features could be of help. the goal of this study was to evaluate the accuracy of two semi-automatic analyses: the vhs 1 and the nca. materials and methods: forty sets of thoracic radiographs of poodles were divided in two groups: (i) images from dogs with no clinical signs of cardiopulmonary disease (n ¼ 10), and (ii) images from patients with proven cardiac disease (n ¼ 30), confirmed by echocardiography to be caused by degenerative mitral valve disease. 74% had severe cardiac morphological changes (la and/or lv enlargement), 6% had moderate cardiac morphological changes, 17% had mild changes and 3% (only one dog) had a normal cardiac silhouette size. vhs and nca measurements were performed in right lateral view with computing techniques, by one experienced veterinary radiologist (a) and two trainees (b and c). all the observers had prior information of each group composition. to determine nca (v 2 ), the contours of the heart and of a square, which base corresponds to one fifth of the distance between five vertebrae (from t 4 to t 8 ), were drawn, and the ratio of both areas was obtained. the interobserver measurements were compared using simple linear regression analysis. the accuracies obtained from the specificity and sensitivity functions were considered. group i vhs measurements of all observers had statistically comparable values and the general average could be used. group ii vhs and area related nca measurements of group i and ii had significant different observer values, in these cases only observations of the experienced radiologist were processed. in groups i and ii the vhs had normal distributions, ranging respectively from 9.18v to 10.70v and from 9.60v to 14.10v, and having a mean value (aesd) of 10.07v (ae0.44) and 11.69v (ae1.31). the optimal threshold value of vhs was 10.7v which corresponded to an accuracy of 85%. in groups i and ii the nca had normal distributions, ranging respectively from 16.51v 2 to 21.20v 2 and from 19.03v 2 to 42.16v 2 , and having a mean value (aesd) of 19.13v 2 (ae1.58) and 27.87v 2 (ae6.58). the optimal threshold value of nca was 21.20v 2 which corresponded to an accuracy of 91.7%. discussion/conclusion: the accuracy of normalized cardiac area was approximately 7% greater than the one of the vhs approach. therefore, the nca appears to be more accurate than semi-automatic vhs method to classify the cardiac silhouette from poodles as normal or enlarged. the use of nca measurement showed more difficulties and variable results in observers of trainee group (b and c.). 1 j.w buchan, j. bücheler. vertebral scale system to measure canine heart size in radiographs. journal of the american veterinary medical association, v. 206, n. 2, p. 194-199, 1995 the aim of this study was to assess the appearance of the feline spleen on abdominal radiographs and to examine intra-and inter-observer agreement. right lateral and ventrodorsal projections of the abdomen of 50 cats with no evidence of splenic disease were independently assessed by 4 individuals (2 specialist radiologists and 2 specialist surgeons) blinded to patient details. assessment criteria comprised borders (not visible, visible but not sharp, sharp), visibility (head, body and tail visible), and shape (not visible, triangular/ folded over/double thickness, rectangular/single thickness). both intra-and inter-observer agreement was calculated using cohen's kappa and the intra-class correlation coefficient. the effect of patient and radiographic variables on the appearance of the spleen was tested using the chi-squared test. the consensus score of the lead assessor assessed that, on the vd and lateral radiographs respectively, the borders were invisible (4 vs 17), visible but not sharp (20 vs 22) and sharp (25 vs 10). the visibility of parts of the spleen were: head (43 vs 21), body (39 vs 14) and tail (34 vs 10). the spleen was triangular/double thickness or rectangular/single thickness for the head, (16 þ 23 vs 20 þ 0), body (19 þ 20 vs 0 þ 14) and tail (4 þ 29 vs 8 þ 2). the shape of the spleen was not visible for head: (7 vs 29), body (11 vs 36) and tail (16 vs 40). there was 1% total disagreement in the consensus score. kappa values ranged from 0.324 to 0.864. of the 7 variables assessed on vd radiographs, agreement was moderate (9) and good (12) and on lateral radiographs, agreement was fair or moderate (9) and good or very good (12). kappa values for inter-observer agreement ranged from à0.006 to 0.852. of the 7 variables assessed on vd radiographs, agreement was poor or worse (6), fair or moderate (29) and good or very good (7) and for lateral radiographs, agreement was poor or worse (6), fair or moderate (24) and good or very good (12), with no difference in these categories between projections (p ¼ 0.409). there was a significant association between age and the visibility of splenic borders (p ¼ 0.047) and the visibility of the head (p ¼ 0.021) on the lateral projection for assessor 3. there was a significant association between the body condition score and the visibility of parts of the spleen on the vd projection for assessors 1 (body, p ¼ 0.001) and 2 (head, p ¼ 0.001; body, p ¼ 0.049; tail, p ¼ 0.015) assessor 4 found the borders of the spleen less visible in sedated/ anaesthetised animals on the vd view (p ¼ 0.048) and assessor 2 found the head more visible on the vd projection (p ¼ 0.030) in sedated or anaesthetised animals. the spleen is commonly visible on radiographs, although its appearance may vary between individuals and according to assessor. j. hall, k. lee, s. priestnall, c.r. lamb. the royal veterinary college, university of london introduction/purpose: pseudoaneurysm is a blood-filled cavity adjacent to an artery following disruption of the arterial wall as a result of iatrogenic or traumatic puncture. a patent channel to the artery lumen persists and blood continues to flow into a pseudoaneurysm, hence rupture may lead to severe bleeding. there are few reports of pseudoaneurysm in animals. pseudoaneurysm has been observed most frequently in humans after femoral artery catheterisation (ahmad et al. 2008 ). pseudoaneurysm following a cat bite puncturing the radial artery of an elderly woman has been reported (levis & garmel 2008). materials and methods: case report results: an 8 year old male neutered maine coon cat presented initially with two puncture wounds on the medial aspect of the carpus, suspected to be the result of a cat bite. the wounds were treated conservatively but there was a persistent, focal 3 â 1.5 cm fluctuant subcutaneous swelling on the craniomedial aspect of the right distal radius. the manus was diffusely swollen. there was no palpable thrill or bruit. there was no lameness, evidence of pain on palpation or regional adenopathy. no bone lesions were found radiographically. aspiration of the fluctuant swelling produced fluid consistent with sterile whole blood. ultrasonography of the antebrachium demonstrated a cavitary swelling with a thin regular wall and content characterised by a sedimentation, producing an anechoic upper layer and a uniformly echogenic lower layer. on the distal aspect of the swelling, pulsatile blood flow with a communication to the adjacent radial artery and swirling of the blood within the cavity were visualised. non-selective angiography of the right antebrachium showed extravasation of contrast from the radial artery into the fluctuant swelling. good collateral blood flow to the distal limb was evident. diagnosis was radial artery pseudoaneurysm. the lesion was exposed surgically and resected after ligation of the radial artery. histopathology demonstrated haemorrhage and fibrin surrounded by a thick fibrous granulating capsule of mature fibroblasts and focal areas of inflammatory cells (lymphocytes, plasma cells and macrophages) consistent with a pseudoaneurysm. at follow-up examination 12 days postoperatively, all swelling had resolved. discussion/conclusion: as in the present case, ultrasonography usually enables prompt, non-invasive diagnosis of pseudoaneurysm. when the ultrasound findings are equivocal or the anatomy is not well defined, angiography or computed tomography may be useful. in humans, a pseudoaneurysm may resolve spontaneously without treatment or may require compression, ultrasound-guided thrombin injection or surgery. in the absence of any veterinary reports on which to base treatment in the present case, surgical resection was considered a safe and definitive option. c.m.r. warren-smith, k. roe, b. de la puerta, k. smith, c.r. lamb. the royal veterinary college, university of london introduction/purpose: tumor seeding is the spread of viable tumor cells as a direct result of an interventional procedure such as surgery or biopsy. tumor seeding along needle tracts in animals is an infrequent event, most often associated with transitional cell carcinoma (1) . seeding from a pulmonary adenocarcinoma has been reported in a cat (2) . materials and methods: case report results: a 7 year old, female spayed cairn terrier had cardiac pacemaker implantation as a treatment for sick sinus syndrome. thoracic radiographs obtained at follow-up examination 6 months later showed a soft tissue mass in the left caudal lung lobe. on computed tomography (ct) the mass appeared as a well-defined rounded structure, approximately 1.7 â 2 cm diameter with a uniform density. no other masses were detected by ct. cytology following ultrasound-guided fine needle aspiration (fna) of the mass was diagnostic of pulmonary carcinoma. the tumor was subsequently resected surgically. no signs of metastasis were seen at surgery. histopathological examination of the resected specimen confirmed adenocarcinoma. at follow-up examination 12 months later a 2.5 cm calcified mass was found radiographically within the soft tissues to the left of the 7th thoracic vertebra. a repeat ct scan showed a large calcified mass affecting the soft tissues of the thoracic wall dorsolateral to the left 5-9th ribs. there was moderate enhancement of the mass following intravenous contrast administration. the location of the mass corresponded exactly to the previous fna tract. repeat fna of this mass was again diagnostic of adenocarcinoma with similar cytological features as the original pulmonary mass. the owners declined further invasive treatment. palliative meloxicam was administered. at 12 month follow-up the dog had developed a cough and mild exercise intolerance. repeat thoracic radiographs showed a marked increase in the size of the mass and lysis of the adjacent ribs. the dog continues to receive palliative meloxicam. discussion/conclusion: as in the present case, tumor seeding in needle tracts usually becomes evident clinically 2-16 months following the original fna (3). in the present case, there was no sign of intrathoracic recurrence or metastatic spread of the pulmonary tumor following surgical resection, hence the needle tract seeding was a significant complication that assumed a prognostic importance. although the risk of tumor seeding following fna is small, it seems prudent to consider this possibility when planning fna or biopsy and to warn owners of this possibility when such procedures are considered a necessary part of a diagnostic work-up. introduction/purpose: mri is the best imaging modality for diagnosis of lesions of the axillary region, but due to limited availability, high costs and necessity for anesthesia less applicable. the aim of the study was to characterize the ultrasonographic appearance of soft tissue masses in the axillary region and to determine the value of ultrasonography (us) in diagnosing tumors of the brachial plexus. a retrospective analysis of soft tissue masses in the brachial plexus region in dogs referred to the small animal surgery at the justus-liebig university giessen between 1989 and 2009 was conducted. dogs with progressive forelimb lameness, neurologic deficits, muscle atrophy, pain, or a combination of these signs, were examined clinically, neurologically and ultrasonographically. linear/convex 7.5-15 mhz transducers were used in conscious and sedated dogs. diagnoses were confirmed by fna, biopsy, necropsy or mri. in 286 patients the axillary region was examined (31 bilaterally) of which 199 axillary regions appeared normal on ultrasound. in 118 (41%) a mass was identified of which 26 showed typical ultrasonographic appearance of lipomas. in the remaining cases, cytology/histopathology revealed 11 fibrosarcomas, 8 metastases of mammary gland carcinomas, 8 undifferentiated sarcomas, 4 histiocytic sarcomas, 3 nerve sheath tumors, 3 carcinomas, 2 lymphosarcomas, 1 melanoma, 1 hemangiosarcoma, and 1 mastocytoma. other diagnoses were inflammatory lymphadenopathy (11), hematoma (6), abscess and traumatic avulsion of the brachial plexus (each 2). twenty nine cases were not available for histopathology. in one case the diagnosis of a peripheral nerve sheath tumor was verified by mri. within the limits of this study, and the available histopathology, there appeared to be no clinically utilizable relationship between the ultrasonographic appearance and the histological interpretation. discussion/conclusion: us detected a lesion in $40% of the examined animals with clinically suspected alterations in the region of the brachial plexus, of which lipoma, fibrosarcoma and inflammatory lymphadenopathy were the most common. us allowed detection of non-clinically evident masses and further evaluation of palpable masses. in addition, us-guided fna or biopsies may allow accurate diagnosis of the lesion, are minimally invasive, and can safely and easily be performed without harm to nearby vessels/nerves. disadvantages of us are the limited evaluation of proximal parts of the nerves, nerve roots and spinal cord due to anatomy. furthermore, a general overview of the region is not possible like on mr images. false negatives can be caused by tumor size, localization and number of involved nerves, experience of the examiner, and quality of the equipment. false positives may occur due to confusion with normal axillary lymph nodes, vessels or artifacts. thus, we conclude that us is an effective and practicable method to examine the axillary region for soft tissue masses. piotr debiak, wojciech lopuszynski 1 , anna lojszczyk-szczepaniak. department and clinic of animal surgery laboratory of radiology and ultrasonography, 1 department of pathological anatomy, faculty of veterinary medicine, university of life sciences in lublin, poland introduction/purpose: among the companion animals and livestock, testicular tumors are most common in dogs. the most frequent are interstitial cell tumor (ict), sertoli cell tumor (sct), and seminoma (sem) 1 . the objective of our study was the imaging modality-based assessment of the frequency and localization of metastases in dogs with testicular tumors. the study involved 42 dogs of different breeds, aged 6-13 years, whose ultrasound evaluation of testes identified focal lesions in the parenchyma. abdominal ultrasound evaluation and thoracic radiographs (in 3 projections) were performed to exclude/confirm metastases. on the basis of diagnostic imaging, dogs without metastases were neutered. dogs with metastatic lesions found in organs were euthanized and necropsy procedures were performed. tissue samples of testicular lesions and altered internal organs obtained during necropsy were divided for routine histology. on the basis of the histopathologic examination, the animals were assigned into 4 groups: i-15 dogs with ict, ii-11 dogs with sem, iii-9 dogs with testicular sct, iv-7 dogs with two histopathologically different testicular tumors (3 dogs with ict/sem, 4 dogs with sem/cst combination). the presence of metastases from the testicular tumors was detected by imaging procedure and confirmed histopathologically in 2 cases (4,8%), namely: one dog with sct (group iii), and one dog suffering from sem/sct (group iv) with seminoma metastases. abdominal ultrasound examination visualized in these dogs irregularly enlarged lymph nodes of the iliosacral lymph center. the dog with sem/sct tumors had enlarged inguinal lymph nodes and those from the lumbar lymph center. the enlarged lymph nodes were of oval shape, nonhomogeneous opacity with no hyperechogenic hilus. the radiographic evaluation of the dog with sct showed the presence of pulmonary metastases. the necropsy revealed micronodular lesions along the testicular cord spreading onto the omentum in both cases. in the case of sct the histopathological assessment has confirmed metastases to the lymph nodes, omentum and lungs. discussion/conclusion: our study indicates a relatively low risk of metastases from testicular neoplasms in dogs. we observed rare metastases from sct and sem tumors, whereas metastases from ict did not occur. however, focal lesions in testicular parenchyma require thoracic and abdominal imaging to exclude metastases. a diagnostic ultrasound examination of sem and sct metastatic altered lymph nodes does not indicate inflammatory reactions. unfortunately, micronodular lesions of the testicular cord may not be diagnosed by ultrasound evaluation. introduction/purpose: in humans, duplication of the ureter is the most common unrinary tract anomaly while it appears to be very rare in domestic animals. in the present report we describe the presence of a duplicated ectopic ureter in a 9 year old labrador. a 9 year old male neutered labrador presented with a history of chronic urinary tract infections, malodorous urine and occasional dribbling of urine noted over the past 2 years. work up prior to referral had shown a recurrent e.coli infection. treatment with antibiotics alleviated the symptoms. on clinical examination no obvious abnormalities were detected and haematology and biochemistry were unremarkable. abdominal ultrasound and intravenous urography (ivu) were performed. abdominal ultrasound showed a tubular fluid filled structure with peristalsis measuring up to 2 cm in diameter. it was extending from the cranial pole of the left kidney towards the bladder neck and no insertion into the bladder was evident; the tubular structure was still visible to the left side of the bladder neck just cranial to the pelvic inlet. this was consistent with an ectopic ureter. the right kidney appeared normal. a contrast urinary study was performed one month and a half later, following resolution of the urinary infection. a pneumocystogram performed previous to the ivu showed an air filled tubular and tortuous structure of up to 3 cm in diameter extending from the region of the prostatic urethra to the left kidney. during intravenous ureterography the left renal pelvis appeared slightly distended compared to the right. the right ureter was normal. a left ureter, with only slightly larger diameter than the right and with normal insertion in the bladder was also seen. the distended air filled structure did not show detectable filling with contrast during the ivu but some contrast reflux could be seen at its caudal aspect during a retrograde urethrogram. a duplicated ureter was suspected. surgery confirmed the presence of a duplicated left ureter with both left ureters terminating at the bladder wall but only the smaller one could be directly catheterised via the ureteral papilla, confirming thus the ectopic termination of the dilated one. the distended ureter was dissected and resected. the dog recovered uneventfully. discussion/conclusion: duplicated ureters are common urinary tract anomalies in humans. but only 2 reports in dogs and 1 in a cat could be found in the literature. in one of the dogs an incomplete ureteral duplication was found while in the other dog a renal and ureteral duplication with normal termination at the bladder was present. so the present report describes for the first time a duplicated ectopic ureter in a dog. ultrasound allowed identification of an ectopic ureter while the pneumocystography and ivu showed the presence of a normal left ureter along with the ectopic one. the combination of both techniques was important to reach the diagnosis. introduction/purpose: detection and characterization of tumor vascularity by using contrast ultrasonography is useful for differential diagnosis of liver tumors. sonazoid is a new ultrasonic contrast agent for detection of liver tumors. in ultrasonic contrast imaging in livers, the arterial vessels including tumor feeding artery are contrasted immediately after sonazoid bolus injection. it is not easy for doctors to distinguish the inflow time of arterial vessels and tumor vessels because of the narrow time window. we therefore proposed a new imaging technique to visualize inflow-time of contrast agents into vessels for further tumor diagnosis. in this study, we established a localized tumor model by inoculating vx2 tumor into rabbit livers. a female japanese white rabbit inoculated with vx2 by intramuscular injection was supplied by sankyo labor service corporation (tokyo, japan) as a tumor source. seventeen female japanese white rabbits, weighing 2-2.5 kg, were used as recipient animals. for tumor implantation into the liver, a small midline incision was made in the recipient animals and the tumor was injected into the exterior left lobe of the liver (sasaki 2006). contrast ultrasound was done on the 14th, 21th, 28th day after tumor implantation. vx2-bearing rabbits were fasted for 24 hours prior to experiments and were anesthetized with a mixture of ketamine and medetomidine. during the experiments, anesthesia was maintained by oxygen and isoflurane. the liver of each rabbit was carefully examined through the skin using a linear diagnostic imaging probe (eup-l65, hitachi medical co., ltd., tokyo, japan) operated by an ultrasonic scanner (eub-7500, hitachi medical co., ltd., tokyo, japan). the contrast images with pulse inversion were recorded at a rate of 15 frames per second immediately after sonazoid bolus injection for 60 seconds. in order to prevent the breathing movement of the liver during recording of contrast images, breathing was suppressed by pancuronium bromide i.v. infusion. animal experiments were conducted in accordance with guidelines for the care and use of laboratory animals, faculty of agriculture, tokyo university of agriculture and technology. to compare the inflow-time of sonazoid into tumor vessels and the other vessels, the time course of grayscale intensity of each pixel was measured and the time (frame) which reached 80% of maximum intensity was calculated by using software (matlab, mathworks, natick, ma). then the inflow-time mapping was constructed by coloring each pixel according to the each inflow time. by inflow-time mapping, the tumor feeding artery and liver artery can be separately observed in vx2 tumor bearing rabbit liver. portal veins and liver veins are also distinguished because of the difference of inflow times. discussion/conclusion: the inflow-time mapping constructed according to the different inflow time of contrast agent into each pixel would be useful to visualize tumor vascularity and helpful for differential diagnosis of liver tumors. references: sasaki k, medan ms, azuma t, kawabata k, shimoda m, umemura s: effect of echo-guided high-intensity focused ultrasound ablation on localized experimental tumor. j. vet. med. sci. 68 (10) in present study, kidney dimensions and volume were measured using radiography and ultrasonography and compared with real measurements immediately after necropsy to determine correlations between radiographic, ultrasonographic and real measurements (real size of kidney). the study was performed on 10 intact mongrel dogs referred to the veterinary teaching hospital for euthanasia for reasons other than abdominal disease. excretory urography was performed with iodixanol (visipaque 320, amersham health cork, ireland) and the renal length and width were measured on ventrodorsal radiographs. length, width, height and volume of kidneys were determined by ultrasonography, in two different positions (lateral and dorsal recumbency) for each kidney. length, width and height of kidneys were measured during celiotomy and after necropsy. volumes of kidneys were measured by water displacement. all of measurements were compared with linear regression analysis (spss package). the gold standard was real dimension of kidneys, measured during celiotomy and immediately after necropsy. there were positive and significant correlations between ultrasonographic and real measurements of renal length, width, height and volume. correlation indices were 0.853 and 0.773 for right and left renal volume, respectively. sonographic dimensions and volumes were smaller compared to the real size during celiotomy and after necropsy. there were also positive and significant correlations between radiographic and necropsy measurements of renal length and width, while radiographic measurements of length and width were greater than the necropsy measurements. there were no significant differences between two positioning of renal ultrasonographic measurements. discussion/conclusion: it is concluded that renal measurements were underestimated using ultrasound and overestimated using radiographs. it seems there is no other study to show correlation between renal volume measured by ultrasonography and real one which compare effect of positioning on renal mensuration in ultrasonography. introduction/purpose: all processes of bone healing depend from blood supply and the revascularization process. related to injury of blood vessels haemorrhage, initial hematoma, and blood clotting cascade and fibrinolysis cause local changes of the physiological blood flow and contribute to initiation of local inflammation. angiogenesis and the significant increase of blood supply in bone fractures of limbs is a continuous subject to research. unsuccessful healing has been a motivator for many scientists and clinicians dealing with this problem to investigate the process of normal bone healing and search for new methods to stimulate the healing process. the objective of this study is to identify and describe changes of blood flow parameters in popliteal artery after experimental osteotomy and osteosynthesis of the tibia in sheep using external bone plate fixation in comparison with blood flow of untouched limb. materials and methods: study group consisted of 8 sheep of both sexes and same breed. the mean age was 3 years (ae0.25) and the study duration was 4 weeks. the measurements were performed in 4 sheep after tibial osteotomy and osteosynthesis with zespol external bone plate method and 4 control sheep that were kept under same conditions. duplex doppler examinations of popliteal artery right before ramification into cranial and caudal tibial artery were made using philips envisor c ultrasound system. radiographic examinations were performed simultaneously with no correlation to the final results as a supplementary study of changes in fracture healing. the study has been approved by the ethical committee of the wroclaw university. changes of resistivity index (ri) depending of time elapsed after surgical procedures were noticed. in the first 24 h obtained values were slightly higher than before surgical procedures reaching values of 1.13 to 1.34 and increasing about 0.01 to 0.19. in the next 48 h ri significantly decreased 0.1 to 0.49 in comparison with presurgical results reaching values of 0.67 to 1.10. peak systolic velocity (psv) values were highly different in each sheep and oscillated between 40 and 91.3 cm/s. discussion/conclusion: results are presented with reference to the values of blood flow in the same vessels of healthy sheep since normal values and their changes in bone fracture and surrounding soft tissue disruption are lacking. initial increase of ri values and its later decrease correspond accordingly to the initial (right after injury) phase and inflammatory phase of fracture healing. in the last 2 weeks of study normalization of obtained values was noticed corresponding to the callus formation and bone remodeling phase of fracture healing. psv values did not follow a specific pattern and no correlation to the time elapsed was found. introduction/purpose: high-resolution ultrasonography is considered an optimal image technique to assess normal anatomy and abnormalities of the peripheral nerves. ultrasound has been used for imaging of peripheral nerves in dogs. however, to the authors' knowledge, information regarding the appearance and ultrasonographic approaches of peripheral nerves in the cat has not been documented. the aim of the present study was to determine the ultrasonographic appearance and approaches to assess the brachial plexus in cats. materials and methods: anatomical nerve study: the dissection of the brachial plexus was carried out in six forelimbs. the landmarks used for anatomical location were axillary artery, scapulo-humeral joint and transverse processes of c5, c6, and c7. vascular injection and criosections: red latex was introduced through the thoracic aorta in two cadavers, which were frozen at à201c for 24 hours and then to à801c for 24 hours more. cross sections were made from cervical spine to elbow. photographs of each slide were taken to provide the interpretation of the ultrasonographic images. ultranosographic nerve study ''in vitro'': the brachial plexus were ultrasonographically evaluated using a 4-13mhz liner array transducer in four fresh feline cadavers. the accuracy of the ultrasound localization was demonstrated by injecting ink around the targeted nerves and immediately dissected. ultranosographic nerve study ''in vivo'': three live healthy adult experimental cats were sedated to perform the ultrasonographic examination of the brachial plexus using the same technique described in the ''in vitro study''. the anatomical dissection of the brachial plexus allowed establishing the anatomical landmarks to locate the nerves by ultrasound. the cervical spinal nerve roots were visualized on paravertebral approach. the brachial plexus was localised using the axillary artery as anatomic landmark on the axillary approach. musculocutaneous, radial, median and ulnar nerves were individually identified on medial humeral approach. the nerves appeared as hypoechoic tubular structures with an echotexture of discontinuous hyperechoic bands surrounded by a hyperechoic rim. discussion/conclusion: the axillary artery was the anatomical landmark used for localization of the brachial plexus. the different components of the brachial plexus were identified as hypoechoic structure with internal echoes and surrounded by a hyperechoic rim on medial humeral approach. references: guilherme s., benigni l. ultrasonographic anatomy of the brachial plexus and major nerves of the canine torathic limb. vet radiol ultrasound. 2008. 49 (6) introduction/purpose: diagnostic ultrasonography has been used to define cutaneous layers and changes of skin thickness in relation to hydration status and fluid distribution. recently, skin thickness has been measured in shar pei dogs, a breed considered unique because of its folding and thickening of the skin, with a linear 13 mhz transducer. 1 it was demonstrated that skin thickness in the shar pei is greater than in beagles. this was caused by abnormal dermal deposition of mucin, a substance predominantly constituted by hyaluronic acid (ha). the aim of the present study was to understand how changes in dermal components could likely contribute to differences in dermal echogenicity and to relate them to the histological findings. materials and methods: 10 shar pei dogs from breeders and 10 beagles from the veterinary faculty of uab used as controls were enrolled in this study. shar pei group included 6 intact females and 4 intact males with a mean age of 2 years, all with the characteristic increase of skin thickness and bundles of wrinkles extending on the forehead, withers and hind legs. beagle group included 7 intact females and 3 males of which one spayed, with a mean age of 5 years. dogs were considered healthy on the basis of clinical examination, cbc count, total proteins analysis and urinanalysis. ultrasonographic examination was made with a 13 mhz linear-array transducer. the ultrasound beam was maintained strictly perpendicularly to the skin surface on frontal, withers, sacral and metatarsal regions. biopsy specimens were taken from the same skin areas and stained by haematoxylin and eosin s(h&e), alcian blue at ph 2.5 and masson's thrichrome stains. in all dogs a defined hyperechoic line was observed at the interface between the coupling gel and the skin, which corresponds to the epidermal entry echo. a homogeneous hyperechogenicity with a fine echotexture was observed in the epidermis-dermis interface in the shar pei group. in 6 out of 10 beagles from the control group a double-layered appearance of the dermis were observed. in these dogs the superficial layer was more echoic than the deeper layer. by h&e, collagen fibers were demonstrated to be separated by a pale grey-pink substance corresponding to mucinous material in shar pei dogs. by alcian blue stain, in the dermis of shar pei scattered collagen fibers were between a network basophilic material, considered a characteristic aspect of acid glycosaminoglycans such as ha, whereas by masson stain collagen fibers stained of a turquoise material. all these findings were not present in the skin of beagles. discussion/conclusion: in shar pei, dermal hyperechogenicity, as in human studies, was related to ha-dependent deposition and its ability to form a viscoelastic network together with collagen and elastic fibers. the presence of a diffuse, intense dermal distribution of ha between collagen fibers was further confirmed by the histological results. in beagles, the distinct bands observed with ultrasound in the epidermis-dermis layers were hypothesized to be related to differences in dermal fluid storage in the interstitium separating the collagen fibers, such as reported in previous studies. 2à3 in conclusion, ultrasound is a useful diagnostic tool to demonstrate dermal changes in shar pei dogs. high frequency ultrasonography could be considered a valid alternative to other more invasive methods of investigation such as biopsies, to measure the degree of ha dermal deposition. introduction/purpose: non-cardiac thoracic changes are common in dogs and imaging is of importance in the evaluation of these animals. ultrasound is a method that can be used to better understand the nature of these lesions, however, prior knowledge of the ultrasonographic anatomy of the region is necessary. this study aims to determine the non-cardiac thoracic structures that can be evaluated by ultrasonography in normal dogs. ultrasonographic study of non-cardiac thoracic structures of two normal dogs obtained in the veterinary department at the federal university of viçosa, minas gerais, brazil. radiographic examination of the thorax of each animal was obtained in order to exclude thoracic abnormalities. the ultrasonographic examination was performed with the animal in both lateral positioning and each intercostal space was evaluated. during ultrasonographic examination of the thorax the following structures were visualized: skin, intercostal muscles, ribs, parietal pleura and pulmonary pleura. the image of normal lungs was a smooth hyperechoic line representing the pulmonary pleura and can be visualized as sliding line under the thoracic wall as the animal breathes (sliding sign). because of the reflection of the sound beam after the pulmonary pleura, reverberation artifacts were visualized as a series of parallel hyperechoic lines. discussion/conclusion: the evaluation of the thoracic cavity is difficult because the normal lung, filled with air, does not allow transmission of sound waves due to the marked difference in acoustic impedance between soft tissue and air. furthermore, the ribs produce a strong posterior acoustic shadow, making the exam more difficult. thus, intrathoracic structures, such as the mediastinum and normal lung parenchyma are not visualized by ultrasound examination. if pleural fluid is present or a disease replaces the air in the lung parenchyma an acoustic window will be provided. introduction/purpose: renal diseases are usual in small animals, especially in cats. there are few reports describing values for hemodynamic renal vasculature derived by duplex doppler ultrasonography in normal cats. the purpose of this prospective research was evaluating normal dopplerfluxometrics parameters of renal arteries (ra), interlobar arteries (ia) and abdominal aorta artery (ao) in adult healthy persian cats. materials and methods: fifty renal units of 25 persian cats (13 females and 12 males, age range 12-60 months, mean 30 months) were selected for normal by clinical and biochemical examination; for normal systemic blood pressure, and normal anatomy with b-mode ultrasonography. it was performed color mapping of renal vasculature and pulsed doppler of both ra, ia in both kidneys at three sites (cranial, middle, caudal), and ao. all vessels were measured resistive index (ri). early systolic acceleration (esa) of ra and ia in both kidneys was obtained at spectral mapping. it was also obtained ratio indices between ar/ao, and ai/ar velocities. results of ao measurements were psv 53.17 ae 13.46 cm/s and mean diameter of 0.38 ae 0.08 cm. the mean ra diameter of fifty renal units was 0.15 ae 0.02 cm. considering velocimetric values in both renal arteries the mean obtained was psv 41.17 ae 9.40 cm/s and ri 0.54 ae 0.07. esa of ra mean obtained was 1.12 ae 1.14 m/s2. superior reference value calculated was 3.40 m/s2. renal-aortic ratio mean obtained was 0,828 ae 0,296. results of ia measurements were psv 32.16 ae 9.33 cm/s and ri 0.52 ae 0.06. esa of all ia mean obtained was 0.73 ae 0.61 m/s2. superior reference value calculated was 2.0 m/s2. renalinterlobar ratio mean obtained was 1.45 ae 0.57. discussion/conclusion: this research was performed with selected cats as normal, healthy and same ranging of age. ri obtained was similar that referred in literature. some conditions that leave to a lost of complacency and increase vascular resistance, can affect the waveform at doppler spectral mapping but not ri. literature suggests that ri have poor sensitivity for detection of renal disease. esa parameter of renal and intrarenal arteries provides information of spectrofluxometric alterations of distal vascular bed. to our knowledge, no reports are available describing normal values of esa to renal vasculature in persian cats. it was established a new ratio between renal artery peak systolic velocity and interlobar artery peak systolic velocity. this index was developed based on well-known doppler identifiable effects of stenosis, undependable of the cause. more studies would be necessary to verify hemodynamic behavior of this index in pathological conditions in cats as the effect of age, nephropathies or systemic pressure in these dopplervelocimetric parameters. introduction/purpose: cerebrovascular diseases in dogs seem to be increasing in advancing age. these diseases are defined as brain abnormalities caused by blood supply impairment. although the strokes are considered important causes of neurological symptoms in dogs, reports in veterinary literature are unusual. the purpose of this study was to describe the underlying causes and ultrasonographic findings of cerebrovascular diseases in small breed dogs, and comparing them to the postmortem findings. 1 medical records of transcranial doppler sonography (tcds) examinations performed between 2007 and 2008 in 512 dogs with clinical signs of central nervous system disease were reviewed. the criteria for selection of the 52 patients reported here were: small breed dogs with no predilection for sex, showing an acute onset of clinical signs 2 (nystagmus, head tilt, hemiparesis, sudden blindness) with no progressive focal cerebral dysfunction and diagnosis of cerebrovascular disease confirmed at postmortem examinations within 30-60 days after imaging studies. tcds examinations were performed by the same operator. dogs were restrained by the owner, without sedation. hair clipping wasn't often required and acoustic coupling gel was applied above the zygomatic arch to obtain temporal and occipital windows in all dogs. tcds examinations were taken to evaluate brain parenchyma anatomy and echotexture as well as identifying the ventricular system and detecting focal lesions. color doppler flow mapping and pulsed doppler spectral analysis were performed to identify the circle of willis and obtaining the resistive index (ri). underlying causes of cerebrovascular diseases were subdivided as follows: 1) cerebral amyloid angiopathy (27%); 2) endocrinopathies (25%) such as diabetes, cushing syndrome and hypothyroidism; 3) coagulopathy (19%); 4) granulomatous meningoencephalitis (11%); 5) schnauzer hyperlipemia (6%); 6) tumors (6%). transcranial ultrasonography detected focal encephalic lesions in 22 dogs; diffuse lesions were observed in 20 dogs and 10 dogs showed no abnormalities on b-mode scan. these dogs presented low ri values for one or more arteries studied. duplex doppler ultrasound detected higher ri values in 43 dogs. only 4 animals showed normal doppler spectral parameters, with ri values ranging from 0,45 to 0,55 3 . discussion/conclusion: results showed that there is no sexual predilection in canine cerebrovascular disease, but older dogs were more often affected, mean age 9 years (range 15 months-18 years). correlation between tcds and histologic results indicates that focal lesions detected by tcds were nonspecific findings and could be associated with several causes including hemorrhagic mass and connective tissue apposition. diffuse decrease in echogenicity was related to inflammatory diseases and/or edema. diffuse increased echogenicity was usually related to aging changes. tumor and granulomatous meningoencephalitis detected by spectral analysis also determined arterial hemodynamic changes. focal lesions were correlated to local artery hemodynamic changes. tcds provided hemodynamic information of cerebral circulation adding to b-mode features. 1 department and clinic of veterinary surgery, the faculty of veterinary medicine cerebrovascular disease. veterinary clinics north america small animal practice transcranial doppler ultrasound analysis of resistive index in rostral and caudal cerebral arteries in dogs introduction/purpose: in compare to other diagnostic techniques, ultrasonogeraphy is relatively inexpensive, noninvasive, and allows definition of ocular and retrobulbar anatomy and, with the advent of doppler imaging, permits measurement of the blood velocity parameters of the orbital and ocular va-sculature1. the aim of this study was to evaluate feline ocular arteries using doppler imaging results: mean psv and edv were 14.3 cm/s and 6.1 cm/s respectively for lpca, 12.8 cm/s and 5.5 cm/ s for spca, 13 cm/s and 4.2 cm/s for aca and 21 cm/s, 8.9 cm/s)for pra. discussion/conclusion: doppler imaging has the potential for determining no invasively and consecutively the blood velocity parameters found in orbital and ocular diseases including orbital inflammations and neoplasia, vascular diseases including systemic vascular disease (hypertension)vasculopathies, anemia doppler imaging of the ophthalmic vasculature of the normal dog, blood velocity measurement and reproducibility key: cord-023134-y665agnh authors: nan title: oral research communications of the 22(nd) ecvim‐ca congress date: 2012-11-20 journal: j vet intern med doi: 10.1111/jvim.12000 sha: doc_id: 23134 cord_uid: y665agnh nan sidestream dark field imaging (sdf) is a technically relatively simple method to visualize the microcirculation. however, current gold-standard, or 'consensus' analysis (ca) of sdf films takes approximately 1 hour per film limiting the application of sdf in a clinical setting, and leading to increased expense due to analysis of low quality films. we developed a subjective point of care scoring system (bedside evaluation of the microcirculation, bem) that could provide real-time intervention points and optimize care for the critical patient. the objective of this study is to evaluate whether the bem-score correctly identifies films of sufficient and insufficient diagnostic quality as defined by ca.twenty variable-length microcirculation films taken at the level of the canine tooth were selected from a database of films with available ca. before the study, three observers were trained using an instruction video to evaluate five quality parameters: stability, content, illumination, focus and pressure. the bem was performed by viewing and scoring each film four times in immediate succession. all five quality parameters were scored (0 perfect, 1 sufficient and 2 insufficient) according to ca analysis. only the fourth viewing was considered for analysis. bem quality analysis was only considered sufficient if no parameter was scored insufficient. repeatability and reproducibility were assessed by assessing all films in a random order three times daily for three days.bem pass-fail assessment matched ca 85.6% of the time with individual observer agreement of 84.4-87.8%. agreement of bem with ca did not change over the study period (84.4%, 87.8% and 84.4% on days 1,2 and 3 respectively) indicating accurate quality analysis after a single bem-score of the training video. the mean cumulative bem quality score (2.51, sd 1.87) was very similar to ca (mean 2.6, sd 1.66). however mean individual bemparameter scores differed from ca reflecting differences in observer interpretation.high levels of inter-observer agreement and the strong correlation with ca for pass-fail assessment demonstrate that bem quality evaluation can produce repeatable and reliable results. variation in individual parameter scores may reflect systematic erroneous assignment of certain parameters or individual bias towards assessment of certain features. nevertheless, this did not impact on the overall evaluation of film quality. this study provides a platform to investigate whether rapid semi-quantitative analysis of the microcirculation itself is similarly feasible. sidestream dark field imaging (sdf) is a straightforward technique to evaluate microcirculation. however, current 'consensus' analysis (ca) of sdf films is time-consuming, thereby restricting the clinical application of sdf. a subjective bedside scoring system (bem) is proposed that could rapidly provide a semi-quantitative assessment. we have previously shown that observers could accurately evaluate film quality parameters. the objective of the present study is to assess the correlation of bem with ca for quantitative microcirculation parameters. three observers were trained using an instruction video to evaluate four quantity parameters: total vessel density (tvd), capillary vessel density (cvd), perfused vessel density (pvd) and microvascular flow index (mfi). fifteen variable-length microcirculation films of sufficient quality, taken at the level of the canine tooth, were selected from a database of films with available consensus analysis. each parameter was scored (1 lowest -5 highest) . the bem was performed by viewing and scoring each film four times in immediate succession with the final score being considered for analysis. bem was performed on each film in random order three times daily for three days. ca scores were divided into quintiles for each parameter and mean bem score were calculated for each. conversely, for each bem score, mean ca scores were calculated, per observer or per day.the mean tvd and pvd bem scores for the 20% quintile were lower than mean bem scores for other quintiles. mean bem score for the 80-100% quintile was the highest mean bem score for pvd only. mean cvd, pvd and mfi values for bem score 1 were lower than mean values for other bem scores. in parallel, although tvd never received a bem score of 1, the mean ca value for films with bem score 2 were lower than for higher bem scores. the mean tvd and mfi values for bem score of 5 were higher than mean values for bem scores [1] [2] [3] [4] . similar values were obtained on each individual study day.this study demonstrates that rapid semi-quantitative assessment of the microcirculation using the bem-score can produce repeatable and reliable results, although significant overlap exists. further studies are required to evaluate the value of this technique in a clinical setting. objective: compare two non-invasive blood pressure (nibp) measurement devices (petmap and doppler) with invasive blood pressure (ibp) measurement in normotensive, anesthetised and awaken dogs. design: prospective clinical study animals: ten female dogs aged between 6 to 60 months (average 20.7 months), and weighting 12.8 to 34.2 kg (average 23.4 kg) undergoing a routine spay. interventions: blood pressure measurement procedures: after the induction of general anesthesia, a catheter (20g) was placed in the dorsal pedal artery and invasive systolic (sap i ), diastolic (dap i ) and mean (map i ) arterial blood pressures were obtained. the nibp cuffs were placed on the ipsilateral front limb. five consecutive measurements were obtained with each indirect device and considered as a mean measure. the ibp was obtained simultaneously to the five nibp measurements and also considered as a mean measurement. measurements on awaken dogs were obtained four hours after surgery. doppler's systolic (sap d ) and petmap's systolic (sap o ), diastolic (dap o ) and mean (map o ) arterial blood pressure were evaluated and compared to the corresponding ibp using the bland-altman analysis. the percentage of paired measurements with a mean difference of 10 and 20 mmhg was also evaluated for the sap o , dap o and sap d . results: agreement between ibp and nibp measurements obtained with the petmap and the doppler was assessed with the bland-altman analysis. on anesthetised dogs, both indirect devices underestimated all direct blood pressures. the petmap bias (standard deviation) were -6.1 mmhg (10.4 mmhg), -6.9 mmhg (6.7 mmhg), and -2.9 mmhg (9.3 mmhg) for sap o , dap o and map o respectively. the doppler bias was -4.1 mmhg (24.7 mmhg) for sap d . on awaken dogs, the petmap underestimated sap i and overestimated dap i and map i . the bias were -5.8 mmhg (16.5 mmhg), 4.6 mmhg (7.9 mmhg) and 3.8 mmhg (8.9 mmhg) for sap o , dap o and map o respectively. the doppler underestimated the ibp and the bias was -15.1 mmhg (21.6 mmhg).on anesthetised and awaken patients, the percentage of values lying within 10 and 20 mmhg of the ibp was higher (or equal) for the petmap compared to the doppler. conclusion: results suggest a better performance of petmap device to predict the ibp in normotensive-anesthetised and normotensive-awaken dogs. intracranial hypertension (ich) is associated with high morbidity and mortality in canine veterinary medicine, yet remains difficult to clinically diagnose. the lack of an easily feasible and available diagnostic method has prevented clinical studies on the prevalence, cause and treatment of ich. recently, transcranial doppler ultrasonography (tcd) has been reported as a noninvasive diagnostic method in humans. however, only a few preliminary reports on the use of this technique in dogs have been published. this study evaluated the repeatability and reproducibility of tcd of the basilar and left and right cranial, cerebral arteries in healthy beagle dogs.tcd was performed using a standard ultrasound machine at the level of the basilar artery, and left and right cranial cerebral artery, in six adult beagle dogs. systolic, diastolic and average velocity, resistance (ri) and pulsatility indexes (pi) were assessed for each vessel. repeatability was evaluated by calculating the intra-class correlation coefficient (icc) between three separate, consecutive measurements in every dog. an icc was also determined for the reproducibility of these measurements on three consecutive days. during the procedures, ecg, blood pressure and clinical parameters were monitored.statistical analysis showed a highly significant repeatability of all measured parameters (systolic, diastolic and average velocity, ri and pi) for all blood vessels (n = 162, icc = 0.89 -0.99, p < 0.01). systolic velocity, ri and pi were also significantly reproducible for the basilar artery (n = 36, icc = 0.80 -0.90, p < 0.01), and the left and right cranial cerebral arteries (n = 72, icc = 0.65 -0.74, p < 0.01). however, no significant correlation was found between basilar and cerebral blood flow velocities.measurement of pi and ri of the basal and cranial cerebral arteries using tcd appeared to have high intra-operator repeatability and reproducibility. however, measurements performed on the basilar artery had higher between day reproducibility. since the technical skill required to assess these parameters subjectively appeared to be less complicated at the level of the basilar artery, tcd in a clinical setting is probably advocated at the basal artery.in conclusion, tcd is characterized by high intra-observer repeatability and reproducibility, making this technique a promising tool for the measurement of pi and ri, which have both been reported to be correlated with intracranial pressure. intracranial hypertension (ich) is associated with high morbidity and mortality in canine veterinary medicine, yet remains difficult to clinically diagnose. furthermore, many patients suspected to suffer from ich are treated with sedatives or anticonvulsants. besides the impact of these molecules on the patients' neurological examination, they variably influence the cardiovascular system. this study evaluated the effect of various sedatives and anticonvulsants on the pulsatility and resistance index (pi) and (ri), as measured by transcranial doppler ultrasonography (tcd) in healthy beagle dogs. additionally, we evaluated the effect of ivabradine, a specific negative chronotrope, on tcd findings. tcd was performed at the level of the basilar artery in six adult, beagle dogs. this technique was performed prior to the injection of molecules (v0), and after administration of acepromazine (ace, 100 lg/kg iv), diazepam (dia, 0.5 mg/kg iv), medetomidine (med, 40 lg/kg im) or ivabradine (iva, 1 mg/kg iv). a wash-out period of at least 24 hours was respected between the administration of each drug. during the procedure, ecg, heart rate (hr) and blood pressure (bp) were monitored. results were analyzed using repeated measures anova. correlations between the clinical parameters, and pi and ri were investigated using pearson's correlation. results were considered significant when p < 0.05. significantly lower pi and ri values [(mean pi; 95% ci), (mean ri; 95% ci)] were obtained for med [(0.75; 0.67 -0.83 (1.85; 1.46 -2.23) ]. pi for dogs after iva was significantly increased compared to dia, but did not differ significantly from other groups.hr was significantly lower after med compared to other groups, and after iva, compared to v0. bp was significantly lower after ace than after iva, med or on v0. these clinical parameters were not significantly correlated with pi or ri. dia and ace did not significantly influence ri or pi, however med significantly decreased ri and pi. these findings agree with previous reports describing a decrease in icp provoked by med, as pi is positively correlated with icp. this effect appears to be independent of the effect of med on hr, since iva also significantly decreased hr, yet did not significantly affect pi or ri. in conclusion, blood pressure and heart rate did not significantly affect pi and ri. hypertrophic cardiomyopathy (hcm) is the most commonly diagnosed heart disease in cats with little documentation of the effects of treatment on outcome in cats with preclinical disease. therefore, this prospective cohort study was undertaken to evaluate the effects of treatment with atenolol on outcome in cats with asymptomatic hcm. we hypothesized that 1) 5-year mortality would be increased in cats with hcm compared to a matched control group of healthy cats, and 2) administration of atenolol would reduce 5-year cardiac mortality in occult feline hcm.cats were enrolled over a 5-year time period (2003 to 2007) in a prospective, open-label, observational study. diagnosis of hcm was based on transthoracic echocardiography. cats were either treated with atenolol (6.25 to 12.5 mg/cat, q12h, po) or did not receive treatment. decision to treat was based on owner preference, suggestion by the clinician, and animal compliance with regard to pill administration. baseline echocardiograms were analyzed, and morbidity and mortality were monitored at 3 and 6 months and thereafter by annual rechecks, and by phone interviews of referring veterinarians and owners over a 5-year time period. groups were compared by a non-paired t-test, introduction: systolic anterior motion of the mitral valve (sam) is a dynamic left ventricular outflow obstruction frequently observed in feline hypertrophic cardiomyopathy (hcm). however, several cases of sam have also been observed in cats without left ventricular hypertrophy (lvh) and this finding could be interpreted as an early stage of the disease not yet accompanied by lvh. alternatively, it could be speculated that the dynamic outflow obstruction could cause intraventricular pressure overload sufficient enough to induce lvh at a later stage. this study was conducted to test the hypothesis that sam can potentially cause myocardial stress and myocardial damage by measuring plasma ntprobnp and serum troponin-i levels in cats with sam not associated with lvh. methods: the study was based on a retrospective analysis of 13 cats who underwent cardiac investigation of a heart murmur, performed by a boarded cardiologist. these cats were diagnosed with sam not associated with lvh (ivsd and lvfwd < 5.5mm, both on bmode and mmode measurements) via colour doppler echocardiography. plasma ntprobnp (cardiopet test, idexx laboratories, inc.) and serum high sensitivity troponin-i (hsctni hs, idexx laboratories, inc) were measured in these patients, as well as serum urea, creatinine and thyroxin concentrations. the association between biomarkers (ntprobnp and hsctni) concentration and echocardiographic values (myocardial thickness, left atrial dimension and aortic peak velocity) was determined by using the spearman correlation coefficient (rho). results: all cats were euthyroid and did not show evidence of renal disease. mean hsctni measurement was not performed in one cat due to an unsuitable sample. eleven out of 13 cats (85%) showed ntprobnp values above the normal reference limit (100 pmol/l) with a median concentration of 393 pmol/l (± 194, range 109-711) . five out of 12 cats (42%) showed hsctni values above the normal reference limit (0.16 ng/ml) with a median concentration of 0.24 ng/ml (± 0.35, range 0.16-0.99). there was a significant positive correlation between aortic peak velocity and ntprobnp (rho=0.657, p=0.0146). conclusion: ntprobnp is increased in cats with sam without lvh, suggesting the presence of myocardial stress, which appears to be proportional to the degree of outflow obstruction derived by the aortic peak velocity. some of these cats also present a degree of myocardial insult suggested by the increased serum hsctni. whether the increased biomarker concentrations are related to sam-induced pressure overload or to an early stage of primary myocardial disease needs to be evaluated with appropriate longitudinal studies. heart murmurs are caused by turbulent blood flow or by vibration of cardiac structures. turbulent blood flow in young animals may originate from congenital structural heart disease or from physiological phenomena. the prevalence of heart murmurs and congenital heart disease in the general (i.e. non referral) feline population are unknown. the aims of this prospective study were to determine the prevalence of heart murmurs in young cats and to determine the prevalence of congenital heart disease in young cats with heart murmurs.in total 2935 domestic shorthair cats aged 2 to 6 months underwent a routine physical examination prior to vaccination between may 1 st 2009 until march 31 st 2012. cats were from adoption programs run by either dierenasiel breda e.o. (2090/2935) or dierenopvangcentrum tilburg (845/2935). in cats with murmurs, the murmur was timed, graded and the point of maximum intensity was determined. subsequently, 2d, m-mode and doppler transthoracic echocardiography with continuous ecg monitoring was performed.heart murmurs were detected in 135 animals (4.6%). congenital heart disease was detected in 47 animals (1.6%), acquired heart disease in 2 animals (0.06%) and no identifiable heart disease in 86 animals (2.9%) with murmurs.in the 86 animals without heart disease dynamic right ventricular outflow tract obstruction was the cause of the murmur in 2 cases, turbulence within the left ventricle was the cause in 6 cases.in 2 animals with acquired heart disease, pulmonary hypertension associated with a. abstrusus was diagnosed.in the 47 cats with congenital heart disease, isolated defects were found in 38 cats, being tricuspid valve dysplasia in 19 cats, dynamic left ventricular outflow tract obstruction associated with mitral valve dysplasia in 13 cats, ventricular septal defects in 3 cats, double chambered right ventricle in 2 cats and pulmonic stenosis in 1 cat. combination defects were found in 9 cats, being ventricular septal defect and tricuspid valve dysplasia in 3, ventricular septal defects and double chambered right ventricle in 2, ventricular septal defect and atrial septal defect in 1 cat, a ventricular septal defect and pulmonic stenosis in 1 cat and mitral and tricuspid valve dysplasia in 2 cats.in conclusion in this prospective study we found the prevalence of heart murmurs to be 4.6% and the prevalence of congenital heart disease to be 1.6% with atrioventricular valve abnormalities and ventricular septal defects being the most common. dynamic left ventricular outflow tract obstruction (dlvoto) is a common cause of heart murmurs in adult cats. the obstruction is caused by systolic anterior motion (sam) of the mitral valve. sam is typically observed in adult cats and humans with hypertrophic cardiomyopathy (hcm). many studies support the concept that structural deformities of the mitral valves and the papillary muscles could be primary causes of sam. congenital malformations of the mitral valve causing sam and dlvoto have been observed in dogs and people and are believed to exist in cats, but have not yet been reported. the aim of this study was to report the clinical and echocardiographic findings in young cats with dlv-oto.thirteen domestic shorthair kittens, between 12-21 weeks of age, presented with a systolic heart murmur between may 1 st 2009 until march 31 st 2012. all cats underwent a physical examination preceding 2d-, m-mode and doppler echocardiography. kittens were from adoption programs run by either dierenasiel breda e.o. (n=8/2090) or by dierenopvangcentrum tilburg e.o. (n=5/845) nine kittens were male, four were female. all kittens were asymptomatic. dlvoto caused by sam was present in all animals, uninterruptedly in eleven, only at high heart rates in two. seven cats had normal left ventricular dimensions (nlvd), six cats had concentric left ventricular hypertrophy (clvh). in five cats papillary muscle abnormalities were noted being an accessory papillary muscle in two and enlarged papillary muscles in three, two of which had clvh.twelve animals were reexamined 2-5 months after the initial examination. all animals were still asymptomatic. a heart murmur and dlvoto could no longer be detected in nine animals, five with initial clvh and four with initial nlvd. in three animals a heart murmur and dlvoto were still present. two of the cats with nlvd developed clvh. one cat with initial clvh continued to have clvh.treatment with atenolol was instigated in three cats which continued to have dlvoto and clvh. two cats were reexamined. treatment with atenolol led to complete reversal of clvh in both cases. interventional radiology techniques have been used to palliate both malignant and non-malignant causes of vascular obstruction for both intrinsic and extrinsic lesions.three dogs presented for with large, non-resectable cardiac masses obstructing venous return to the right atrium. venous return to the heart was severely obstructed leading to congestion with subsequent ascites (2) or head swelling and pleural effusion (1) . due to the extensive nature of the disease, an interventional palliative approach was pursued. transatrial self-expanding metallic nitinol stents were placed from the cdvc to the crvc in order to restore venous return to the heart via blood flow through the stent interstices.in all cases, stent placement was successful and resolution of clinical signs achieved. two dogs required additional stent placement in 14 months and 6 months, respectively, for stent occlusion. in both cases, restenting resulted in ascites resolution or substantial reduction. one dog was euthanized 21 months following initial stent placement for general systemic decline and return of moderate ascites. the second dog remains alive 22 months following initial stent placement on diuretic therapy with moderate ascites present. the third died of undetermined causes 5 1/2 months later.to the authors' knowledge, this is the first report of longterm palliative transatrial stenting for cardiac tumors affecting venous return to the heart. the stents were well tolerated in these canine patients for which surgical options were not possible. the irish wolfhound (iw) dog has a high prevalence of heart diseases, particularly dilated cardiomyopathy (dcm) and atrial fibrillation (af).during a prospective longitudinal study, 1438 irish wolfhounds were investigated by one veterinary cardiologist (2) between 1990 and 2012. dcm was diagnosed in about 26% of dogs. in 88% of cases, dcm was accompanied by af. af without evidence of dcm was diagnosed in 31 dogs (2%).for pathological investigations, hearts of 19 iws were collected and fixed in 4% buffered formalin. based on the most recent results from cardiovascular examination, five groups were established: normal hearts (group 1, n=4), dcm with sinusrhythm (group 2; n=2), dcm with af (group 3; n=6), dcm with af and congestive heart failure (chf) (group 4; n=4), af and left ventricular reverse remodeling (lvrr) due to medical therapy after diagnosis of dcm (group 5; n=3).all hearts were evaluated by one pathologist (1) who was blinded to the clinical diagnosis. gross inspection included measurments of weight, size, and architecture of left (lv) and right (rv) ventricles. three sites of each lv, rv, and interventricular septum (ivs) were histologically evaluated, and the extend of myocardial fibrosis, adipocyte infiltration, and angiosclerosis were graded semiquantitatively.iws with dcm and chf (group 4) had died significantly younger (5.0±2.0 years) than dogs with normal hearts (7.0±1.9 years) (p=0.05). concerning gross pathology findings in hearts with clinical dcm diagnosis, lv chambers were dilated in 3/4 cases of group 4, while in groups 3 and 5, the papillary muscles appeared grossly prominent and not flattened as in group 4.histopathologically, in control dogs lv and rv myocardium showed no (3/4) or mild (1/4) interstitial collagen deposits, no or single adipocytes, and normal vessels. in contrast, heterogeneous findings were seen in groups 2-5: most hearts (10/15) showed mild to moderate multifocal myocardial fibrosis and up to moderate diffuse infiltration of adipocytes within the lv myocardium, and mild angiosclerosis, but five hearts were histologically normal. only in one dog attenuated wavy fibers were seen in the apical region of lv. ivs was normal in 9 out of 15 cases. rv showed mild interstitial fibrosis and mild to moderate adipocytes in most cases (14/19) of all groups.in conclusion, pathological findings in hearts of iws affected with dcm are different from other breeds. furthermore, the gross and histological findings are variable and do not correspond to the clinical diagnosis in all cases. the irish wolfhound (iw) dog has a high prevalence of heart diseases, particularly dilated cardiomyopathy (dcm) and atrial fibrillation (af). during a prospective longitudinal study, 1438 irish wolfhounds were investigated by one veterinary cardiologist (2) between 1990 and 2012. dcm was diagnosed in about 26% of dogs. in 88% of cases, dcm was accompanied by af. in addition, af without evidence of dcm was diagnosed in 31 dogs (2%).for pathological investigations, hearts of 23 iws were collected and fixed in 4% buffered formalin. based on the most recent results from cardiovascular examination, three groups were established: dcm with af (group 1; n=12), af without evidence of dcm (group 2; n=7), normal hearts (group 3, n=4).aim of this study was to investigate the histopathological findings in left and right atria of iws with atrial fibrillation compared to normal hearts of iws. all hearts were evaluated by one pathologist (1) who was blinded to the clinical diagnosis. hearts were inspected grossly and two cross sections of each left (laa) and right (raa) atrial appendage were embedded in paraffin wax and stained with h&e and picrosirius red. myocardial fibrosis and adipocyte infiltration in both atria were graded semiquantitavely.mean age ± sd of all dogs at time of death was 6,9 ± 1.7 yrs. the gross and histopathological findings of the left atrial appendage (laa) were not significantly different among groups, but dogs in both af groups had raa dilation (3.6 to 22.0 ml, median 11 ml volume) compared to controls (3.5 to 9.6 ml volume, median 5.34ml). histopathologically, raa in control dogs showed small amounts of interstitialcollagen and single adipocytes. in contrast, raa in dogs affected with af with andwithout evidence of dcm, had mild to moderate multifocal or diffuse myocardial fibrosis, and diffuse infiltration of adipocytes which was statistically significant different from normal hearts (p=0.005). in general, fibrosis and number of adipocytes were significantly increased in raa compared to laa in both groups with af (p=0.014).fibrosis and the accumulation of adipocytes within the myocardium are described toresult in electrical inhomogeneity predisposing to arrhythmia.on a cellular level, right atrial fibrosis and adipocyte accumulation might be the changes responsible for the development of atrial fibrillation and atrial dilatation in this breed of dogs with an exceptional high prevalence of af and dcm. asynchronous ventricular contraction causes deterioration in cardiac function and reduces the response to medical therapy in people with heart disease. various echocardiographic techniques have been used in humans for evaluation of left ventricular (lv) synchronization in order to assess whether cardiac resynchronization therapy (crt) would be beneficial. prior studies using tissue doppler imaging (tdi) derived strain imaging have not detected lv dyssynchronization in doberman pinschers with dilated cardiomyopathy (dcm). a newer 2d imaging modality, speckle tracking strain analysis, is more effective in detecting lv dyssynchrony in humans. this technique has not been previously evaluated in doberman pinschers with dcm. this study therefore aims to evaluate lv synchrony in doberman pinschers with dcm using 2d speckle tracking strain. 52 client-owned doberman pinschers were included. standard echocardiography was used to evaluate systolic function and left ventricular dimension in order to categorize the dogs as normal or abnormal, depending on systolic and diastolic ventricular dimensions, ejection fraction and/or shortening fraction. the operator was blinded to the echocardiographic diagnosis of each dog. each parameter was measured in triplicate. radial (rs) and circumferential strain (cs), using right parasternal short axis at the level of the papillary muscles were calculated. synchrony was assessed by measuring the difference in the qrs onset time-to-peak strain between the septal anterior and posterior left ventricular segments. to evaluate differences between groups a nested effect anova (beat within group) was performed. a p<0.005 was considered significant. based on the echocardiographic parameters mentioned above, 19/52 dogs were considered normal and 33/52 had dcm. significant differences were found in the time to peak rs (p= 0.0009) between normal dogs and dogs affected with dcm. mean in time to peak rs was 43.8 ms (+/-35.8) in normal dogs and 69.21 ms (+/-48.9) in dogs with dcm. time to peak cs did not exhibit significant differences between groups (p= 0.62) and was 55.6 ms (+/-49.91) in normal dogs and 60.66 ms (+/-63.2) in dogs with dcm. the results of this study show that the delay between the anteroseptal-to-posterior wall peak radial strain is greater in doberman pinschers with dcm than in normal dogs. further studies would be required to evaluate whether there is a difference in synchrony between dogs in occult stage and overt dcm. ventricular dyssynchrony could negatively affect systolic dysfunction as it does in people, and crt may therefore be helpful for the treatment of dobermans with dcm. patent ductus arteriosus is often treated with intra-arterial coilembolization or implantation of an 'amplatz canine duct-occluder'. for both procedures an arterial access is necessary. because of the high risk of developing a fatal hemorrhage from the arterial puncture site after removal of the large-bore introducer sheath upon completing the intervention, cardiologists generally use surgical cut-down with subsequent ligation of the femoral artery instead of percutaneous arterial puncture using seldinger's technique. the effect of commercially available chitosan patch has been tested in 10 experimental dogs with the approval of the institute's ethical committee. on the first 4 beagles the committee required a terminal experiment. for the selection of an appropriate introducer-sheath (i.e. smaller than the arterial diameter), the femoral artery was first imaged with ultrasound. under general anesthesia introducer-sheaths were placed in both femoral arteries using seldinger's technique. after their removal a chitosan-patch was applied on the wound according to the manufacturer's instructions: 10-minute manual pressure. the dogs were monitored with direct arterial blood pressure measurement, ecg, pulse oxymetry and capnography. all 4 dogs were kept under general anesthesia for several hours and the legs were moved vigorously every 30 minutes to mimic movements of awake animals. no macroscopic hemorrhage was noticed on the 8 puncture sites and no signs of severe subcutaneous bleeding was suspected as the blood pressure and heart rate remained stable. after several hours the dogs were euthanized. after having shown the effective working of chitosan-patch, a permission was granted for survival experiments in 4 beagles. in 2 of these dogs one femoral artery was punctured with an introducer whose thickness exceeded the diameter of the femoral artery. in both dogs an uncontrollable subcutaneous hemorrhage occurred immediately after the 10-minute manual compression time with severe drop of blood pressure and development of tachycardia. both dogs were euthanized during anesthesia. in another 2 beagles the size of the introducer was smaller than the diameter of the artery (80-100%). no relevant hemorrhage took place in these dogs and they recovered from the procedure without any complications. similarly good outcome was found in two 9-month-old boerboels. from this pilot study we concluded that chitosan-patch can effectively control hemorrhage from a femoral arterial puncture site if the introducer-sheath is thinner than the artery's lumen. using seldinger's technique allows a less invasive and quicker cardiac catheterization and preservation of the femoral artery for the leg's blood supply and for repeated intra-arterial interventions. objectives were to examine longitudinal change in echocardiographic left heart chamber dimensions and cardiomyopathy classification in cats with primary myocardial disease.clinical records from 2004-2012 were reviewed for cats with 2 or more echocardiographic examinations at least 6 months apart and a diagnosis of primary myocardial disease. for each study (2 from each cat), left heart chamber dimensions were measured by a single blinded trained observer in random order, and the cardiomyopathy type was classified according to predefined criteria. paired comparisons were made using the wilcoxon signed rank test and fisher's exact test.63 cats met the inclusion criteria. cardiomyopathy type at entry consisted of hypertrophic cardiomyopathy (hcm, n=60), dilated cardiomyopathy (dcm, n=1), arrhythmogenic right ventricular cardiomyopathy (arvc, n=1) and 1 normal cat. overall, median left ventricular (lv) diastolic diameter and median long axis left atrial (la) diameter both increased (p=0.007, 0.001 respectively) whereas lv diastolic wall thickness and lv fractional shortening did not change, but changes in dimensions of >10% were seen in both directions. change in cardiomyopathy type was documented in 3 cats: hcm to normal; normal to hcm; hcm to the endomyocardial form of restrictive cardiomyopathy.the inclusion criteria led to a bias towards clinically stable cats with hcm. despite this, changes in cardiomyopathy phenotype were observed. either our criteria for the different feline cardiomyopathies should be defined more precisely, or phenotypic expression in cats with cardiomyopathy changes over time. transesophageal echocardiography (tee) has proven useful in evaluating patent ductus arteriosus (pda) morphology thereby guiding appropriate device selection. additionally, tee, in combination with fluoroscopy, has been used to guide the transcatheter coil embolization and for deployment of amplatz canine ductal occluder (acdo) in dogs. recently, we described the use of transthoracic echocardiography (tte) guidance during transcatether pda occlusion with acdo without the use of fluoroscopy, but observed problems of deployment in patients with sub-optimal acoustic windows. however, tee, can overcome issues of suboptimal tte acoustic windows and provides higher image resolution of cardiac and vascular regions. therefore, we hypothesized that tee could be used to successfully visualize the vascular structures and interventional devices to safely perform pda occlusion with acdo without requiring fluoroscopy.we recruited 5 dogs with patent ductus arteriosus (pda) for tee-guided percutaneous ductal occlusion with an acdo. dogs were anesthetized, positioned in right lateral recumbency and the right femoral artery was accessed percutaneously (modified seldinger technique). the tee probe was advanced to a midesophageal position with minimal force to obtain a long axis 4-chamber view (transverse plane). the probe was then retroflexed and withdrawn to a cranial esophageal position until a cross section of the descending aorta was seen. to visualize pda to the probe was slightly straightened and turned counterclockwise, and the ultrasonic beam was oriented between 60 and 120 degrees.in all dogs, the guide wire and a long introducer-sheath were guided from the aorta through the pda into the main pulmonary artery by tee monitoring. the acdo was advanced through the introducer-sheath until the flat distal disk was visualized within the main pulmonary artery by tee monitoring. the distal disk was positioned against the pulmonic ostium and the coupled proximal disk was deployed within the ductal ampulla while being monitored by tee visualization.the guide wires, long introducer-sheath and acdo appeared hyperechoic on tee images and tee guidance provided images of sufficient quality to clearly monitor the procedures in real-time. real-time monitoring also allowed for immediate corrections to guide wire, catheter or device positioning. the procedures were successful and without complications in all patients.we have demonstrated that tee monitoring, like tte monitoring, can guide every step of transcatheter acdo embolization procedures without requiring fluoroscopy, thereby avoiding radiation exposure, and provides an alternative to tte-based guidance, especially when tte visualization of the pda is insufficient for safe and timely acdo deployment. right ventricular (rv) dysfunction occurs in human patients with left-sided cardiac disorders because of the mechanism of ventricular interdependence. doppler echocardiographic indices of diastolic function of the right ventricle are good prognostic markers during left ventricular (lv) failure secondary to ischemic and dilated cardiomyopathy.the aims of the present study were: to assess lv and rv diastolic function by conventional doppler and pulsed-wave tissue doppler imaging (pw-tdi) in dogs with mitral valve disease (mvd), with or without pulmonary hypertension (ph); to test if echocardiographic parameters of lv and rv diastolic dysfunction correlate to the doppler-estimated pulmonary artery systolic pressure (pasp).114 dogs were prospectively evaluated, including 86 dogs with mvd. for each dog, a complete echocardiographic evaluation was carried out. dogs with mvd were divided in 3 groups according to the acvim classification of heart failure. using the cut-off value of tricuspid regurgitation (tr) peak velocity of 2.8 m/s, presence or absence of ph was considered, standard echocardiographic and mitral and tricuspid doppler parameters (e wave and a wave), and pw-tdi parameters (systolic wave, sa; early diastolic wave, e'; late diastolic wave, a'; e'/a' ratio; e/e' ratio) for lateral and septal mitral annulus, and lateral tricuspid annulus were measured. the echocardiographic data were compared by use of anova and multiple contrast t-test with bonferroni correction. the relationship between left-sided echocar-diographic parameters and rv diastolic parameters was examined by correlation analysis. the correlation of pasp with lv and rv diastolic parameters was examined by multiple linear regression. a value of p<0.05 was considered significant.dogs were classified as follows: 28 healthy dogs; 36 dogs in class b1; 28 dogs in class b2; 22 dogs in class c and d. no differences were found among groups regarding rv conventional doppler and pw-tdi parameters. however, a significant, weak correlation was found between some left-sided echocardiographic parameters (left atrial dimension; end diastolic and end systolic volume indexes; peak e wave and a wave velocities and e:a ratio) and some rv pw-tdi parameters. ph was diagnosed in 48 dogs, while 54 dogs were deemed without ph. dogs with ph had significantly different trans-mitral e and a wave peak velocity and e/e' ratio of lateral and septal mitral annulus. these two latter parameters were also correlated with pasp (r 2 = 0.241 and 0.338, respectively).our findings highlight the importance of considering ventricular interdependence in dogs with mvd, particularly those with ph. during the cardiac cycle, the left ventricle (lv) undergo a complex deformation, consisting of overall shortening accompanied by increase in wall thickness and twisting due to the helical orientation of the myocardial fibers. using speckle tracking echocardiography (ste), the circumferential strain (cs) as well as the twisting motion of the lv, calculated as the net difference between lv apical and basal rotation angles during the cardiac cycle, can be quantified by post-processing 2-dimensional short axis images of the lv. thus, the aim of this study was to evaluate the global cs and the twisting motion in 101 small-medium sized dogs with varying severity of mr attributable to myxomatous mitral valve disease (mmvd). using a vivid-i ultrasound system, all dogs underwent echocardiography including parasternal short axis views at the basal and apical level for offline analysis of rotation, and at the midpapillary muscle level for analysis of global cs, using commercially available software (echopac). twisting motion during systole (twist sys ), early (untwist early ) and late (untwist late ) diastole were calculated as well as their rates computed as the time derivatives. furthermore, time to onset of untwist, calculated from start of electromechanical activation until peak twist sys was measured. associations between twist and untwist variables, global cs and conventional echocardiographic indices of mr severity and lv remodeling were examined by multiple linear regression analyses including dog characteristics such as heart rate (hr), sex, breed, body weight and age. global cs increased with increasing mr (p <0.0001). untwist early (p = 0.0004) and its rate (p = 0.0008) also increased with increasing mr, albeit in co-variation with certain baseline dog characteristics. time to onset of untwist increased with mr (p = 0.0005), left atrium to aortic root ratio (p = 0.0007) and lv internal diameter in diastole (p <0.0001). in conclusion, untwist early and global cs gradually increased and the onset of untwist appeared to be delayed with increasing mr severity in small-medium sized dogs with spontaneous mmvd. this hyperdynamic stage with a delay in untwist may represent lv adaptation to loading conditions in mmvd, but might also indicate mid-and subepicardial compensation for an early lv dysfunction, as reflected by delayed onset of relaxation, as timing of contraction-relaxation cross over is the most vulnerable period of myocardial fiber mechanics. systolic anterior motion (sam) of the mitral valve is the mechanical correlate of left ventricular outflow tract dynamic obstruction (lvotdo) and has been associated with hypertrophic cardiomyopathy (hcm) in most instances. however, sam without left ventricular hypertrophy or with regressing hypertrophy is increasingly recognised.the echocardiographic studies of 12 cats, diagnosed between 06/2006 and 03/2012, with sam without hcm (7 cats) or sam with regressing hypertrophy (5 cats) were reviewed for evaluation of the anatomic and mechanic alteration contributing to lvotdo .ventriculo-aortic and aorto-septal malalignment were suggested by the significantly narrower aorto-mitral (am) and aorto-septal (as) angle compared to a group of 14 control cats (as = 120 versus 152 -p = 0.007; am = 14 versus 157 -p = 0.014).other anatomic alterations of the lvot constituents included false tendon inserted on the septal crest (11/12), basal septal angulation (7/12), apical displacement of the postero-medial or antero-lateral papillary muscle (5/ 12), bifid/accessory papillary muscle (1/12), aberrant chordal insertion on the septal crest (3/12), severe papillary muscle hypertrophy (3/12), and thickened aortic cusps with sub-aortic ancillary echoes (5/12).mechanisms of lvotdo included obstructive mitral septal leaflet (2/12), protrusion of a displaced papillary muscle (2/12), impingement of an angulated basal septum into the lvot (7/12), and mechanical lvot narrowing from hypertrophic papillary muscle (1/12). basal septal impingement into the lvot was associated with apical ballooning (5/7) without obvious apical akinesis.out of 7 cats with lvotdo without hypertrophy, 4 had follow-up studies; only 1 cat responded positively to atenolol treatment. all the cats with initial hypertrophy had reverse remodelling and normalised aortic ejection velocities within 1 month to 11 month after atenolol treatment initiation; 1 cat developed aortic regurgitation.these data indicate that sam is not exclusively related to hcm and may be a cause, not a complication, of left ventricular hypertrophy. therapies that delay the onset of congestive heart failure (chf) in dogs with dmvd at risk of disease progression would be clinically beneficial. increases in la/ao, lveddn and serum nt-probnp and ctni concentrations are associated with decreased survival times. activation of the renin-angiotensinaldosterone system is implicated in cardiac remodeling in canine dmvd. we hypothesised that administration of spironolactone to dogs with compensated dmvd demonstrating the above risk factors would reduce the rate of cardiac remodeling associated with progressive dmvd. dogs with acvim class b dmvd were recruited to a randomized, blinded, placebo-controlled pilot study. no dogs were receiving medications for cardiac disease. all dogs demonstrated at least one of the following risk factors: echocardiographic evidence of cardiomegaly, nt-prob-np>550pmol/l, ctni>0.025ng/ml. no dogs had evidence of other cardiac disease or renal disease, hypoadrenocorticism, hyperkalaemia, or hyponatraemia. dogs were randomized to receive spironolactone (2mg/kg orally) or placebo sid for 6 months. comparisons between groups were made using mann-whitney tests. repeated measures linear models were constructed to compare the rate of change of variables over time. significance was set at p<0.05. data were analysed based on the intention to treat. twenty dogs of varying breeds were enrolled. ten dogs demonstrated 3 risk factors, 7 dogs 2 risk factors and 3 dogs 1 risk factor. ten dogs received placebo; age range 6.3-11.7 years (mean ±sd, 9.0±1.7 years), body weight range 4.5-17.0kg ( one dog in this group died suddenly, 1 progressed to chf and 2 received suboptimal spironolactone dosage. nt-probnp was significantly higher in the spironolactone group at baseline. (p=0.010). nt-probnp (p=0.002), la/ao (p=0.002) and lveddn (p=0.001) increased over time in the placebo, but not in the spironolactone, group. the rates of change of nt-probnp (p=0.059), la/ao (p=0.062) and lveddn (p=0.072) approached, but did not reach, significant differences between groups. in conclusion, treatment with spironolactone might slow the rate of increase in cardiac size in dogs with acvim class b dmvd showing risk factors for poor outcome. decreasing the rate of increase in cardiac size might delay the onset of chf. further studies are warranted to investigate these hypotheses. cardiac dysfunction is a concern in human systemic inflammatory response syndrome (sirs) patients, where increased cardiac biomarkers and decreased cardiac function have already been described. in a previous canine sirs study, an increase of cardiac biomarkers (nt-probnp, ctnt and lactate) and their prognostic value has been established. the present study evaluated the kinetics of basic echocardiographic parameters (fractional shortening (fs) and left ventricular ejection fraction (lvef), which both reflect systolic function; and the ratio of the left atrium to the aorta (la/ao), which reflects preload) in canine sirs. our hypotheses were that (1) fs, lvef and la/ao are altered in canine sirs and (2) that these parameters carry prognostic information.dogs with sirs, without primary cardiac disease, presenting to the emergency service were prospectively included from january until august 2010. cardiac ultrasonography was performed by two veterinarians in a standardized fashion at initial presentation, after 6 (t6), 12 (t12), 24 (t24), 72 (t72) hours of hospitalization until discharge or death and at a control visit (t1m) over one month after discharge. dogs were classified according to their underlying disease process: infection, neoplasia, trauma, gastric-dilation and volvulus (gdv), other gi diseases, and miscellaneous diseases. statistical analysis was performed with sas. univariate analysis was used to assess normal distribution. a mixed procedure and a logistic procedure was performed accordingly (p < 0.05).thirty seven dogs (infection, n=6; neoplasia, n=4; trauma, n=4; gdv, n=4; other gi, n=4 and miscellaneous diseases, n=15) were included. twenty-eight patients survived, while 9 did not (died, n=3; euthanasia for financial reasons, n=2; euthanasia for prognostic reasons, n=4). eleven dogs had control visits, 12 owners declined a control echocardiography, 2 patients were lost to follow-up and 3 died before control visit. fs and la/ao were significantly correlated with survival to discharge, however lvef was not. additionally, lvef and fs did not change significantly during hospitalization; neither compared to t1m. la/ao did however increase significantly during hospitalization. la/ao at t0 (1.03; 0.76-1.74) differed significantly from values at t12 (1.12; 0.84-1.68), t72 (1.20; 0.8-1.54) and t1m (1.19; 1.05-1.59).unexpectedly, surviving dogs had lower fs (35.7%; 19-64) than non-survivors (44.3%; 33-53). la/ao was associated with survival and increased rapidly after hospitalization to values similar to t1m, which probably reflects the efficacy of fluid therapy in emergency cases.in this population of canine sirs patients, no echocardiographic evidence of cardiac dysfunction was demonstrated. reports from first-opinion practice of feline arterial thromboembolism (ate) are scarce. our aim was to describe and evaluate the outcome in cats with ate presenting to three first-opinion clinics.clinical records of cats presenting with ate between 2004-2012 were reviewed for history, clinical findings, presence of congestive heart failure (chf) and outcome. kaplan-meier and log rank analysis was performed to evaluate associations with survival.during the study period, 242 cats presented with ate; an overall feline incidence of 0.26%. most cats were male (58.5%) and non-pedigree (92.1%). signs of cardiovascular disease prior to ate included cardiomyopathy (12%), a heart murmur (23.5%), a gallop sound/arrhythmia (3.3%) and hyperthyroidism (7.1%). most cats presented within 6 hours of clinical signs (72.6%). median age at presentation was 12 atrioventricular block (avb) is an arrhythmia resulting from conduction abnormalities through the atrioventricular node that leads to severe signs and sudden death.the aim of this study was to evaluate long-term intrinsic rhythm variations in dogs undergone pacemaker (pm) implantation. ninety-two dogs of different breeds with 3 rd degree avb (3avb) (59.8%), advanced 2 nd degree avb (2avb)(14.1%), paroxysmal 3avb (12%), 2:1 avb (7.6%) and 3avb with atrial fibrillation (6.5%) were retrospectively analyzed. forty-nine (53.3%) were males and 43 (46.7%) females with a mean age of 8.8 + 3.3 (sd) years and a mean body weight of 26.5 kg + 12.8. the intrinsic rhythm was evaluated the day of pm implantation (t0), after 1 day (confidence interval 95% [ci 95%] 1-2) (t1), 33 days (ci 95% 28-35) (t2), 105 days (ci 95% 98-156) (t3), 275 days (ci 95% 221-380) (t4). according to the avb grade at different controls, the rhythm disturbance was considered advanced, regressed or unchanged. shapiro-wilk and kolmogorov-smirnov tests were used to test normalcy, f-test to compare means in a generalized linear model and chi-squared to examine the association between categorical variables and status at each control. sixty (65.3%) dogs had no intrinsic rhythm changes, 20 (21.7%) had avb progression and 12 (13%) had avb regression. forty-eight cases of 3avb remained unchanged, while 4 regressed to sinus rhythm, 2 to 2:1 avb and 1 to advanced 2avb. eight advanced second degree avbs progressed to complete avb, 2 regressed to sinus rhythm, 1 to 2:1 avb and 2 remained unchanged. five paroxysmal 3avbs progressed to complete avb, 3 to 2:1 avb and 3 remained unchanged. four 2:1 avbs progressed to complete avb, 2 regressed to sinus rhythm and 1 remained unchanged. all avbs with atrial fibrillation remained unchanged. chi-square test showed that changes of intrinsic rhythm were associated with the type of avb (v 2 4.5, p<.03) and the time of controls (v 2 64.4, p<.0001), while other factors were not statistically significant. regression occurred within 30 days while progression occurred at any times. the results showed that the degree of avb at the moment of pm implantion should not be considered a definitive diagnosis since more than 1/3 of the cases could present progression or regression. because of their potential progression, pm implantation should be considered as first choice treatment also in cases of low 2avbs, and further studies are needed to evaluate the cause of transitory high grade avbs. tissue doppler imaging (tdi) is a complement to conventional echocardiography for assessment of myocardial function. the aims of the study were to investigate breed differences and intraobserver-variability of colour tdi variables in healthy dogs.fifty-three privately-owned male dogs were prospectively recruited. dogs were declared healthy by physical examination, blood pressure measurement, ecg, analyses of urine and blood (haematology and biochemistry), and conventional echocardiographic 2d and doppler examination, as part of the eu-funded lupa-project. directly following these extensive examinations, the tdi acquisition was performed by the same experienced echocardiographer using a philips hd11xe with a s8-3 (small dogs) and s3-1 (large dogs) mhz probe. cineloops were acquired from the right short-axis view and radial colour tdi variables at the endocardium and epicardium of the left ventricular free wall were later analysed. six other healthy dogs were included in a substudy aimed at evaluating the effect of 6 sources of variation using a hierarchial random-effects model.ten of the 53 examined dogs were excluded due to breathing artifacts and poor quality of the curves, leaving 43 dogs in the study; labrador retrievers (19), cavalier king charles spaniels (19), and dachshunds (5), with a mean age of 3.4 ± 1.2 years (sd). a p-value < 0.05 was considered significant in the statistical analyses. kruskal-wallis one-way analysis of variance showed that labrador retrievers had significantly higher values for endocardial and epicardial systolic (s) waves, and longer time to peak for both endocardial and epicardial e-and a-waves, compared to the other two breeds. labrador retrievers also had significantly lower heart rate (hr). further analysis of the breed differences using multiple regression analysis showed major effects of body weight and hr on endocardial and epicardial s waves, while time to peak, both endocardial and epicardial, for the e-and a-waves, were primarily affected by hr. dog was the variance component having the major effect on variability of tdi variables.in conclusion, time to peak of both diastolic waves was longer in labrador retrievers compared to the small-breed dogs. furthermore, higher s-wave values in labrador retrievers might indicate a different contractility pattern in largebreed dogs, and warrants further investigation. mitral regurgitation (mr) is the most common heart disease in dogs.dogs with a more advanced stage of this disease are likely to develop pulmonary edema of heart failure. the aim of this study was to evaluate the plasma c-reactive protein (crp) concentra-tion in dogs that underwent mitral valve repair for mr.all dogs were operated between october 2006 and october 2010. the dogs were categorized according to the international small animal cardiac health council (isachc) classification, and physical examination, thoracic radiography, and 2d color flow doppler echocardiography were performed before and after surgery. the plasma crp concentration and white blood cell counts were also determined before and after surgery. cardiogenic pulmonary edema was diagnosed on the basis of clinical examination and thoracic radiography.overall, 44 dogs (mean body weight, 5.6 ± 3.1 kg and mean age, 9.3 ± 2.2 years) were enrolled; 11 of these dogs had cardiogenic pulmonary edema. the dogs breeds were chihuahua (n = 10), cavalier king charles spaniel (n = 10), maltese (n = 7), yorkshire terrier (n = 3), shih tzu (n = 2), miniature dachshund (n = 2), and others (n = 10). no significant difference was found for age and body weight. the vertebral heart size and la/ao ratio significantly decreased after surgery compared with the preoperative values. before the operation, crp concentration and white blood cell counts in isachc class iiib dogs (2.5 ± 2.3 mg/dl and 21288 ± 7656 /ll, respectively) were higher than those in class ib (0.2 ± 0.3 mg/dl and 11500 ± 1320 /ll, respectively), class ii (0.1 ± 0.1 mg/dl and 8822 ± 2243/ll, respectively), and class iiia (0.7 ± 1.8 mg/dl and 11731 ± 4620/ll, respectively) dogs. additionally, crp concentration and white blood cell counts in class iiib dogs significantly decreased after surgery compared with preoperative values. crp concentration and white blood cell counts in the dogs with cardiogenic pulmonary edema significantly increased compared with those with non-pulmonary edema. furthermore, cardiogenic pulmonary edema disappeared within 3 months after surgery, and the crp concentrations and white blood cell counts became normal.in conclusion, crp concentration increases in dogs with mr and cardiogenic pulmonary edema. it is widely recognized that inflammatory reaction plays a key role in the development of heart failure. consequently, these data indicate the importance of strict management for pulmonary edema and inflammation. the neurotransmitter serotonin (5-hydroxytryptamine, 5ht) has recently been suggested to have a role in development of myxomatous mitral valve disease (mmvd) in dogs.the aim of this study was to investigate whether serum 5ht concentration was associated with mmvd severity in dogs, and to assess potential associations between serum 5ht concentrations and dog characteristics, echocardiographic variables, heart rate, systolic blood pressure, and platelet size (mean platelet volume) in the study population.120 client-owned dogs with naturally acquired mmvd of varying severity were prospectively recruited for the study. dogs were classified according to mmvd severity (breeds predisposed to early onset of mmvd, but without echocardiographic evidence of the disease, or mild, moderate or severe disease). serum 5ht concentrations were analyzed using an elisa assay.lower serum 5ht concentrations were shown in dogs with severe mmvd, compared with dogs predisposed to mmvd (p = 0.0025) and dogs with mild mmvd (p = 0.0011). unilinear and multiple regression analyses showed that serum 5ht concentrations decreased with increasing left atrial to aortic root ratio (la/ao), were higher in cavalier king charles spaniel (ckcs) dogs compared to dogs of other breeds, and were higher in female dogs than in male dogs. the la/ao was the variable most strongly associated with serum 5ht concentration.in conclusion, the finding of higher serum 5ht concentrations in dogs predisposed to mmvd (ckcs) and dogs with mild mmvd suggests that alterations in 5ht signaling might play a role in progression of early stages of mmvd. hypersomatotropism (hs) can be a common reason for development of diabetes mellitus in the cat. remission of diabetes can be achieved with an accurate diagnosis of the hs, although diagnosis is hampered by the relative complexity of confirming hs, requiring a combination of insulin growth factor-1 (igf-1) or feline growth hormone measurement and intracranial imaging. unfortunately, all three have limitations as diagnostic aid and when evaluating the success of therapy, particularly radiotherapy (rt). consequently, more precise markers of hs are required. the current study aimed to evaluate physiological behaviour and diagnostic potential of serum ghrelin in feline hs. as ghrelin is an endogenous ligand of the gh secretagogue receptor, it was hypothesised production of ghrelin might be suppressed in hs and subnormal serum levels could be a marker for increased gh activity.fasted (pre-insulin) serum samples were collected from 20 normal (age matched control), 20 uncomplicated diabetic (dm) and 33 hypersomatotrophic diabetic (hsdm) cats. cats were categorized into the hsdm-group on the basis of elevated igf-1 (>1000ng/ml) and demonstration of a pituitary lesion on imaging. in 13 cats additional serum samples were obtained following rt. cats were categorized into the dm-group on the basis of low igf-1 (<700ng/ml) and normal insulin requirements (<1.5 iu/kg). serum ghrelin was determined using a total ghrelin elisa system validated for the cat. data were tested for normality and concentrations compared between groups using unpaired t-tests and a paired t-test for the before and after rt hsdmgroups.serum total ghrelin was not different between the hsdm (7.9ng/ml+/-3.3) and dm-group (6.7ng/ml+/-2.3, p=0.14). a significant difference was present between the control group (12.9ng/ml+/-6.8) and both the hsdm (p=0.0007) and the dmgroup (p=0.0004). serum ghrelin concentrations in the hsdm cats undergoing rt (n = 13) were significantly higher following completion of treatment (6.7ng/ml+/-1.9 versus 8.6ng/ml+/-2.2, p=0.006).the results suggest feline serum total ghrelin is suppressed to similar levels in both the diabetic and diabetic hypersomatotrophic state compared to healthy subjects. consequently, it appears serum ghrelin levels are not helpful in determining the presence of hs in the diabetic cat. however, the results do indicate treatment of hs with rt results in an increase in serum ghrelin, suggesting the presence of an independent inhibitory effect of excess gh on serum ghrelin production. this potential inhibitory effect of gh might render serum ghrelin measurement useful as an additional tool to assess hypersomatotrophic remission after rt. further studies are however indicated. to date only few studies characterized histopathological features of the endocrine and exocrine pancreas in cats with diabetes mellitus. loss of b-cells is a consistent finding but no detailed data about the presence and types of inflammatory cells are available. we recently observed that hyperglycemia increases neutrophils in the exocrine pancreas. the aims of the present study were to assess whether diabetic cats have pathological evidence of islet inflammation or pancreatitis and to define islet lesions in comparison to a well-matched control population.formalin-fixed, paraffin-embedded pancreatic samples were collected from post-mortem examination performed on diabetic and control cats died due to any disease at the clinic for small animal internal medicine, university of zurich (switzerland) between 1997 and 2009. control cats were selected to be matched for age, sex, breed and body weight. sections were routinely stained with hematoxilin-eosin, and doublelabeled immunohistochemistry was performed for the following markers: insulin and myeloperoxidase (neutrophils), insulin and cd3 (t-lymphocytes), insulin and cd20 (b-lymphocytes), insulin and pcna (proliferation marker), and glucagon and ki-67 (proliferation marker). light-microscopic cell counting and morphometric analyses were performed manually and with software (image-j), respectively. data were analyzed with contingency tables and ttests.thirty-seven diabetic cats and 20 controls were included. the mean insulin-positive cross sectional area was approximately 40% lower in diabetic than control cats (p<0.01), that of glucagon was similar. proliferation of insulin-positive and glucagon-positive cells and the average counts of neutrophils, t-and b-lymphocytes in the islets did not differ between groups. interestingly, the presence of (t and b) lymphocytes in general tended to be more frequent in diabetic (8/37=21.6%) than control (1/20=5.0%) cats. in the exocrine pancreas, a trend towards increased presence of necrosis and fibrosis was observed in diabetic cats (6/37=16.2% vs. 0/20=0%; p=0.07) but inflammatory infiltration did not differ. proliferation of acinar cells was 3-fold increased in diabetic cats (p<0.01), notably nearby islets (6-fold, p<0.001).the results confirm previous observations that loss of b-cells occurs in diabetic cats. in addition, a subset of diabetic cats shows lymphocytic infiltration of the islets that might have contributed to b-cell loss. increased necrosis and fibrosis of the exocrine tissue may suggests that the diabetes leads to pancreatitis in some cats. the increased proliferation rate of acinar cells deserves further investigation. in humans this finding has been associated with chronic pancreatitis as well as transdifferentiation into islet cells. on the basis of a relatively high prevalence of hypersomatotropism (hs) amongst cats with a diagnosis of diabetes mellitus, as well as the possible subtle phenotype of these patients and the significant implications on prognosis and treatment, screening diabetic cats for hs could be advocated. for most veterinarians serum total insulin-like growth factor-1 (igf-1) assessment represents the most feasible and accessible means of performing screening. however, hepatic igf-1 production is dependent on presence of sufficient portal insulin, which can be deficient in newly diagnosed diabetic cats, resulting in false negative results. additionally, elevation of igf-1 has been reported in non-acromegalic diabetic cats. alternative or additional diagnostic tests for hs, as well as to evaluate the success of treatment of hs, are therefore desirable. since feline hs is associated with tissue growth, serum type iii procollagen propeptide (piiip), a peripheral indicator of collagen turnover, was hypothesised to be a useful indicator of active disease or growth hormone bioactivity. fasted, pre-insulin, serum samples were prospectively collected from 16 uncomplicated diabetic (dm) and 16 hypersomatotrophic diabetic (hsdm) cats. cats were categorised into the hsdm-group on the basis of elevated igf-1 (radioimmunoassay, >1000 ng/ml), followed by demonstration of a pituitary lesion on intracranial imaging and into the dm-group on the basis of low igf-1 (<600 ng/ml) and modest insulin requirements (<1.5 iu/kg). an elisa system for piiip was developed for use in the cat. data were tested for normality and concentrations compared using the mann whitney test. correlation with serum igf-1 was assessed by calculating a spearman's correlation coefficient (rho).dilutional parallelism using cat serum with high and low piiip-activity indicated validity of the elisa system. intra-assay coefficient of variation calculation proved adequate precision at high and low concentrations (7.3% and 7.8%) and the assay detection limit was found to be 2.7 ng/ml. median serum piiip was 24.3 ng/ml (range: <2.7-44.7) in the hsdm group, versus 4.9 (range: <2.7-8.0) in the dm group (p<0.001). there was a significant correlation between serum igf-1 and piiip (rho=0.60, p<0.001).in conclusion, serum piiip can be measured in the cat. additionally, serum piiip seems an alternative measure of growth hormone bioactivity in cats, given the significant elevation in concentration in feline hs. further evaluation of piiip in cases with hs will help determine the exact added value of evaluating this parameter in the diagnosis of hs, as well as in the assessment of treatment efficacy. glucose and galactose are transported across the brush border membrane (bbm) of enterocytes by sodium/glucose cotransporter-1 (sglt1), coded for by slc5a1. sglt1is the sole route for intestinal glucose absorption and its level of expression dictates bbm transport capacity for glucose. the relevance of sglt1 expression in predisposition to diabetes mellitus and obesity was investigated in dogs. the aims were to assess the effect on promoter function of known snps in the 5' flanking region of canine slc5a1, and to search for novel snps in well defined samples of dogs with varying risk for diabetes or obesity. caco-2/tc7 cells were shown to express sglt1 in vitro. the 2kbp fragment of canine slc5a1 5' flanking region from -1974 to +25 relative to the transcription start site was cloned from canine genomic dna, ligated into pgl3 basic plasmids bearing firefly luciferase as a reporter gene, used for transient transfection of caco-2/tc7 cells, and shown to drive luciferase production significantly above control (p<0.001). to determine the effect of the three known snps in this region on promoter function, new promoter/reporter constructs (all possible permutations of these three snps) were created using site-directed mutagenesis. these constructs were used for transient transfection of caco-2/tc7 cells using renilla luciferase as an internal control. no significant differences in promoter function were seen, suggesting that these three snps do not have a significant effect on the constitutive transcription of sglt1 mrna in dogs.a search for novel snps in this region in dogs was made in two breeds predisposed to diabetes mellitus (samoyed, cairn terrier), two breeds that rarely develop diabetes (boxer, german shepherd dog), and two breeds predisposed to obesity (labrador retriever, cocker spaniel). genomic dna from 10 healthy individuals of each of these breeds was obtained from the uk companion animals dna archive, with kind permission. the slc5a1 5' flanking region was amplified from each individual by high-fidelity pcr using breed-labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing (454 sequencing, gs flx, roche). the sequence of the slc5a1 5' flanking region in all individuals of all breeds tested was identical. on this evidence, variations in slc5a1 promoter sequence between dogs do not influence the pathogenesis of diabetes or obesity in these breeds. remission of diabetes mellitus may be achieved in up to 50% of cats. for remission to occur, recovery of b-cell function and possi-bly of b-cell mass is required. a novel class of antidiabetic drugs that act via the incretin system increase b-cell proliferation and glucose-stimulated insulin secretion in rodents. two strategies of incretin-based therapies, glp-1 analogues (e.g. exenatide shortacting (ex-sa), exenatide long-acting (ex-la)), and dpp-4 inhibitors (e.g. sitagliptin), are successfully used in human diabetics. knowledge about the use of incretins in cats is scarce. it was demonstrated that ex-sa and a dpp-4 inhibitor (nvp-dpp728) increase insulin secretion after intravenous glucose stimulation in healthy cats. the effects of these drugs after meal stimulation and the use of ex-la have not been explored in cats so far.the aims of the study were to test whether ex-sa (byetta ® , q12h, sc), ex-la (bydureon ® , q5d, sc) and sitagliptin (januvia ® , q24h, po) can be safely used in cats, and to identify the most effective drug and dose in a dose-escalation study.nine healthy cats were used. ex-sa was given to 3 cats at 0.2, 0.5, 1 and 2 lg/kg for 5 consecutive days each. ex-la was given to 3 other cats at 40, 100, 200 and 400 lg/ kg with single injections each. sitagliptin was given to 3 cats at 1, 3, 5 and 10 mg/kg for 5 consecutive days each. a washout period of 2 weeks was allowed between doses. on day 5 of each treatment block, a meal response test (mrt) was performed in all cats after a 16 h fastby feeding 50% of daily energy intake with subsequent blood sampling at timepoints 0, 15, 30, 60, 120 and 300 minutes. insulin and glucose area under the curves (auc) were calculated for each drug dose.gastrointestinal side effects of 1 to 3 days duration were observed in 2 cats of each group, irrespective of the dose. well-being and appetite were otherwise conserved. ex-sa increased insulin auc by 320%, 364%, 547% and 198%, respectively, compared to insulin auc during mrt without drug administration. ex-la and sitagliptin increased insulin auc by 127%, 169%, 178%, 95% and 43%, 101%, 70%, 56%, respectively. auc for glucose was similar in all cats, irrespective of the drug and dose. we conclude that ex-sa, ex-la and sitagliptin can be safely used in healthy cats and that ex-sa increases insulin secretion more effectively than ex-la and sitagliptin. insulin detemir is a synthetic long acting insulin analogue designed to maintain basal levels of insulin in humans with diabetes mellitus (dm).pharmacokinetic studies in dogs indicate that insulin detemir has a greater effect than other types of insulin, requiring a lower dose. the objective of our study was to evaluate its efficacy and the frequency of hypoglycemia in dogs with dm treated with insulin detemir. eight dogs were included into the study. median (range) age was 9 years (7-12), 7 were female (2 intact, 5 spayed), and 1 was intact male; median (range) body weight was 8.5kg (3.7-42.0). dogs with relevant concurrent diseases (e.g. hypothyroidism, hypercortisolism, neoplasia, renal insufficiency) and dogs with prior administration of diabetogenic drugs were excluded. all dogs received insulin detemir bid for at least 6 months, re-evaluations were performed after 1, 2, 4, 12 and 24 weeks and included clinical signs, blood glucose curves (bgc) and fructosamine concentrations. median (range) insulin dose was 0.12 u/kg (0.07-0.13) bid at admission which was not significantly different after 24 weeks of therapy (0.12 u/kg, range 0.05-0.34). initially, all dogs had markedly elevated blood glucose (21.9 mmol/l, 20.5-29.6 mmol/l), and elevated fructosamine concentrations (547 lmol/l, 430-702 lmol/l). mean(±sd) glucose concentrations (mmol/l) of the bgc at each re-evaluation were 12.2±5.1, 15.1±5.3, 14.0±6.9, 17.9±8.3, 14.0±3.7; median (range) of the glucose nadir values (mmol/l) were 9.4 (2.3-11.9), 10.9 (3.6-18.1), 6.6 (2.7-8.0), 10.3 (3.9-24.8), 6.2 (3.4-12.1) and fructosamine concentrations (lmol/l) were 509 (274-683), 470 (314-749), 445 (379-460), 501 (417-653), 480 (382-571), respectively. glucose concentrations (mean) were significantly (p<0.001) lower after 24 weeks therapy than before treatment. hypoglycemia (glucose nadir <5 mmol/l) was a consistent problem, identified in 5 dogs (23% of the bgc of the 8 dogs). there were 6 episodes (in 4 dogs) of owners reporting clinical signs (lethargy, weakness, unsteady gait) that could have been caused by hypoglycemia. based on owner opinion, clinical assessment of the veterinarian and bgc after 6 months of therapy, good control of the disease was obtained in 5 (62%) and moderate control in 3 dogs (38%). according to our preliminary results insulin detemir is effective in controlling hyperglycemia in dogs with dm. it is more potent than other types of insulin which are also used bid and therefore lower doses were used. nevertheless hypoglycemia was a common finding especially in small dogs. therefore insulin detemir should be used with great caution and dilution could be considered. spontaneous hypercortisolism is due to acth-independent hypersecretion of cortisol by an adrenocortical tumor (at) in about 15% of the cases. although the understanding of growth and hormonal activity of ats has expanded in recent years, the pivotal factors/acts in the pathogenesis of the at remain undisclosed.the canonical wnt-pathway plays a role in cell survival and cell cycle progression and has been shown to be involved in many different tumor types, including human and mouse ats. central in this pathway is ß-catenin. cytoplasmic and/or nuclear accumulation of ß-catenin has been demonstrated in human cortisol-secreting ats. this has been partly explained by mutations in exon 3 of ß-catenin.in our study, the activation of the canonical wnt-pathway was investigated in 11 adrenal adenomas and 25 carcinomas of dogs with acth-independent hypercortisolism. fifteen normal canine adrenals served as control tissue. the mrna expression was measured for wnt-ligands (wnt 2, wnt 3, wnt 4, wnt 5a, wnt 5b variants 1 and 2, wnt 6, wnt 7a, wnt 10b), wnt-ligand inhibitors (dkk 3, wif 1, sfrp 1) and for wnt target genes (cmyc, axin2 and cyclind1). in addition, the coding region of the mrna of ß-catenin was sequenced and the localization of ß-catenin was evaluated by immunohistochemistry (ihc).the results of expression analysis of wnt-ligands and wnt-ligand inhibitors demonstrated a significant downregulation of wnt 4 and wnt 5b variants 1 and 2. wnt target gene cyclin d1 was significantly downregulated in adenomas, while cmyc and axin 2 mrna expression did not differ between ats and normal adrenals.sequence analysis of ß-catenin revealed a mutation in 12/36 of the ats: 9 silent mutations, most probably single nucleotide polymorphisms (snps), 1 neutral mutation (arginine to histidine), 1 nonsense mutation resulting in a premature stop codon and 1 deletion of 26 basepairs combined with a neutral mutation. the amino acid change appeared not to be tumorassociated whereas the stop codon was. there were no activating ß-catenin mutations. the ihc demonstrated accumulation of ß-catenin in the cytoplasm of part of the ats, while nuclear staining for ß-catenin was rarely present.we conclude that the canonical wnt-pathway is most likely not involved in the pathogenesis of canine cortisol-secreting ats. trilostane is the treatment of choice for canine pituitary-dependent hyperadrenocorticism (pdh) however there is controversy about the ideal treatment regime. the objective of this study was to evaluate efficacy and safety of sid vs bid trilostane treatment for canine pdh.this prospective randomised study included 32 dogs with pdh, 16 dogs treated with trilostane sid (initial dose 1-6 mg/kg/24 hrs) and 16 with trilostane bid (0.5-3 mg/kg/12 hrs). a history, physical exam, haemogram, biochemical profile, acth stimulation test and urinary cortisol to creatinine ratios (uccr) were performed before treatment, and at one week (sid 8-12 hrs post trilostane; bid 4-6 hrs) and 1, 3, 6 and 12 months after treatment (8-12 hrs post medication)the median (±sd) daily dose throughout the study was similar in dogs receiving sid (3.7±2.1 mg/kg/day) or bid (3.8±0.8 mg/kg/day) trilostane. baseline cortisol concentration (mean±sd) before trilostane and at the five reevaluations in the sid group were 6.9±2.4, 3.9±2.5, 4.3±1.9, 4.4±1.5, 5.0±2.0 and 2.2±0.7 and in the bid group were 6.4±3.9 lg/dl, 3.1±1.4, 3.7±2.1, 3.6±1.7, 3.3±1.6 and 2.5±1.5. baseline cortisol was significantly higher (p=0.023) in the sid group only at six-month evaluation. post acth cortisol concentrations in the sid group were 29.8±13.2, 10.3±5.6, 12.1±5.8, 10.3±2.2, 8.8±4.9 and 4.2±2.2 and in the bid group were 32. 7±12.4, 8.6±6.8, 8.6±4.8, 8.3±4.4, 8.9±4 .9 and 7.6±7.2. no statistically significant differences were found at any evaluation. uccr in dogs in the sid group were 480±509, 241±163, 306±201, 224±104, 186±119 and 107±66.8 and in the bid group 270±278, 225±172, 222±178, 237±108, 157±62 and 102±11. similarly no statistically significant differences were found throughout the study. in each of the five re-evaluation times, clinical signs (polyuria, polydipsia and/or polyphagia) persisted in 73% (11/15), 62% (8/13), 57% (8/14), 58% (7/12) and in 63% (5/8) of dogs in the sid group. persistence of clinical signs occurred in 46% (6/13), 27% (3/11), 41% (6/15), 25% (4/16) and 0% (0/12) of dogs in the bid group. mild adverse effects occurred in 10/16 and in 9/16 of the dogs treated with sid and bid trilostane, respectively.based on this study, we conclude that the results of acth stimulation test and uccr in dogs with pdh treated with both protocols are similar at most re-evaluation times. however, clinical signs resolved in a greater proportion of dogs receiving trilostane bid. using trilostane twice daily will help to reduce the number of dogs that do not have a good clinical response. the effectiveness of trilostane therapy is monitored by regular acth stimulation, which is time-consuming and expensive. therefore, a monitoring system without a stimulation protocol and with less client expense would be preferable.the aim of our study was twofold: firstly, to evaluate, if baseline cortisol, endogenous acth (cacth) or the baseline cortisol to acth ratio (cortisol/acth ratio) could replace the acth stimulation test; secondly, to evaluate, if baseline cortisol provides additional information than post-acth cortisol alone or if its measurement could be abandoned.forty-one trilostane-treated dogs with pdh diagnosed between april 2006 and december 2011 were included in the study. a total of 148 acth stimulation tests with baseline, post-acth and delta cortisol (post-acth cortisol minus baseline cortisol) and 75 cacth results and cortisol/acth ratios were analyzed.control of adrenal gland function was classified according to the target range of post-acth cortisol concentration as: excessive (< 41 nmol/l; group 1), optimal (41-149 nmol/l; group 2), or inadequate (> 149 nmol/l; group 3). in a second step, control of adrenal gland function was reclassified according to baseline cortisol values only and the new classification was compared with the original one.there was a significant correlation between baseline cortisol and post-acth cortisol and a significant difference of baseline cortisol between the groups, however, with a large overlap. reclassification of the adrenal gland function on the basis of baseline cortisol revealed a misclassification in 53/ 148 (36%) tests.endogenous acth did not correlate with baseline or post-acth cortisol and did not differ between the groups. the baseline cortisol:acth ratio differed significantly between group 1 and 3 and between group 2 and 3, but again with a large overlap.to determine if measurement of baseline cortisol gives additional information than post-acth cortisol alone, the delta cortisol values were analysed. delta cortisol correlated significantly with post-acth cortisol but not with baseline cortisol and differed significantly between the groups, the overlap however was large.the large overlap using only single values without taking post-acth cortisol into consideration leads to an unacceptably low correct differentiation of control of adrenal gland function. therefore, the acth stimulation test and determination of post-acth cortisol cannot be replaced by baseline cortisol, endogenous acth or the cortisol/acth ratio. however, as baseline cortisol concentration does not give additional information than post-acth cortisol alone, its determination can be abandoned. in humans, recombinant human thyrotropin (rhtsh) enhances radioactive iodine uptake (raiu) in patients with differentiated thyroid cancer. this property is particularly interesting in dogs because high doses of radioiodine-131 ( 131 i) are used for the treatment of this disease. no studies have been performed in veterinary medicine to optimize 131 i treatment of thyroid cancer.the aim of this study was to evaluate the effect of rhtsh on the uptake of 123 i in dogs with thyroid tumors.nine dogs with thyroid neoplasia were included in this prospective cross-over study. six dogs had unilateral tumors, 1 dog had bilateral tumors and 2 dogs had ectopic tumors. diagnosis was based on physical examination, cytology, cervical scintigraphy and, when available, histopathology. in 6 dogs 123 i was administered for a baseline raiu determination in week 1. in week 2 (after a wash out period of 2 weeks), these dogs received rhtsh (100 lg iv) 24h before 123 i injection. in 3 patients the order of the protocol was reversed. for each scan, the dogs received 37 mbq (1mci) of 123 i iv and planar scintigraphy was performed 8h and 24h thereafter for tumor raiu calculation. blood samples were taken at baseline and at 6, 12, 24 and 48h after rhtsh administration for measurement of serum total thyroxine (tt4) and serum thyrotropin (tsh) concentrations.rhtsh caused no statistical significant change on thyroid tumor raiu at 8h (p=0.89) or at 24h (p=0.98). despite the lack of overall statistical significance, after rhtsh administration the 8h raiu increased in 5 tumors and the 24h raiu increased in 4 tumors. when an increased raiu was observed, 123 i uptake with rhtsh ranged 1.2 to 3.8 times baseline uptake. in 3 patients, the post-rhtsh raiu more than doubled compared to baseline raiu. the raiu of 2 thoracic metastases from 2 patients could be calculated. in 1 thoracic metastasis the raiu doubled after rhtsh; in the other the raiu decreased after rhtsh.in euthyroid patients, rhtsh induced a significant increase in tt4 concentrations (p=0.01), confirming the biological activity of rhtsh.this study suggests that iv administration of 100 lg rhtsh 24h before 123 i has an inconsistent effect on thyroid tumor raiu, with a marked increase in uptake in some tumors and a decrease in others. further studies are necessary to determine the best dosage, route and timing of rhtsh to optimize thyroid tumor raiu.this study was partly funded by the dutch animal cancer foundation transdermal methimazole has been suggested as an alternative treatment option of hyperthyroid cats. in a previous study we could show a good clinical effectiveness of a pleuronic lecithin organogel (plo-) based product on a twice-daily basis. a reduced dose frequency is known to improve owner compliance, however no study has yet evaluated long-term treatment responses after once daily administration.objectives of the present study were to assess whether once daily administration of transdermal methimazole in its original formulation of (plo) was an effective alternative to the twice-daily treatment during a follow-up period of up to 18 months and to evaluate t4 courses during a 10-hour period after methimazole application in selected cats.twenty client-owned cats with newly diagnosed hyperthyroidism and with available follow-up information were included in the study. methimazole was formulated in plobased vehicle and was applied to the pinna of the inner ear at a starting dose of 5mg/cat q 24 hours. cats were rechecked 1-2 weeks, 3-4 weeks, 1-2 months, 2-3 months, 3-6 months, 6-12 and 12-18 months after starting therapy. additionally, in 10 cats t4 concentrations were measured every 2 hours after gel application over a 10 hour period 1 week after starting therapy. after 1-2 weeks, clinical improvement was observed in all animals. a change of treatment to oral medication due to erythema of the internal pinna of the ears was necessary in one cat, while none of the other animals showed any side effects during the follow-up period. significant decreases in t4 concentrations were determined at all rechecks compared to pre-treatment concentrations. methimazole dosage was increased in 5, decreased in 11 and remained unchanged in 4 cats. two cats with a decrease in the dose later had to be re-increased, while one cat was changed to q12 hours. there was no significant change in t4 during the 10-hour period and fluctuations corresponded to variations of precision in series.these results are in accordance with those of our previous study using the twicedaily regimen. we could show that once daily administration of transdermal methimazole in its original formulation of plo is an effective treatment option for long-term management of feline hyperthyroidism. further, timing of blood sampling after gel application is not important when assessing response to treatment. increase in the prevalence of large thyroid tumors, intra-thoracic thyroid thyroid scintigraphy provides valuable information regarding both thyroid anatomy and physiology and plays an integral role in the diagnosis, staging, and management of feline thyroid disease. in this study, we performed thyroid imaging on 1,572 consecutive hyperthyroid cats that were referred for radioiodine therapy between january 2009 and december 2011. scintigraphy was performed as part of our staging protocol in which thyroid volume is estimated for 131 i dose estimation (vet radiol ultrasound 1996; 27:141) . in each cat, the location of each area of increased radionuclide uptake (iru) was also recorded (cervical, thoracic inlet, chest). finally, each scan was evaluated for features suggesting malignancy (multiple, extensive areas of iru, heterogeneous pattern of iru with irregular, spiculated margins, extension of tumor through thoracic inlet into the thorax, and metastasis to regional lymph nodes or lung).of the 1,572 cats, most had been recently diagnosed. in 228 cats, however, the interval between diagnosis and 131 i treatment ranged from >1 to 6.1 years; almost all of these cats had received long-term antithyroid drug treatment. the 1,572 cats were divided into 5 groups based on interval from diagnosis to 131 i treatment: group 1 (0-1 year), 1,344 cats; group 2 (>1-2 years), 114 cats; group 3 (>2-3 years), 62 cats; group 4 (>3-4 years), 29 cats; and group 5 (>4-6.1 years), 23 cats. when the estimated thyroid volumes in the 5 groups of cats were compared, a progressive, significant (p< 0.001) increase in median tumor volume occurred: 2.1 cm 3 (group 1); 4.1 cm 3 (group 2); 5.7 cm 3 (group 3); 6.1 cm 3 (group 4); and 7.7 cm 3 (group 5). the prevalence of cats with areas of iru within the thoracic cavity also increased progressively: 5.1% (group 1); 7.9% (group 2); 14.5% (group 3); 17.2% (group 4); and 26.1% (group 5). finally, the prevalence of suspected thyroid carcinoma (25 of the 1,572 cats) also increased progressively: 0.7% (group 1); 3.5% (group 2); 6.5% (group 3); 10.4% (group 4); and 21.7% (group 5). in contrast, no increase in prevalence of ectopic thyroid tissue was found: 2.9% (group 1); 2.8% (group 2); 3.2% (group 3); 6.9% (group 4); and 4.3% (group 5). in conclusion, our results indicate that hyperfunctional thyroid tissue continues to grow and enlarge over time. thyroid carcinoma is extremely rare in cats with recently diagnosed hyperthyroidism, but the prevalence increases dramatically over time, suggesting that transformation from benign disease is common in cats controlled medically. measurement of plasma renin activity (pra) is considered the gold standard for monitoring mineralocorticoid substitution in humans with primary hypoadrenocorticism (ph). it is the most sensitive parameter to reflect insufficient as well as inappropriate high replacement. in dogs with ph mineralocorticoid substitution is currently monitored mainly by serum potassium and sodium concentrations. the role of pra for monitoring mineralocorticoid replacement has not been investigated. the aims of the study were to measure and compare pra in dogs with newly diagnosed ph and dogs with diseases mimicking ph, and to evaluate pra in dogs with ph treated with different mineralocorticoid substitution regimes.the following groups of dogs were included in the study: 5 dogs with newly diagnosed ph (group 1), 10 dogs that were already treated for ph (group 2), and 10 dogs with diseases mimicking ph (group 3). in group 1 pra was measured before treatment and 1-3 weeks, 4-6 weeks, and 7-10 weeks after start of therapy. in group 2 pra was measured at least twice every 1 to 6 months. in group 3 pra was measured once at initial presentation. three dogs of group 1 and 7 dogs of group 2 were treated with fludrocortisone (florinef ® , bristol-myers squibb). two dogs of group 1 and 3 dogs of group 2 were treated with docp (percorten ® -v, novartis). pra was measured with an enzymatic assay via trapping of angiotensin i in the service d'angiologie, chuv-nes, lausanne. results were analysed by means of non-parametric methods (p < 0.05).pra before treatment was significantly higher in group 1 (1.6-36.0 ng/ l/h, median 22.5) than in group 3 (0.5-2.8 ng/l/h, median 1.0). average pra during therapy ranged from 0.47 to 29 ng/ml/h (median 8). pra did not decrease significantly in dogs treated with fludrocortisone. pra of dogs treated with docp (0.11-10.61 ng/ml/h, median 5) was significantly lower compared to dogs treated with fludrocortisone (0.47-38.3 ng/ml/h, median 16.5). all dogs treated with docp had normal serum sodium and potassium at all re-checks, whereas dogs treated with fludrocortisone had mild to severe electrolyte abnormalities at several occasions. there was a weak correlation between pra and serum potassium.measurement of pra is a promising tool for monitoring mineralocorticoid substitution in dogs with ph. according to our preliminary results docp is superior to fludrocortisone for mineralocorticoid replacement. phenobarbital is widely used to control epilepsy in dogs. use of phenobarbital induces hepatic enzyme activity, and may decrease serum total and free thyroxine (tt4 and ft4), with the exact mechanisms and prevalence of this phenomenon being unknown. the aim of the present retrospective study therefore was to investigate how many dogs treated with phenobarbital show a decrease in thyroid hormones and to give insight into potential mechanisms. for this, tt4, ft4, and tsh were measured in 746 canine serum samples submitted for assessment of therapeutic concentrations of phenobarbital. in a smaller subset of dogs, albumin, total protein, and transthyretin (ttr) were also measured at the diagnostic center for population and animal health, michigan, usa.according to thyroid results, dogs were classified as 'non thyroidal illness' (nti) (tt4 < 11 nmol/l, ft4 ! 6 pmol/l, tsh 0.48 ng/ml); 'equivocal' (e) (tt4 < 11 nmol/l, ft4 < 6 pmol/l, tsh 0.48 ng/ml); 'hypothyroid' (ht) (pattern of low tt4 and ft4, tsh > 0.48 ng/ ml); and 'normal' (n) (tt4 ! 11nmol/l; ft4 ! 6 pmol/l and tsh 0.48 ng/ml).forty-five dogs were classified as nti (6.0%), 52 dogs were classified as e (7.0%), 38 were classified as ht (4.7%), and 552 as n (74%); 52 dogs did not fit in any defined category. there was no statistically significant difference in mean phenobarbital concentrations between nti, e, ht, and n (94.2 ± 35.1, 100.6 ± 43.9, 86.8 ± 29.4, and 84.3 ± 31.9 lmol/l, respectively). twenty dogs of each group were analyzed for albumin and total protein and compared to healthy dogs that were not receiving phenobarbital. no statistically significant difference was noticed (p>0.05). the attempt to measure ttr concentrations via commercially available elisa (tsz elisa, framingham, ma, usa) gave us inconsistence performance. in conclusion, 6% of dogs on phenobarbital treatment were classified as nti. if group e would be included, this value could be as high as 13%. neither phenobarbital, nor total protein or albumin concentrations had predictive value for thyroid hormone concentrations. this study was able to define the prevalence of the phenomenon that some dogs on phenobarbital therapy have low thyroid hormone concentrations, but did not give insight into the pathogenesis. ideally, demonstration of hypothyroidism in dogs receiving phenobarbital should include assays of thyroid hormones and tsh. pheochromocytoma (pheo) is a rare malignant catecholamine-secreting tumor of the adrenal medulla. catecholamines and metanephrines in plasma and in 24-h urine are approved biomarkers for the detection of the disease in humans, however, the question which of the tests is best is controversial. we previously demonstrated that measurement of urinary catecholamine and metanephrine to creatinine ratios is helpful for the diagnosis of pheo in dogs and that urinary normetanephrine to creatinine ratio may be the best test to discriminate between pheo and hypercortisolism (hc).knowledge on plasma catecholamines and metanephrines in dogs is scarce and no comparison between urinary and plasma parameters has been performed. the objective of the study was to measure urinary and plasma catecholamines and metanephrines in dogs with pheo, hc and in healthy dogs and to determine the test with the least overlap between the groups. six dogs with pheo, 9 dogs with hc (6 with ath, 3 with pdh) and 10 healthy dogs were included. urine samples were collected into hcl containing tubes to ensure a ph < 2, blood samples were collected on ice, centrifuged at 4°c and immediately snap frozen in liquid nitrogen. all samples were stored at -80°c. urinary epinephrine (u-epi), norepinephrine (u-norepi), metanephrine (u-meta) and normetanephrine (u-normeta), and plasma epinephrine (p-epi), norepinephrine (p-norepi), free and total metanephrine (pf-meta and pt-meta) and free and total normetanephrine (pf-normeta and pt-meta) were analysed by hplc. urinary catecholamines and metanephrines were expressed as ratios to urine creatinine concentrations. data were analysed by non-parametric tests (p< 0,05).similar to our previous findings u-epi, u-norepi, u-meta and u-normeta were significantly higher in dogs with pheo and u-norepi and u-normeta were significantly higher in dogs with hc compared to healthy dogs. comparison between dogs with hc and dogs with pheo revealed significantly higher u-meta and u-normeta in the latter group. u-normeta was the only parameter with no overlap.in dogs with pheo p-norepi, pf-meta, pt-meta, pf-normeta, pt-normeta were significantly higher and in dogs with hc p-norepi, pf-normeta and pt-normeta were significantly higher than in healthy dogs. comparison between dogs with hc and dogs with pheo showed significant higher pf-meta, pt-meta, pf-normeta, pt-normeta in the pheo group. overlap was present with all 4 parameters, but was least with pf-normeta and pt-normeta.according to our preliminary results u-normeta, pf-normeta and pt-normeta are valuable parameters for the diagnosis of pheo, so far u-normeta has performed better than the plasma parameters. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to ckd progression. vascular endothelial growth factor (vegf) is a marker of hypoxia. the aim of this study was to evaluate urinary vegf as a prognostic marker in cats with ckd compared with the established progression factors, proteinuria and hyperphosphataemia. cats were recruited through geriatric clinics held at two first opinion london practices between 1999 and 2010. diagnosis of ckd was based on concurrent findings of plasma creatinine >2mg/dl and usg <1.035, with persistence of azotaemia for at least 2 weeks. vegf was measured in urine samples taken from cats at diagnosis of ckd and indexed to creatinine giving vegf: creatinine ratios (uvc). survival was compared among low (<0.3lg/g), medium (0.3-0.6lg/g) and high (>0.6lg/g) categories of uvc using the log-rank test. multivariable binary logistic regression was used to assess whether uvc was associated with an increase in plasma creatinine concentration of at least 25% within 1 year of diagnosis. cats which did not demonstrate progression but were followed for <1year were excluded from the study. cases which developed hyperthyroidism, received ace inhibitors, had gross haematuria, urinary tract infections, nephrotic syndrome or evidence of bladder neoplasia were also excluded. survival data are presented as median [95% confidence interval] and other descriptive data are presented as median (25 th , 75 th percentile). significance was set at p<0.05. cats with low uvc (n=49) had survival of 840 [574, 1106] days, while those with medium (n=30) and high uvc (n=10) had survival of 863 [385, 1341] days and 260 [0, 554] days respectively. there was no difference in survival time between cats with low and medium values of uvc (p=0.414), but cats with both low (p<0.001) and medium (p=0.002) uvc had significantly longer survival than cats with high uvc. plasma creatinine concentration increased by 52(33, 109)% in cats which progressed (n=40) and 6(0, 14)% in cats with stable renal function in the year following diagnosis (n=39). uvc was also associated with progression of azotaemia (p=0.008) independently of upc and plasma phosphate concentration. the progressive group had uvc of 0.36(0.26, 0.54) lg/g, while the stable group had uvc of 0.26(0.20, 0.38) lg/g. if high uvc indicates renal hypoxia, the results of this study support the hypothesis that hypoxia is associated with progression in cats with ckd. however, further studies evaluating renal hypoxia directly would be required to verify this. the term 'triaditis' is used to describe concurrent inflammation of the liver, pancreas, and small intestine, although occasionally only two of these organs may participate. the aim of this study was to investigate the frequency of coexistence of different combinations of cholangitis, pancreatitis and/or inflammatory bowel disease in cats, and describe the clinical, clinicopathological, and histopathological findings.initially 67 cats were included in the study. thirty-nine cats had a suspicion of 'triaditis' based on clinical signs (depression, anorexia or polyphagia, vomiting, diarrhea, and/or weight loss), while 28 cats clinically healthy, were considered as controls. each cat on presentation underwent cbc, biochemistry profile, serum total t4, spec fpl ® and ftli blood examinations. cats diagnosed with intestinal parasitism, infectious disease, neoplasia, and/or hyperthyroidism were excluded from the study. biopsies from the liver, pancreas and small intestine (duodenum, jejunum, and ileum) were collected from each sick cat during laparotomy as part of the diagnostic investigation. biopsies from healthy cats were collected during laparotomy for ovariohysterectomy. all owners had signed a consent form and the study protocol was approved by the university's and state's ethics committee.of the 39 sick cats with a suspicion of triaditis, 12 were excluded because of neoplasia or other conditions. of the 28 clinically healthy cats, 20 had histopathological evidence of inflammation in their liver, pancreas, and/or intestine and were eventually grouped together with the sick cats. collectively, histopathological evaluation of the biopsies revealed 47 cats (27 with clinical signs and 20 without) with inflammatory lesions in at least one organ. of those 47 cats, 40 (85.1%) had histopathological evidence of ibd, 30 (63.8%) of cholangitis, and 12 (25.5%) of pancreatitis. thirteen cats (27.6%) had only ibd, 8 (61.5%) of which were symptomatic. six (12.7%) cats had only cholangitis, 2 (33.3%) of which were symptomatic. one (2.1%) had only pancreatitis and was symptomatic. sixteen cats (34%) had concurrent ibd and cholangitis, 6 (37.5%) of which were symptomatic; 3 (6.4%) had ibd in combination with pancreatitis, 2 (66.7%) of which were symptomatic, while 8 (17%) had ibd, cholangitis and pancreatitis and were all symptomatic. common biochemical findings were increased activities of alt (11/47), alp (11/47), and increased total bilirubin (14/ 47), ftli (3/47), spec fpl (12/47) concentrations.the results of our study indicate that different combinations of concurrent inflammation of the liver, pancreas, and intestine do exist in cats. it also appears that these conditions may be subclinical in many cases. several potential pathogens are found in feline faeces, ranging in significance from incidental to pathogenic. treatment recommendations are based on accompanying clinical signs. real time pcr has enabled rapid screening of small quantities of faeces for potential pathogens, with high sensitivity and specificity. co-carriage of feline faecal pathogens may reflect a symbiotic relationship or related pathogenesis, in which case identification of common cocarriage patterns may influence treatment decisions and prognosis. the primary objective of this study was to identify co-carriage of selected feline faecal pathogens. secondary objectives were to evaluate the prevalence of individual pathogens in feline faeces, and their association with pedigree status, and a history of diarrhoea. results of a commercial 8-way feline diarrhoea realpcr tm panel (idexx reference laboratories, uk) from june 2010 to january 2012 were evaluated. real time pcr was performed for tritrichomonas foetus, giardiaspp., cryptosporidiumspp., toxoplasmagondii, salmonellaspp., clostridium perfringens enterotoxin a gene, feline coronavirus, and feline panleukopenia virus. additional data was recorded when available, including age, gender, breed, and history of diarrhoea. weak or borderline positive results and those from pooled faecal samples were excluded. associations of the carriage of pairs of pathogens were evaluated with a chi-squared test, statistical significance set at p < 0.05.results of 1882 pcr panels were evaluated. the prevalence of faecal pathogens was 17.8% (tritrichomonas foetus), 19.3% (giardia), 24.8% (cryptosporidium), 0.8% (toxoplasma gondii), 0.5% (salmonella), 54.7% (clostridium perfringens), 57.1% (feline coronavirus), and 22.6% (feline panleukopenia virus).salmonella was the only faecal pathogen significantly associated with a history of diarrhoea. faecal samples from pedigree cats were significantly more likely than dsh to be positive for tritrichomonas foetus, giardia, clostridium perfringens and feline coronavirus.significant co-carriage was identified for feline coronavirus with all other pathogens except salmonella. there was significant co-carriage of tritrichomonas foetus with clostridium perfringens and giardia, and also for giardia with panleukopenia virus, cryptosporidium, and clostridium perfringens. finally, there was significant co-carriage of cryptosporidium with clostridium perfringens, panleukopenia virus with cryptosporidium, and toxoplasma gondii with salmonella.in conclusion there was a moderate to high prevalence of all feline faecal pathogens tested except toxoplasma gondii and salmonella. positive pcr results were more likely in pedigree cats, and salmonella was associated with a history of diarrhoea. significant co-carriage of faecal pathogens was common, possibly reflecting common environmental risk factors, a shared pathogenesis, or a symbiotic relationship. mycoplasma spp have been identified as causative pathogen in feline lower airway disease and are not thought to colonize the lower airways of clinically healthy cats.to challenge this hypothesis, 30 domestic shorthair cats aged 1-10 years (median, 4.5 years), without signs of respiratory disease, housed in a shelter, underwent transoral lower airway washing with sterile saline under general anaesthesia. retrieved bronchalveolar lavage fluid (balf) was subjected to microbiological and cytological analysis.during preanaesthetic clinical examination, only minor alterations were discovered: tartar in 7, conjunctivitis in 4, flea infestation in 3 and dirty auditory canals in 3 cats. one animal each showed dandruff, corneal ulceration and a heart murmur 2/6.balf-cultures of 26 animals (86.7%) were positive for mycoplasma spp. 66.7% of examined cats had mycoplasma felis in their balf (light growth in 11, moderate growth in 4 and heavy growth in 5 animals), 50% ureaplasma felinum/cati (light growth in 8, moderate growth in 7 cases), 23.3% mycoplasma gateae (light growth in 4, moderate growth in 2 and heavy growth in 1 cat) and 3.3% mycoplasma feliminutum (light growth in 1 animal). using aerobic bacterial culture, pasteurella multocida (46.7%), a-haemolytic streptococci (33.3%), haemolytic e. coli (3.3%) and acinetobacter iwoffii (3.3%) were detected, with only 7 samples (23.3%) yielding negative results.median balf-total nucleated cell count was 550/ll (range, 4-7790) with a median mononucleated cell percentage of 73%, a median neutrophil fraction of 15.5% and a median eosinophil proportion of 1.5%. only 2 samples were neutrophil-dominated (92.5 and 97.5%), indicating purulent inflammation. both specimens were positive for mycoplasma felis (1 light growth, 1 heavy growth), and one showed moderate growth of pasteurella multocida. like all of the animals included in this study, the corresponding cats remained clinically healthy during a 2-week followup period.balf-samples showed neither cytological nor microbiological signs indicative of upper airway contamination.in contrast to earlier studies, we conclude that -at least in cats housed in shel-ters and subjected to high infection pressure -in addition to bacteria like pasteurella spp, streptococcus spp and e. coli, mycoplasma spp can occur in the feline lower airways without causing respiratory signs. inflammasomes are intracellular multi-protein complexes that coordinate the maturation of interleukin (il)-1b and il-18 in response to pathogens and metabolic danger signals. both cytokines are vital for the maintenance of the intestinal homeostasis and have been linked to chronic intestinal inflammation in humans. both il-1b and il-18 are produced as inactive proforms and undergo subsequent maturation through cleavage into their active forms by caspase (casp)-1, which is in turn activated by the inflammasome complex. the best characterized inflammasome subtype in human inflammatory bowel disease (ibd) is nlrp3, which seems to be crucial for the regulation of intestinal homeostasis. defective nlrp3 signaling has been suggested to contribute to ibd. additionally, il-1b, il-18 and casp-1 are upregulated on the mrna and protein level in human ibd. so far, no study has investigated the role of the inflammasome and respective down-stream cytokines in canine ibd. thus, the goal of the current study was to investigate the expression of inflammasome components in duodenal tissues from dogs with ibd compared to healthy controls. rna extraction from endoscopic biopsies (ibd group n = 25, control group n = 20) and reversetranscriptase quantitative pcr was performed in a sybrgreenbased assay using specific primers for the following canine genes: nlrp3, casp-1, il-1b and il-18. a 10-fold dilution of plasmid controls for each gene was used to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). comparison between groups was performed using mann whitney u tests (graph pad prism 5). significance was set at p < 0.05. eight samples (5 ibd, 3 controls) had to be excluded due to poor cdna quality (inadequate expression of reference genes). when comparing the remaining samples in both groups, casp-1 (p = 0.001) and nlrp3 (p < 0.001) expression was significantly lower in ibd dogs than controls. in contrast, il-1b (p = 0.138) and il-18 (p = 0.903) expression was not different between groups.down-regulation of nlrp3 and casp-1 could be part of a negative feedback loop in the pro-inflammatory environment in ibd. alternatively, this could represent general disturbances in intestinal homeostasis or failure to up-regulate "danger-signaling pathways"in inflammation. final conclusions might be difficult to draw without matching protein data, as assessing il-1b and il-18 mrna levels cannot give insight into the ratio of "pro-cytokines"to active cytokines. thus, investigating il-1b and il-18 protein content of canine ibd tissues is warranted in the future. the trefoil factor family (tff) comprises a group of small peptides produced in goblet cells, which are crucial for epithelial restitution and maintenance of tight junction function in the gut. in humans with inflammatory bowel disease (ibd), tff expression is up-regulated, which is thought to represent an unspecific repair mechanism. however, tffs have also been shown to be involved in the local control of disease in rodent models and in humans. so far, there has been no study investigating tff expression in the canine intestine. thus, the goal of this study was to assess tff expression in gastrointestinal tissues from dogs with ibd and healthy dogs. rna was extracted from endoscopic duodenal (ibd n = 22, healthy controls n = 18) and colonic biopsies (ibd n = 12, controls n = 11) and cdna generated. quantitative reverse-transcription pcr was performed for canine tff1 and tff3 in a sybr-green-based assay. a 10-fold dilution of plasmid controls for each gene was used to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). for statistical analysis, significance was set at p < 0.05. overall, tff1 expression was significantly different across groups (kruskal wallis p < 0.0001): in control dogs, tff1 expression was higher in the colon than in the duodenum (mann whitney p < 0.0001). when comparing ibd cases to controls, duodenal tff1 was significantly up-regulated (mann whitney p = 0.0001). tff3 expression was not different across groups (kruskal wallis p = 0.056). however, separate analysis of the intestinal location showed significant down-regulation of tff3 in the colon of ibd dogs compared to controls (t-test p = 0.018).this study demonstrates evidence for dysregulation of tff gene expression in canine ibd. a lack of tff1 could contribute to defective epithelial barrier function, causing "leakiness"of tight junctions. this increased intestinal permeability could lead to an increased antigenic load from microbes or food antigens, which could perpetuate faulty immune recognition of microbe-associated molecular patterns, thus leading to increased intestinal inflammation. up-regulation of tff3 in the colon could in turn signify a compensatory repair-mechanism in inflammation. further investigation of tff gene or protein expression is warranted in canine ibd. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb 10415 e1707 (ef) on gene expression and protein production in canine duodenal biopsies. samples from 11 healthy beagles and 8 ibd dogs were cultured ex-vivo with ef (1x10 8 cfu/ml) or sterile nutrient broth/ pbs (negative control) for 5 hours. rna extraction from biopsies and reverse-transcriptase quantitative pcr was performed in a sybrgreen-based assay using specific primers for the following canine genes: tlr2, tlr4, tlr5, tlr9, il-17a, il-22, il-10, tgfb, il-4, ifnc and tnfa; using a 10-fold dilution of plasmid controls for each gene to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). protein content of ifnc, il-17a and tnfa was measured in culture supernatants using commercially available canine-specific elisas. a linear mixed model for each genewith disease group and treatment set as fixed parameters -was chosen for statistical analysis using spss software. significance was set at p < 0.05.no significant interaction between disease group and treatment was observed for any gene. only expression of il-4 was significantly increased in ibd dogs compared to healthy dogs (p = 0.022) in unstimulated samples. all other significant differences were independent of disease group, but depen-dent on treatment with ef. expression of the following genes was reduced by ef treatment: tlr 4 (p = 0.003), tlr5 (p = 0.015), tlr9 (p = 0.005), tgfb (p = 0.006), ifnc (p = 0.032) and tnfa (p = < 0.001). no significant amounts of ifnc, il-17a or tnfa protein were detected in culture supernatants.this is the first study demonstrating a profound effect of ef treatment on gene expression in ex vivo cultured canine duodenal biopsies. down-regulation of several genes of innate immune receptors and pro-inflammatory cytokines was observed, with the most significant effect on suppression of tnfa expression. effects were seen both in healthy and in ibd dogs. whether this translates to a beneficial effect in a clinical situation needs further investigation. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of cats. there is mounting evidence that microbes play an important role in the pathogenesis of chronic enteropathies, as compositional changes of the intestinal bacterial ecosystem have been associated with chronic intestinal inflammation in humans and dogs. the aim of this study was to characterize the bacterial microbiota in cats with chronic enteropathies.fecal samples were obtained from healthy cats (n=16) and cats with histologically confirmed chronic enteropathies (n=8). the bacterial composition was analyzed by massive parallel 16s rrna gene 454-pyrosequencing (yielding 194,000 sequencing tags), and selected quantitative pcr assays. differences in microbial community structure between healthy and diseased cats were assessed by the phylogenetic unifrac distance method, followed by analysis of similarity (anosim) of the distance matrix. differences in bacterial groups between the disease groups were analyzed using mann-whitney u tests. the resulting p-values were corrected for multiple comparisons using the benjamini & hochberg's false discovery rate. an adjusted p<0.05 was considered for statistical significance.the unifrac distance metric indicated no significant clustering according to disease status. however, the relative proportions of sequences belonging to bacteroides spp. were significantly decreased in the cats with chronic enteropathies compared to the healthy cats (p=0.02). the employed qpcr assays confirmed the sequencing results and also showed a significant decrease in proportions of bacteroides spp. in the diseased cats. in addition, qpcr revealed also a significant decrease in turicibacter spp. in cats with chronic enteropathies compared to the healthy cats (p=0.04). no significant differences in diversity indices were observed between healthy and diseased cats.in conclusion, the here used molecular approach revealed significant reductions in bacteroides and turicibacter spp. in cats with chronic enteropathies. future studies are necessary to evaluate if these microbial changes correlate with functional changes in the intestinal microbiota. campylobacter spp. represent a common cause of gastroenteritis in humans with c. jejuni and c. coli considered responsible for the majority of clinical cases. many human diagnostic laboratories use the prospect ® campylobacter microplate assay (eia) as a sole or screening test for detection of c. jejuni and c. coli as this technique is easier, faster and cheaper than campylobacter spp. culture. c. upsaliensis can cause a diarrhoeal illness similar to that caused by c. jejuni in humans but methods routinely used for c. jejuni and c. coli detection, including the eia, are not optimised or have not been evaluated for the detection of other campylobacter species. c. upsaliensis and c. helveticus have been more commonly isolated from dogs and cats than c. jejuni and c. coli in most studies. campylobacter spp. from pets are potentially zoonotic while the pathogenicity in pets remains uncertain. the significance of c. upsaliensis and c. helveticus infections may be underestimated in human medicine due to detection methods applied.the aim of this pilot study was to assess if the eia detects c. upsaliensis and c. helveticus isolated from different pets in spiked human clinical samples. in addition, the ability of eia to detect campylobacter spp. in dog and cat faeces was assessed. ten clinical human faecal samples and two healthy dog and cat faeces confirmed negative by eia were tested by a range of culture methods to exclude presence of campylobacter spp. dilutions of eight c. upsaliensis, five c. helveticus cultures, and one c. jejuni and c. coli culture, all pcr confirmed, were added to aliquots of faecal samples to obtain a range of viable bacteria from 10 3 to 10 8 cfu/ml. eia was performed following the manufacturer's instructions and in duplicates. fifty out of 82 samples tested positive by eia at various dilutions: 33/52 c. upsaliensis, 15/26 c. helveticus, 1/2 of each c. jejuni and c. coli. detection limit varied between the isolates and was lower in watery than semi-solid faeces.in conclusion, eia detected c. upsaliensis and c. helveticus in spiked faeces from human clinical cases and all campylobacter spp. tested in healthy pets. eia should not be used as a sole detection method and culture methods selected following a positive eia result should enable detection of a wide range of campylobacter spp. in addition, eia may be a useful test in pets to rule out campylobacter spp. infection and in epidemiological investigations. high-resolution manometry (hrm) is the gold standard for the evaluation of functional esophageal motility disorders in humans. no information on this non-invasive technique is available in dogs. the aims of this study were to evaluate the feasibility of hrm in dogs, generate first normal values, and to examine the influence of a standard sedation on the esophageal pressure profile.the study population consisted of 20 healthy adult beagle dogs (mean bw 12.85 kg), the study protocol was approved by the local ethics committee. hrm was performed in sitting position after a 6-h fast, lidocaine jelly at 2% was used as a lubricant. a solid-state catheter (sierra scientific, 2.75 mm diameter) with 36 circumferential pressure sensors spaced 1 cm apart was inserted intranasally, measurements were started after 5 minutes adaptation time. real-time pressure imaging during catheter intubation enabled accurate placement. each manometric protocol included 5 water and 5 canned food bolus swallows. the procedure was repeated 30 minutes after standard im sedation with acepromazine and buprenorphine. data were analysed using manoview software and results (awake vs. sedated) were compared using the wilcoxon test. statistical significance was set at p<0.05.hrm could successfully be performed in 12/20 dogs. reasons for unsuccessful examinations were: defence reactions (5), inability to pass through the ventral meatus (2), reverse sneezing (1). upper esophageal sphincter (ues) characterisation comprised: baseline pressure, residual pressure (nadir of the ues relaxation during swallowing), and relaxation duration. tubular esophageal function characterisation comprised: peristaltic con-tractile integral (pci; amplitude x duration x length of the contraction wave) and the bolus transit time (btt). lower esophageal sphincter (les) characterisation comprised: baseline pressure and residual pressure (lowest continuous 3 second mean les pressure relative to intragastric pressure during swallow induced relaxation).the median values for water/food bolus swallows in awake (sedated) dogs were calculated i) ues: baseline pressure 11.5 mmhg (16.1), residual pressure -2.74/2.73 mmhg (-2.03/2.53) and relaxation duration 192.5/245 ms (230.25/324) ii) tubular esophagus function: pci 478.15/ 1012 mmhg-cm-s (525.08/1605.4) and btt 3.7/4.08 ms (4.85/ 5.82) iii) les: baseline pressure 36.85 mmhg (16.8), residual pressure 7.2/5.64 mmhg (10.83/7). significant differences were found for the ues relaxation duration (water) (p=0.023) and btt (food bolus) (p=0.008).in conclusion, hrm is a feasible technique for the evaluation of esophageal function in dogs. patients that require sedation can still be examined, however at this point it is not clear if sedation would affect the assessment of motility disorders in dysphagic dogs. in humans ambulatory intraesophageal ph-monitoring utilizing the bravo ® capsule is the standard test for establishing pathological gastroesophageal reflux (ger). this technique not only provides information on esophageal acid exposure, but is also able to assess symptoms associated with ger. in dogs ger is poorly understood and it is not clear if ger actually represents a clinically relevant problem.the goals of this study were to examine the canine esophageal ph milieu in health and to examine esophageal ph in dogs presenting with signs commonly attributed to ger in the veterinary literature.thirteen client-owned dogs (cod) of various breeds (median bw 20.3kg, 6.1-45; median age 5y, 1-11) were included. clinical signs ultimately leading to ph-monitoring comprised: lipsmacking (6), repeated swallowing motions (3), chronic vomiting (3), cough (3), retching (2), regurgitation (2), sudden discomfort (2), excessive surface-licking (2), ptyalism (2), presumed postprandial pain (2), refusal to eat despite interest (2), history of esophageal foreign bodies (1), halitosis (1) . each dog showed a median of 3 (1-4) signs, 3 dogs had additional diarrhea. six healthy beagles (median bw 13.9kg, median age 1.5y) with unremarkable gastroduodenal evaluation served as controls (c). no prior antacid or prokinetic treatment was allowed. the capsule was endoscopically placed 4 cm above the lower esophageal sphincter, ph data were transmitted every 4s to a receiver attached to the dog's collar. owners were instructed to press the individually predefined symptom-buttons on the receiver whenever indicated, and not to change the daily routine. data were analysed using the rapidph ® software, reflux was defined as a single ph-measurement < 4. results between groups were compared using non-parametric tests.the median ph-monitoring period (cod/c) was 47.49/ 43.17h. the following parameters (median, range for cod/c) were evaluated: number of refluxes: 12 (1-86)/13.5 (1-65), number of longest (> 5min) reflux: 1 (0-14)/1 (0-4), duration of longest reflux (min): 7 (1-18)/6.5 , and fraction time ph < 4 (%): 0.5 (0.01-5.6)/0.5 (0-3.2). there were no differences between groups. the median number of button pushes was 6 (0-35), 3 dogs had reflux-positive pushes (2.8, 11, and 17.6% of pushes). mild distal esophagitis was noted in 1 dog. final diagnoses were: food-responsive ibd (6), steroid-responsive ibd (2), allergic skin disease (2), chronic laryngotracheobronchitis (2), muscular dystrophy (1) .dogs presenting with historical and clinical signs interpreted as ger may not have relevant reflux episodes. considering normal values established in humans, none of the dogs would have been classified as abnormal. the aim of the present study was to evaluate the changes of some biochemical and ultrasonographic (us) parameters in a group of dogs with naturally occurring acute pancreatitis (ap) during the therapeutic follow-up.dogs with clinical signs and abdominal us findings suggestive of ap associated with increased serum canine pancreatic lipase (cpl) activity were included into the study. in these dogs, the serum concentration of c-reactive protein (crp), amylase and lipase were also measured. severity indexes were established to semi-quantitatively evaluate the severity of clinical and us findings. in particular, a clinical score (0-3) for each of the following clinical parameters was given: presence and frequency of vomiting, appetite and general condition; an us score (0=normal, 1=abnormal) was assigned per each of the following parameters: pancreas (echogenicity, volume, echotexture and echogenicity of the mesentery), gastrointestinal tract, biliary ducts, lymph nodes and abdominal effusion (total score 0-14). all dogs were treated with fluid therapy, ampicillin-sulbactam, 15mg/kg iv q8h, buprenorphine, 0.01mg/kg q8h, and, if needed, maropitant 1mg/kg sc q24h. the two severity scores, serum crp, amylase and lipase concentrations were measured at diagnosis (t0) and after 1 (t1), 3 (t3), and 5 (t5) days, and at discharge (td) and 1 week after discharge (td1).nine client-owned dogs were included with a median (range) age of 10 years (8-14 years). median (range) clinical and us scores were 8 (6-9) and 8.5 (3-13), respectively, at t0, and 0 (0-1) and 1 (0-3), respectively, at td1. a significant, positive correlation was found between the clinical and us score (p<0.001, r=0.68). the median (range) serum concentration of crp (mg/dl), amylase (u/l) and lipase (u/l) was 9.46 (3.36-30.4), 2,995 (776-6,458) and 889 270) , respectively, at t0, and 0.64 (0.01-3.07), 704 (563-1,002) and 302 (94-566), respectively, at td1. on admission, serum crp, amylase and lipase levels were increased in 100%, 77%, and 55% of dogs, respectively while they were increased in 50%, 12.5% and 0% of dogs, respectively, at td1. serum crp and amylase, but not lipase, concentrations decreased during the follow up and were significantly (p< 0.05) lower at td1 compared to t0.results suggest that us findings, and crp and amylase concentrations are correlated with the recovery from the ap. further studies are warranted to evaluate the usefulness of these parameters in the follow-up of ap in a wider population of dogs. enzyme replacement therapy is the mainstay therapy for exocrine pancreatic insufficiency (epi) in dogs. 'enteric-coated' preparations have been developed to protect the enzyme from degradation in the stomach, but their efficacy has not been critically evaluated. the hypothesis of the current study was that enteric coating would have no effect on the efficacy of pancreatic enzyme supplements for dogs with epi.thirty-eight client-owned dogs with naturally occurring epi were included in this multicentre, blinded, randomised controlled trial. dogs received either an enteric-coated enzyme preparation (test group) or an identical preparation without the enteric coating (control group) over a period of 56 days.there were no significant differences in baseline characteristics between test and control treatment groups. body weight and body condition score increased in both groups during the trial (p<0.001) but the magnitude of increase was greater for the test treatment compared with the control treatment (p<0.001). by day 56, mean body weight increase was 17% (95% confidence interval 11-23%) in the treatment group and 9% (95% confidence interval 4-15%) in the control group. the dose of enzyme used increased over time (p<0.001) but there was no significant treatment group difference at any time point (p=0.225). clinical disease severity score decreased significantly over time for both groups (p=0.011) and no significant difference was noted between groups (p=0.869). no significant adverse effects were reported, for either treatment, for the duration of the trial.adding an enteric coating to a pancreatic enzyme supplement conveys a therapeutic advantage, when treating dogs with epi. when liver injury is caused by toxins that inhibit hepatocyte replication, or when the proliferative potential of hepatocytes is exhausted due to the chronic condition of the disease, hepatocyte-dependent liver regeneration is strongly impaired. in this situation another cell compartment is activated to regenerate the liver: the hepatic progenitor cell compartment. bipotent hepatic progenitor cells (hpcs) can differentiate into hepatocytes or cholangiocytes. not only are hpcs activated during severe liver disease, they may well be the cells of origin for subtypes of hepatocellular carcinoma (hcc).the polycomb group protein bmi1 is involved in murine hpc activation and has prognostic relevance in hcc. therefore we investigated the expression of bmi1 in canine liver diseases including hcc. functional consequences of deregulated bmi1 expression are included to reveal its feasibility in liver regenerative medicine.immunohistochemistry (ihc) and laser microdissection followed by gene expression analysis were used to investigate the expression of bmi1 in activated hpcs and hcc. to elucidate the role of bmi1 in hpcs, in vitro gene silencing experiments followed by gene expression analysis, western blot analysis, and a proliferation assay (edu-incorporation) were performed in the human hpc-like cell-line heparg.ihc and gene expression showed a strong nuclear expression of bmi1 in activated hpcs in canine liver diseases. keratin 19 is a marker for hpcs and cholangiocytes, and high expression is associated with poor prognosis in hccs. bmi1 staining was more intense in highly malignant hepatocellular carcinomas positive for keratin 19 compared to keratin 19 negative hccs. bmi1-silencing in vitro studies revealed a significantly reduced hpc proliferation and suggested a role of bmi1 in differentiation (gene expression) of hpcs.these results indicate that bmi1 is expressed in activated hpcs in all liver diseases tested. bmi1 is needed for proliferation of hpcs in vitro and is potentially involved in hpc differentiation. expression of bmi1 in hccs suggests a role in tumour development, potentially due to a persistent activation of hpcs. therefore, enhanced activation of bmi1 will lead to more progenitor cells (beneficial) but most likely also to more aggressive hcc with a hpc-signature. hepatic progenitor cells (hpc) are bipotential, forming cholangiocytes or hepatocytes. as a result, they express markers of both cell types. various markers have been used to select for hpc's yet there is no specific cell marker. cd133, a transmembrane glycoprotein, has been identified as a organ stem cell marker and cancer stem cell marker, and used to select hpc's in rodents and humans. canine hpc have been shown to have gene expression of cd133. this study aimed to assess if cd133 selection could enrich for canine hpcs.a wedge of liver from a healthy dog euthanased for behavioural reasons was digested by collagenase perfusion. hepatocytes were removed by pelleting at 50g for 5 mins. the remaining non-parenchymal cells (npc) were then pelleted at 200g for 5 mins. magnetic assisted cell sorting (macs) for cells positive and negative for cd133 was performed according to the manufacturer's instructions. cd133 positive and negative npc were cultured on bovine type i collagen-coated plates with hepatocyte culture media (lonza). cell morphology was monitored. rna was extracted and cdna produced from cell pellets immediately after separation and also after 3 weeks of culture. relative gene expression of cd133, cholangiocyte marker keratin 7 (k7) and hepatocytes markers k18, albumin and cyp1a1 was performed using b2mg and rpl8 as reference genes.the cd133 positive fraction was 38% of the npcs. morphologically, cd133 positive cells formed more colonies of cells with a small cytoplasm to nucleus ratio, which then transformed to hepatoblastic appearance while the negative cells produced more fibroblastic cells. compared to cd133 negative cells, initially cd133 positive cells showed a 2-fold increase in cd133, and 2.5-fold increase in cholangiocyte marker k7 as well as modest increases in hepatocyte markers albumin and cyp1a1 and a modest decrease in k18. after 3 weeks culture, positive cells expressed more albumin, cyp1a1, k7 & k18 compared to cd133 negative cultures. cd133 expression had reduced to a 1fold increase after culture.the cd133 positive fraction expressed cholangiocyte and hepatocytes markers compared to negative cells initially after sorting. after 3 weeks culture, cd133 positive cells formed hepatocyte-like colonies, as well as continuing to have greater hepatocyte and cholangiocyte gene expression. cd133 expression decreased, consistent with differentiation.a large percentage of the npc were positive for cd133. cd133 sorting also labels haematopoietic and mesenchymal stem cells therefore subsequently using facs for other markers may further enrich for specifically the hpc compartment. until now the hepatic neoplasms in dogs are classified as hepatocellular adenomas and carcinomas, cholangiocellular adenomas and carcinomas, mixed hepato-and cholangiocellular carcinomas and hepatic carcinoids. over the past decade, many advances have been made in the characterization of primary liver tumours in man. this knowledge has resulted in a proposal for a new morphological and immunohistochemical classification of primary liver tumours which facilitates the diagnosis and categorization of these tumours including their aggressiveness and prognosis. the purpose of this study was to investigate the presence and relative incidence of the various morphological types of primary hepatic neoplasms in the dog and to determine whether the new human classification also can be applied to canine hepatic neoplasms. for this study 93 canine primary liver tumours were examined histologically and classified using several immunohistochemical markers including keratin(k)19 (hepatic progenitor (stem) cell/bile duct epithelium marker), heppar-1 (hepatocyte marker), ema (muc1; mucine producing biliary epithelium marker), pcea (canalicular, ductular, and bile duct epithelium marker), nse and chromogranin-a (neuro-endocrine markers). in addition, the tumours were graded according to cellular and nuclear pleiomorphism and mitotic index (grade:0-3) and staged with respect to absence or presence of invasive growth, intrahepatic and/or distant metastases (stage:0-2). of the 93 primary liver tumours, 80 had a hepatocellular origin (86%). these hepatocellular tumours could be subdivided in hepatocellular tumours with <10% positivity for k19 (65%) and tumours with >10% positivity for k19 (22%). the hepatocellular tumours with >10% positivity for k19 were histologically poorly differentiated and often revealed lymphatic and vascular invasion in portal tracts and all showed intrahepatic and/or distant metastasis. in contrast the hepatocellular tumours with <10% positivity for k19 were almost always well differentiated and well demarcated and did not have evidence for vascular invasion, intrahepatic and/or distant metastasis. eight of the 93 tumours were from cholangiocellular origin (9%). all of the cholangiocellular tumours were poorly differentiated (grade 2 and 3) and showed intrahepatic and/or distant metastases. the third group of primary liver tumours had neuroendocrine characteristics, consisted of five tumours (5%) and were classified as carcinoids. they all were histologically poorly differentiated and had intrahepatic and/or distant metastases. in conclusion, the morphological types of the primary hepatic neoplasm in the dog, including their aggressiveness and prognosis, are highly comparable to the situation in man. these finding indicate that the new human classification of primary hepatic neoplasms is applicable in the dog. doberman hepatitis (dh) is a rare inflammatory liver disease characterized by female preponderance, elevated serum transaminase activity and increased hepatic copper content. immune system involvement is suggested by the presence of lymphocyte infiltration, female predisposition, abnormal expression of major histocompatibility complex (mhc) class ii antigens by hepatocytes and association of homozygosity for the mhc ii risk allele drb1*00601 of the dog leukocyte antigen system genotype.we investigated the possibility that autoantibodies are involved in dh. serum samples from 25 subclinical and 13 clinical dh patients and 17 clinically healthy control dobermans were included in an elisa assay for detection of igg autoantibodies against histones (aha). the cut-off value for positivity in the anti-histone elisa was 0.87, determined using the mean absorbance +2 sd of samples from healthy controls. the values for subclinical dh cases (mean ± sd:1.36 ± 0.6; 95% confidence interval (ci) 1.11 to 1.61) and clinical dh cases (mean ± sd:1.46 ± 0.49; 95%ci 1.17 to 1.76) were both significantly higher than values for controls (mean ± sd:0.51 ± 0.18; 95%ci 0.42 to 0.6; p<0.05). no seropositivity was noted in the control group.autoantibodies are the serological hallmark of autoimmune disease. normally, the immune system has an extraordinary capacity for preventing self-antigens to stimulate an inflammatory reaction. the presence of autoantibodies is therefore the consequence of a breakdown or failure of b-cell tolerance toward the corresponding autoantigens. since the appearance of aha indicates possible presence of autoimmunity, our results support the assumption that dh is an autoimmune disease. man's best friend (canis lupus familiaris) is an ideal model organism for a broad variety of naturally occurring diseases. dog breeds have a population structure suited for genetic studies of complex disorders. haploblocks within dog breeds extend up to100 times longer distances than in human populations. related dog breeds often share phenotypes and causative mutations, allowing for finemapping and validation. the inbreeding and bot-tlenecks of dog populations has led to an increased incidence of genetic diseases which remained incidental in the panmictic human populations.extrahepatic portosystemic shunts (ehpss) are large abnormal venous blood vessels connecting the portal vein with a major systemic vein. it results in almost complete diversion of the portal blood past the liver, leading to lack of liver function and liver growth, and hepatic encephalopathy due to brain neurotransmitter dysfunctions. there are only 173 reported human cases, whereas the disease is widespread in dogs. shunts are more frequently diagnosed in purebred dogs than in crossbred dogs. the inheritance is complex without sex effect.a genome wide association studied with 50k snps was peformed on 48 cairn terrier cases and 48 controls of the same breed. after quality control 42 cases and 39 controls were analyzed in 47702 snps. allelic association was calculated with plink and grammas of the genabel package was used to correct for genetic kinship. several chromosome regions were detected with close to significant association. overlap between the two analysis methods was found on three genomic regions covering about 8.4 mb. finemapping was conducted by analyzing more snps in a larger group of cairn terriers and in cases and controls of 8 other breeds. the finemapping resulted in two genomic regions covering 300 and 997 kb that were associated with significant pvalues. comparison of haplotypes of cases from different breeds confirmed the association signals. the regions of interest are analyzed by ngs of 48 cases and 48 controls. historically, doxycycline has been the first line drug for the treatment of canine monocytic ehrlichiosis (cme). some studies have shown that dogs may remain carriers despite doxycyxline treatment, therefore investigation for other anti-ehrlichial agents may be warranted. rifampin was suggested as a promising alternative for the treatment of cme, though its efficacy in clearing the infection has not been thoroughly evaluated. the purpose of this study was to assess the efficacy of rifampin in achieving clinicopathological recovery and clearing the ehrlichial infection from the blood and other tissues in dogs with experimentally-induced acute cme. of the 16 purpose-bred beagle dogs that were included in the study, 5 dogs with acute experimental cme were treated with rifampin (10 mg/kg, sid, po, for 3 weeks), 9 infected dogs received no rifampin (infected controls) and two dogs served as uninfected controls. fourteen days after the completion of rifampin treatment, dexamethazone (0.5 mg/kg, iv, once) was given to the 5 rifampin-treated dogs. clinical score, platelet counts, anti-e. canis immunofluorsecent antibody titers (ifa) and polymerase chain reaction (pcr) detection of e. canis-specific deoxyribonucleic acid in the blood, bone marrow and spleen aspirates were evaluated between the treated and untreated infected dogs on day 21 post-inoculation (pi) (start of rifampin), on day 42 pi (end of rifampin) and day 98 pi (end of post-treatment monitoring). by day 21 pi, all infected dogs became clinically ill and thrombocytopenic, seroconverted and became pcr-positive in at least one tissue. the median clinical score and ifa titers did not differ between the treated and untreated dogs at any of the three time points. μedian platelet counts were significantly higher in the treated compared to the untreated infected dogs on day 42 pi (251,000/ll versus 168,000, p=0.0233) and day 98 pi (254,000/ll versus 205,000/ll, p=0.0195) (two-sample wilcoxon rank-sum [mann-whitney] test). on day 42 pi, 2 treated and 2 untreated infected dogs were pcr-positive, while on day 98 pi, 3 treated and 6 untreated infected dogs remained pcr positive in at least one of the tissues tested. as administered in this study, rifampin hastened hematological recovery of the infected dogs, but was inconsistent in clearing the experimentally-induced acute e. canis infection. acute phase proteins (app) are considered one of the hallmarks of the inflammatory response. among their major functions, apps seem to modulate innate immune system efficiency. in cats, serum amyloid a (saa) and a1glycoprotein (agp) are two major positive apps that are increased during inflammation. this rise is presumed to be secondary to various cytokines that are involved in the innate inflammatory response.recombinant feline interferon-x (rfeifn-x) is an immune-modulator drug that is commonly used in cats naturally infected with retroviruses, namely feline immunodeficiency virus (fiv) and feline leukemia virus (felv). several studies have been performed to clarify the clinical benefits of rfeifn-x therapy in naturally infected fiv and/or felv cats. our group has previously described that c-reactive protein (crp) increased in naturally retroviralinfected cats under rfeifn-x therapy. however, the role of apps such as saa, agp and crp in the innate immuneresponse, remains unknown.the aim of this study was to evaluate saa, agp and crp serum levels in naturally retroviralinfected cats under rfeifn-x therapy.sixteen naturally retroviral infected cats (7 fiv, 6 felv and 3 co-infected fiv/felv stray cats) housed in a lisbonanimal rescue shelter were submitted to rfeifn-x therapy. the licensed protocol was used: 3 courses of 1mu/kg sc administered once daily for 5 days, beginning on days 0, 14 and 60. blood samples were collected for saa, agp and crp quantification before, during and after treatment (at d0, 10, 30, 65) . saa was quantified by elisa (phase saa, tridelta) and agp was determined by single radial immunodifusion (agp,tridelta). feline crp was quantified by elisa (kamiya biomedical company).app serum levels were compared before and after rfeifn-x therapy. a statistically significant increase of saa and agp (p=0.0005 and p=0.012 respectively -friedman test) was observed at d65 in comparison to d0. these findings corroborate the significant increase of crp serum levels previously described (p <0.0001-friedman test).all the apps tested behaved similarly, showing an evident increase in their serum values after rfeifn-x therapy. these results suggest a possible immune modulation effect induced by rfeifn-x which seems to maximize the efficiency of innate immune response. further studies correlating these findings with the cytokine profile will extend our knowledge about the efficiency of rfeifn-x therapy in naturally retroviral infected cats. canine theileriosis is a tick-born disease caused by protozoan parasites of the genera theileria that has been associated with anaemia and/or thrombocytopaenia. the clinical manifestation of this disease in the dog is poorly described. this disease of emerging importance has been diagnosed in several dogs, emphasizing the need to elucidate the specific pathogenic species and characterize the clinical manifestations of the disease.this retrospective study describes the clinical characteristics, diagnostic tests, treatment and outcome of six client owned clinically ill dogs diagnosed with canine theileriosis at the onderstepoort veterinary academic hospital in south africa during 2010-2011. canine theileriosis was diagnosed by polymerase chain reaction (pcr) on whole-blood followed by a reverse line blot (rlb) hybridization assay. other tickborn diseases and neoplastic conditions were excluded.the most common clinical findings were pale mucous membranes (5/6 dogs), lethargy (3/6 dogs) and oral bleeding (3/6 dogs). all dogs had thrombocytopaenia with a median of 59.5 x 10 9 /l (range: 13 -199) and 5/6 dogs had anaemia with a median haematocrit of 18% (range: 5 -32). the anaemia was regenerative in 3/5 dogs and non-regenerative in 2/5 dogs. one dog was positive on an in saline auto-agglutination test. bone marrow cytology and core biopsies were performed in two dogs with severe non-regenerative anaemia and showed myelofibrosis. theileriasp. were detected in four dogs and theileria equi in two dogs. imidocarb dipropionate was administered in all dogs as treatment of choice for the theileriosis. five dogs that received the complete treatment achieved clinical cure. pcr post-treatment was performed in three dogs and was negative. prednisolone and azathioprine was administered in all dogs for suspected immune-mediated haematological disorders secondary to the theileriosis. one dog was euthanased one week after diagnosis.canine theileriosis should be considered a differential diagnosis for dogs with thrombocytopaenia and/or anaemia in endemic tick-born disease areas. pcr is a versatile tool for diagnosis and treatment monitoring in theileriosis. in our study, imidocarb dipropionate was effective in sterilizing the parasitic infection. the bleeding tendency seen in the theileriosis cases is most likely secondary to the thrombocytopaenia and/or concurrent thrombocytopathy as described in canine ehrlichiosis. further studies are required to determine the possible links between thrombocytopaenia, anaemia and myeloproliferative disorder observed in canine theileriosis. a.l. proksch 1 , s. unterer 1 , u. truyen 2 , r.s. mueller 1 , k. hartmann 1 . 1 clinic of small animal medicine, lmu university of munich, munich, germany, 2 institute for animal hygiene and veterinary public health, university of leipzig, leipzig, germany canine parvovirosis still is a common and severe disease, especially in puppies, and there is a need for drugs that can decrease severity of symptoms and accelerate recovery. the paramunity inducer pind-orf stimulates the immune system. therefore, the aim of the study was to investigate whether pind-orf, used as an additional drug, leads to faster recovery in dogs with parvovirosis.in total, 40 dogs with parvovirosis were randomly assigned to two groups (20 dogs each). in all dogs, infection with canine parvovirus was diagnosed by fecal elisa or polymerase chain reaction (pcr). also, all dogs had clinical signs. the study was performed as prospective placebo-controlled, double-blinded trial. all dogs received either pind-orf or placebo, and additional standardized treatment for canine parvovirosis. clinical scores, complete blood count, and serum protein and albumin were evaluated daily (day 0 -7) and at day 14. viral shedding was measured on day 0, 3, 7, and 14 by fecal pcr and virus isolation.no significant difference could be found in clinical scores, most blood parameters, and duration of virus shedding when comparing dogs receiving pind-orf and dogs receiving placebo. the only significant difference was an increase in lymphocyte counts observed in the pind-orf group. three dogs receiving placebo did not survive, but no significant difference between groups was determined concerning survival rate.in this study, no significant influence of the paramunity inducer pind-orf on the course of parvovirosis was determined. therefore, there is no indication to recommend pind-orf therapy in canine parvovirosis. the microscopic agglutination test (mat) is currently considered as the gold standard for the diagnosis of leptospirosis in dogs. however, it is not a perfect tool to predict the infecting serogroup as cross-reactions and paradoxical reactions do exist. the objective of this study was to determine the infecting serovar in 29 dogs with a positive polymerase chain reaction (pcr) on one or more biological sample(s) by molecular typing and to compare the results with those provided by the highest mat titer for each dog.forty-one positive pcr biological samples (20 urine samples, 19 blood samples, one kidney sample and one cerebrospinal fluid -csf-sample) from 29 dogs with a clinical suspicion of leptospirosis between 2008 and 2011 and at least one positive pcr on blood, urine, kidney or csf (kit taqvet pathogenic leptospira, lsi, france) were submitted to molecular typing. the genomospecies were first determined by partial rpob or partial 16s rrna gene sequencing. the serovar was further identified by multiple loci variable number tandem repeat analysis (mlva). three variable number tandem repeat (vntr) loci were used as markers for serovar identification.the genomospecies were leptospira interrogans sensu stricto in 36 samples belonging to 25 dogs, leptospira borgpetersenii in three samples (three dogs) and leptospira kirschneri in two samples (two dogs). interestingly, for one dog, we found a different species in the blood and in the urine. mlva could unequivocally determine infecting serovar in 14 samples belonging to 13 dogs: australis in one dog, muenchen in three dogs, fugis in one dog, canicola in two dogs, autumnalis in six dogs. for only two dogs among those 13, the infecting serovar identified by mlva belonged to the infecting serogroup identified by mat. for 16 samples belonging to 15 dogs, mlva failed to identify the infecting serovar because one or more marker(s) could not be amplified. for 11 samples belonging to 9 dogs, amplification profile suggested coinfection or infection with a serovar that had not been previously characterized by mlva. it must be noted that for some dogs, we were able to characterize the serovar in a single kind of sample.despite lack of sensitivity of mlva applied to clinical strains in our study, this rapid method could contribute to a better knowledge of the epidemiology of canine leptospirosis with some adaptations in the choice of markers. recently, two trichomonads have been identified in diarrheal stool of puppies: pentatrichomonashominis (ph) and tritrichomonas foetus (tf) [1] . tf infection in cats results in a chronic colitis. however in dogs, pathogenicity of tf and ph has never been studied. so the objective of this study was to evaluate the association between trichomonads' infection in puppies and the presence of diarrhea.faecal samples were collected prospectively from 273 puppies (4 to 13 weeks of age) in 3 french breeding kennels. for each puppy, a rectal swab was performed and the fecal consistency was evaluated using a 15-point numerical scale (1 = liquid feces, 15 = hard feces) [2]. detection of trichomonads was performed by using a commercially available system "in pouchtm tf test"(biomed diagnostics, oregon usa). evaluation of this media was done as already described [3] . observation of motile trichomonads organisms in the system culture was considered as a positive result. nine positive culture systems, from one kennel, were frozen and single-tube nested pcr assays were performed on them in order to sequence and identify the trichomonads.29.7% (81/273) of the cultures were positive. 32.6% of the puppies (89/273) had gastrointestinal troubles. puppies infected by trichomonads had significantly more digestive problems than puppies not infected (49.4% vs 25.5%; p < 0.05). ph was systematically isolated in the 9 positives cultures.ph was the only trichomonads isolated in the nine culture system tested by single-tube nested pcr assay. these results show the poor specificity of the medium to distinguish tf from ph. this observation underlines the necessity to use pcr for precise identification of type of trichomonads. trichomonads are significantly associated to the presence of digestive disorders in puppies. some etiological studies on the subject (by considering the co-infection with fly snapping, fly-biting or jaw snapping are names given to a syndrome in which dogs appear to be watching something then suddenly leaping and snapping at it. fly-biting dogs are generally referred to neurologists or behaviourists because the abnormalities are often interpreted as focal seizures or as obsessive compulsive disorder (ocd). there is one published case report of fly biting presumably caused by dietary intolerance in a cavalier king charles spaniel.the aims of this case series were 1) to characterize fly biting, 2) perform a complete medical evaluation of dogs presented with fly biting, and 3) evaluate the outcome of this behaviour following appropriate treatment of the underlying medical condition.seven dogs presented for fly-biting behaviour (fb) were assessed. all dogs underwent a complete medical and behavioural history as well as physical and neurological examinations. further investigation was performed if an abnormality was found on examination or if the history was suggestive of an underlying problem. based on clinical presentation, physical examination, neurologic examination, and laboratory test results, a diagnosis was made and a specific treatment recommended. response to treatment was monitored and evaluated following phone conversations with owners at day 30, 60 and 90 from onset of treatment. many gastrointestinal disorders were found in fb dogs which included eosinophilic and lymphoplasmacytic infiltration of the stomach and small bowel, delayed gastric emptying and gastroeosophageal reflux. complete resolution of the fb was observed in 5/6 dogs diagnosed and specifically treated for the underlying gastrointestinal (gi) disease. one dog was diagnosed with chiari malformation and responded temporarily to pain management.in conclusion, this prospective case series indicates that fly biting behaviour may be caused by an underlying medical disorder, gi disease being the most common. resolution of this behaviour is possible following specific treatment of the underlying medical condition. although several diagnostic tests have been developed to diagnose fip, there are still difficulties to differentiate between fip and diseases with similar clinical appearence in vivo. fip is an inflammatory disease, therefore, as in other inflammatory conditions, the concentrations of apps are expected to be increased. the aim of this study was to evaluate the ability of the apps to distinguish fip from other diseases.serum samples from 88 cats presented with effusion were obtained for measurement of three apps. the diagnostic work-up was performed with respect to fip (jvim 2011; 25: 1505) and further diagnostic procedures were performed depending on the medical condition. 20 cats were diagnosed as having fip and 68 cats had another disease (cardiac 22, tumor 24, other 22). serum amyloid a (saa) and haptoglobin (hp) were measured by automated analyser using feline validated assays (eiken and tridelta, respectively), alpha-1-acid glycoprotein (agp) was measured using a manual method based on single radial immunodiffusion (tridelta).the median concentrations of saa, hp and agp were significantly (p<0.001) higher in cats with fip compared to cats without fip (saa: 98.5; range 1.3-163.4 lg/ml; 7.6; range 0.1-163.8 lg/ml,), (hp: 2.0; range 2.0-9.0 mg/ml; 1.8; range 0.0-2.0 mg/ml,), (agp: 2900; range 960-5040 lg/ml; 690, range 120-4500 lg/ml).all major feline apps seem to be useful diagnostic tools to help in differentiating fip from other diseases. however, the concentration of apps in some diseases (septic processes, disseminated neoplasias) was as high as in fip. protothecosis is an uncommon disease of people and animals caused by prototheca spp., an unicellular aerobic algae. to date, about 30 cases have been described in dogs in north america and australia. in europe, only 4 cases have been documented in the last 2 decades (poland, italy, greece and spain). affected dogs show signs referable to the gastro-intestinal tract, particularly the colon, but ocular and neurologic signs are also reported. the disease has an insidious onset, a slow progression and fatal course. the aim of the present study is to describe clinical and laboratory findings in 4 cases of canine protothecosis from the north of italy, diagnosed between 2009-2011. medical records were retrieved and information pertaining history, clinical and instrumental data, as well as follow-up, were collected.the median age of the 4 dogs was 8 years (range: 5-11), 3 of them were female and 2 were boxers. major complaints were chronic large bowel diarrhea with hematochezia and weight loss observed since a median time of 3 months (range: 1 to 7). previous treatment with gastrointestinal diets, antiparasitic drugs and antibiotics yielded no improvement. additionally, 2 dogs developed uveitis during the disease course. in all dogs a complete blood count, a serum biochemical profile, including protein electrophoresis, and abdominal ultrasound were performed; serum ctli, folate and cobalamin were available in 3 dogs and urinalysis in one. the results of the above laboratory tests were normal. ultrasonography was unremarkable in 2 dogs and showed increased colon wall thickness in the other 2. definitive diagnosis was obtained from endoscopic biopsies of the colon and/or rectal scrapings in 3 dogs and from biopsies of the colon at necropsy in one. in each case spheroid, ovoid or irregularly-shaped organisms suggestive of prototheca spp. were observed. different treatments were attempted without benefit in 3 dogs. in one dog transient improvement was obtained with itraconazole. the median survival time was 5 months (range: 2-12).the present work indicates that protothecosis should be included in the list of differential diagnosis in dogs with large bowel diarrhea, especially in those with chronic refractory colitis or developing ocular signs. dogs infected with prototheca spp. have a guarded prognosis. diagnosing protothecosis in 4 dogs over a 2-year period may suggest that the disease is emerging in some southern european countries. idiopathic pulmonary fibrosis (ipf) is an interstitial fibrotic pulmonary disease, mainly described in the west highland white terrier (whwt). the diagnosis is challenging and ultimately relies on lung histopathology. identification of biomarkers specific for the disease would be very helpful. ccl2 (mcp-1) is a chemotactic cytokine for monocytes. it is a known biomarker in human ipf. in dogs with ipf, increased ccl2 gene expression has been described in lung tissue. the aim of the present study was to compare serum ccl2 concentration in dogs with ipf versus healthy dogs and dogs with other chronic pulmonary diseases.thirteen dogs with ipf (ten whwts, two scottish terriers, one yorkshire) mean age 12 years, range 8-15), nine dogs with eosinophilic bronchopneumopathy (ebp) (various breeds, 5 years, 1-12), ten dogs with chronic bronchitis (cb) (various breeds, 9 years, 1-13) and ten healthy whwts (9 years, 3-14) entered the study. diagnosis was established after clinical, radiographical, bronchoscopic (and balf analysis) examinations, as well as, in dogs with ipf, either lung high resolution computed tomography (six dogs) or histopathology (three dogs) or both (four dogs). ccl2 concentration in serum was determined by elisa (canine cll2/mcp-1quantikine ® , r&d sytems). results in the different groups were then compared using non-parametric test (mann-whitney rank sum test).serum ccl2 concentration was elevated in dogs with ipf (median; interquartile range = 528.8 pg/ml; 444.7-692.0) compared to healthy whwts (344.0; 254.5-415.5), (p<0.001). serum ccl2 value in ipf dogs was higher than in ebp dogs (281.6; 163.9-416.5) (p=0.009) and than in cb dogs (277.7; 137.3-364.7) (p=0.003).the present study shows that (1) idiopathic pulmonary fibrosis (ipf) is a progressive interstitial fibrotic disease, described in humans and in dogs. etiology and pathogenesis of ipf are poorly known in both species, even if a genetic basis is suspected in dogs because of the predisposition of the west highland white terrier (whwt). serum transforming growth factor beta 1 (tgfb1) concentration is elevated in both healthy whwts and whwts with ipf, as compared to healthy dogs of various breeds. in human ipf, pathways involving tgfb1, a cytokine with profibrotic properties, seem to be central in the pathogenesis and are considered as potential therapeutic targets. tgfb1 is produced as a pro-protein and usually stored as a latent complex. activation of the latent complex is an important step that regulates tgfb1 function. multiple activation mechanisms have been identified including binding to integrins and thrombospondin 1 (thbs1).the aim of the present study was to quantify tgfb1 expression, as well as expression of proteins involved in tgfb1 activation, by quantitative rt-pcr, in lung tissue from dogs with ipf versus control dogs.total rna was extracted from lung tissues from 14 dogs with ipf (12 whwts, 1 scottish terrier, 1 lhassa apso) and 11 control dogs (various breeds). ipf was confirmed by histopathology on all samples. expression of tgfb1, 2 integrins (itgb6 and itgb8) and thbs1 was measured by qrt-pcr. for each gene, a relative copy number was calculated for each sample and results were normalised using two stably expressed housekeeper genes (rps18 and tbp). statistically significant differences between the groups were assessed using a student t-test or a mann-whitney rank sum test with significance defined as a p < 0.05.expression of tgfb1 and itgb6 was not statistically different between the two groups. expression of itgb8 was significantly lower (p<0.001) while thbs1 expression was significantly higher (p=0.016) in the ipf group relative to controls.results of the present study could not confirm that increased gene expression of tgfb1 by lung tissue is the source of the high circulating tgfb1 level in ipf. this study highlights different activating pathways of tgfb1 in ipf lungs compared to control lungs with a shift toward an increased activation via thbs1 in canine ipf. obesity is the most common nutritional problem in dogs, and its detrimental effect on basal lung function parameters has been recently shown using whole-body barometric plethysmography. the 6-minute walk test (6mwt) has been recently demonstrated to be a non-invasive easy-to-perform test in clinical settings, able to discriminate between healthy dogs and dogs with pulmonary disease.the aim of this study was to investigate the effect of body weight loss (bwl) on pulmonary function assessed by 6mwt and arterial blood gas values.six experimental beagles and 6 privately-owned dogs, all obese but otherwise healthy, were enrolled in a diet-induced bwl program. physical examination, bw and body condition score (bcs) assessment, arterial blood gas analysis and 6mwt were performed when dogs were obese (bcs 8-9/9), and repeated with animals in the middle of their bwl program (overweight, bcs 6-7/9) and at the end of it (lean, bcs 5/9). for the 6mwt, dogs were walked for 6 minutes, along an inside 53m-long hallway. heart rate (hr) and oxygen saturation (spo2) were measured by pulse oximetry before the test (pre-test value), after three minutes of walk (mid-test value) and at 0, 1, 2, 3 and 5 minutes post-test.all dogs concluded the bwl program (initial bw: 27,3±2,9 kg; final bw: 20,85±2,9, means±se, p 0,001). bwl caused a significant increase in the walked distance (lean: 581,8±40,9m; overweight: 582,7±44,4m; obese: 500,4±40,0m; means±se, p 0,05) and a decrease in pretest respiratory rate (rr) (lean: 33±3,3/min; overweight: 40±3,6/ min; obese: 49±4,1/min; means±se, p 0,05). resting arterial blood gas results were not influenced by bwl and neither did the pre-test hr and spo2 values measured by pulse oximetry. obese dogs showed significant higher hr mid-test values compared to overweight and lean dogs (lean: 122,0±7,5/min; overweight: 119,8±7,2/min; obese: 167,2±8,2/min; means±se, p 0,001). moreover, spo2 values recorded at 0 and 1 minute post-test were significantly higher in overweight and lean dogs, compared to obese dogs. also, hr values registered at 1, 2, 3 and 5 minutes post-test were all lower in overweight and lean dogs.in conclusion, obesity negatively affects the blood oxygenation level during and shortly after physical exercise in dogs, with subsequent hr increase. bwl induces a significant decrease in resting rr and it improves pulmonary function during exercise, even before achieving the targeted ideal bw. the 6mwt, but not pre-test arterial blood gas values, is an efficient tool to demonstrate the efficacy of bwl. there are few diagnostic laboratory methods available for evaluation of platelet function and contribution to thrombotic events in the clinical setting. the impedance whole blood platelet aggregometer multiplate ® has recently become available. although it is being marketed for monitoring effect of antiplatelet therapy, it can also be used for assessment of platelet aggregation in response to various agonists, reflecting platelet function, activity and reactivity in response to disease. the purpose of this study was therefore to investigate multiplate ® as a diagnostic tool for detection of variations in platelet aggregation in dogs with diseases known to predispose to hypercoagulability and thrombosis and to evaluate whether there is a correlation between multiplate aggregation response and the maximal amplitude (ma) measured by thromboelastography (teg).twenty clinically healthy dogs and eighteen diseased dogs with neoplasia, generalized inflammation or protein losing enteropathy ornephropathy admitted to the university hospital for companion animals, university of copenhagen, were included in the study. citrated and heparinised blood samples were collected. multiplate ® aggregations were performed on diluted heparinised whole blood for 12 minutes using adp, collagen (col) and arachidonic acid (aa) as agonists and nacl as buffer control. results were recorded as area under the curve (auc). dilute (1:50000) tissue factor teg analyses were performed on citrated whole blood.diseased dogs had significantly increased auc compared to healthy dogs for nacl buffer control (p=0.0005), adp (p<0.0001) and col (p=0.0048) whereas no significant difference was obtained for aa as agonist (p=0.3116). teg-ma was significantly higher (p=0.0114) in diseased dogs compared to healthy dogs. a significant correlation was not found between teg-ma and multiplate auc using adp (p=0.1720, r=-0.3366), col (p=0.2274, r=-0.2994) or aa (p=0.4304, r=-0.1982) .these results demonstrate that multiplate ® aggregation responses are significantly increased in a population of diseased dogs with diseases known to predispose to hypercoagulability and thrombosis, but results are not significantly correlated to teg-ma. this suggests that the multiplate method can be used to detect increased platelet reactivity in dogs with diseases known to predispose for hypercoagulability and thrombosis and that multiplate provides additional information on platelet function than teg alone in this patient group. further studies are needed to determine how multiplate and teg-ma results correlate to thrombosis and whether there may be an added benefit of using them in combination. hypoalbuminaemia is a commonly identified biochemical dyscrasia. the clinical impact of this can be far reaching, particularly in severely affected animals. problems recognised to ensue include altered colloid osmotic pressure and cavity effusions, clotting abnormalities, altered carriage of drugs, hormones and electrolytes, along with acid-base disturbances.the aim of the study was to determine the incidence of side effects encountered when administering 20% intravenous human albumin to dogs suffering with hypoalbuminaemia. animals were presented to a specialist referral centre in hampshire, uk for various conditions resulting in hypoalbuminaemia. hypoalbuminaeamia was considered to be present when the serum albumin concentration was <20g/l. a total of 69 albumin infusions were given to 60 dogs presenting with a serum albumin concentration of 17g/l or lower. albumin was given as an intravenous infusion of 800mg (4ml)/kg following premedication with chlorphenamine. the duration of administration was between 30 minutes and 4 hours. the dogs were monitored for potential adverse reactions during the administration of albumin and in the post-infusion period. potential adverse reactions included hypotension, hyperthermia, tachycardia, tachypnea, peripheral oedema, agitation/restlessness and collapse. none of the animals showed adverse reactions during the administration of albumin or in the post-infusion period. one animal demonstrated mild hyperaesthesia that resolved prior to completion of the infusion.of the animals presented, 39 survived to discharge, 15 animals were euthanased and 6 died. each dog received an average of 1.23 (sd 0.46) albumin infusions during the period of hospitalisation. there was no significant difference in the starting albumin concentrations between the survivors and nonsurvivors. there was no significant difference in the increase in albumin concentration post-infusion between survivors and non-survivors. there was no significant difference in the number of albumin infusions between the survivors and non-survivors. the concentration of albumin pre-infusion did not negatively impact on survival and discharge from the hospital. the results of this study demonstrate no significant complications during or following administration of 20% human albumin solution in dogs. there was no association between the administration of albumin, number of albumin infusions or amount of albumin administered in any patient and outcome. albumin infusion may improve the chances of severely hypoalbuminaemic dogs surviving to be discharged from the hospital. the use of human albumin for this purpose enables the limited supplies of canine plasma to be reserved for dogs needing plasma transfusion for other reasons. recently, metabolic syndrome has gained attention in human medicine given its associations with development of diabetes mellitus and cardiovascular diseases. canine obesity is associated with the development of insulin resistance, altered lipid profiles, and mild hypertension, but the authors are not aware of any existing studies examining the existence of ms in obese dogs. thirty-five obese dogs were assessed before and after weight loss. the guidelines of the international diabetes federation were modified in order to produce a definition for canine metabolic syndrome (cms), which included a measure of adiposity (using a 9-point body condition score [bcs]), systolic blood pressure, plasma cholesterol, plasma triglyceride, plasma glucose, and urine protein:creatinine ratio (upcr). by way of comparison, total and regional body fat mass were assessed by dual-energy x-ray absorptiometry, whilst adiponectin, insulin, and high-sensitivity c-reactive protein (hscrp) were also assessed with validated assays.systolic blood pressure (p=0.008), cholesterol (p=0.003), triglyceride (p=0.018), insulin (p<0.001), and upcr (p=0.034) all decreased after weight loss, whilst plasma adiponectin increased (p=0.001). however, hscrp did not change with weight loss. prior to weight loss, 10 dogs were defined as having cms. there was no difference in total or regional body fat mass between these dogs and those who did not fit the definition of cms. however, plasma adiponectin concentration was less (p=0.004), and plasma insulin concentration was greater (p=0.01) in cms dogs.in this study, up to a third of obese dogs suffer from cms, and this is characterized by hypoadiponectinaemia and hyperinsulinaemia. these studies can form the basis of further investigations to determine pathogenetic mechanisms and the health significance for dogs, in terms of disease associations and outcomes of weight loss. cystourethroscopy has greatly enhanced the diagnosis and treatment of numerous conditions in veterinary medicine. rigid telescopes used in female cytoscopy provide well-illuminated highly detailed images using rod lens system technology. the flexible ureteroscope has been used for male urethrocytoscopy but is limited in image quality and procedural abilities due to the fiberoptic system, diminished illumination, and smaller working channel. the purpose of this study was to describe the technique, efficacy, and complications using a novel percutaneous perineal approach to the male urethra in order to gain access for rigid cystoscopy.the perineal approach was performed ten times in nine dogs for ectopic ureter laser ablation of idiopathic renal hematuria sclerotherapy. the dogs were placed in dorsal recumbency. using fluoroscopic guidance, an 18 gauge renal access needle was advanced trans-perineally into the pelvic urethra. guidewire access was obtained and the access site dilated to accept a 16fr peel-away sheath. a rigid cystoscope was then placed through the sheath to perform the procedures. urinary catheters were placed following three of ten procedures for three to eighteen hours. the only identified peri-operative minor complication included urination form the perineal site approximately six hours post-operatively once in a single dog. no signs of stranguria or pollakiuria or incisional complications were identified in any of the 9 dogs post-operatively at follow-up examination or contact (range 4 to 1248 days).the percutaneous perineal approach in male dogs for rigid cystoscopy appears to be a safe and effective means of facilitating endoscopic procedures. irh results in chronic upper urinary tract bleeding. in humans, ruptured renal pelvic hemangiomas/angiomas are typically the cause. although benign, anemia, ureteral and urethral obstruction(s) can ensue. with the advent of endourology renalsparing therapies like ureteropyeloscopic-guided electrocautery or sclerotherapy has replaced ureteronephrectomy. the objective is to describe the use of endoscopic-fluoroscopic-guided sclerotherapy for the treatment of irh in dogs and report the first clinical outcomes.each uvj was identified cystoscopically. once the bleeding was confirmed a retrograde ureteropyelogram was performed. a ureteropelvic junction balloon was used for ureteral occlusion and pelvis filling volumes were recorded. four dwells were performed (2 5% povidone iodine mixture; and 2 sterile liquid 0.5-1% silver nitrate). a double-pigtail ureteral stent was placed.seven dogs had sclerotherapy. five unilateral, 1 bilateral, and 1 developed contralateral bleeding (n=9 units). five were right and 4 left-sided. there were 6 males and 1 female. the median age and weight was 6 years and 27.5kg, respectively. median procedure time was 150 minutes. there was 1 complication of severe renal discomfort and pyelectasia in an unstented dog.cessation of hematuria occurred in 6/9 renal units (median 12 hours). two had recurrence within 3 weeks; both resorted to intermittent mild hematuria. two failed treatment. median follow-up time was 5 months (range, 1.5-19) .overall, topical sclerotherapy for irh can be safe and effective. this is the first report of local sclerotherapy for irh in dogs and could be considered a valuable endoscopic-guided therapy prior to ureteronephrectomy. further investigation is required. percutaneous nephrolithotomy (pcnl) is considered the standard -of-care for removal of nephroliths >1.5 cm in people, minimizing morbidity and preserving renal function. success rates are reported to be 90-100%. most veterinary nephroliths remain clinically silent and removal is only recommended for complicated stones. morbidity of nephrotomy can be severe. the objective is to describe endoscopic-guided nephrolithotomy (enl) in canine and feline patients and report clinical outcomes, hypothesizing it is safe and effective. patients that had either pcnl or surgicallyassisted endoscopic nephrolithotomy (senl) were retrospectively evaluated. a renal puncture needle and balloon-dilation-sheath combination was used for tract formation. a nephroscope provided visualization for intracorporeal lithotripsy. stone fragments were removed and a ureteral stent was placed. nine dogs and 1 cat (12 renal units) were included. four had pcnl and 6 senl. indications included recurrent utis (4), worsening azotemia (4), and ureteral-outflow obstructions (2). median weight was 8.2 kg (3. 1-26.9 ). stone composition was calcium oxalate (6), mixed struvite (2), urate (1), and cystine (1). median stone size was 2 cm (0.7-5). median pre-and 3 month post-operative creatinine was 1.3 (0.8-9.1) and 1.1 mg/dl (0.6-6.1), respectively. the median procedure time was 165 minutes. successful removal of all stones were documented in 11/12 (91.6%). procedure-related complications occurred in 3 units, all were easily managed. median follow-up time was 150 days (4-2007). four patients are still alive. no patient died from the procedure. overall, enl can be safely performed in dogs and cats, yielding similar success rates to people. advanced endourologic experience is recommended. fgf-23 is a key regulator of plasma phosphate concentration. it is elevated in cats with naturally occurring ckd and increases as renal function declines. elevated fgf-23 concentrations are an independent predictor of survival time in human haemodialysis patients, but this association has not previously been examined in cats with ckd. this study investigated if fgf-23 was independently associated with survival time (all cause mortality) in cats with newly diagnosed ckd.cats diagnosed with ckd at two london-based first opinion practices between 2000 and 2011 were identified. ckd was defined as plasma creatinine concentration >177lmol/l with concurrent urine specific gravity (usg) <1.035 or plasma creatinine concentration >177lmol/l on two consecutive visits. cats were excluded if no residual edta plasma was available to measure plasma fgf-23 concentration within 42 days of the date of diagnosis of ckd. fgf-23 concentrations were measured using a previously validated human intact fgf-23 elisa. plasma fgf-23, creatinine, phosphate and total calcium concentrations, packed cell volume (pcv), systolic blood pressure (sbp), usg and age were entered into univariable cox regression models of survival time. fgf-23 concentrations were logarithmically transformed due to a highly skewed distribution. statistical significance was defined as p<0.05. variables with p<0.05 were carried forwards into a backwards, stepwise multivariable cox regression analysis of survival time. ninety-one cats were included in the study. at the end of the follow-up period (february 2012), 64 cats had been euthanased or died (median (range) survival time 236 (0 -1766) days) and 27 cats were alive or lost to follow-up (median (range) survival time 696 (42 -1311) days). median (range) age at diagnosis (n=85) was 14.0 (6.6 -18.3) years. univariable cox regression analysis indicated that plasma creatinine (p<0.001), phosphate (p<0.001) and log-fgf23 (p<0.001) concentrations were negatively associated with survival time, and that pcv (p<0.001) and usg (p<0.001) were positively associated with survival time. in the multivariable model (n=82), plasma logfgf23 (hazard ratio (hr)=1.327, 95% confidence interval (ci) for hr=1.072-1.644; p=0.009) and creatinine concentration (hr=1.003, 95% ci for hr=1.002-1.004; p<0.001) were negatively associated with survival time, and pcv (hr=0.936, 95% ci for hr=0.885-0.990; p=0.020) was positively associated with survival time.plasma fgf-23 concentration is a novel prognostic indicator in feline ckd, independent of other factors including plasma creatinine concentration and pcv. future studies should investigate whether belgium ragdoll breeder organizations often forewarn ragdoll cat owners that renal problems may develop due to polycystic kidney disease (pkd), chronic interstitial nephritis, familial renal dysplasia or nephrocalcinosis. in several european countries, screening of ragdoll cats for kidney disease is already performed for years, without scientific evidence. therefore, we aimed to investigate if ragdoll cats are predisposed for kidney disease based on the laboratory parameters, one ragdoll cat was diagnosed with iris stage 2 chronic kidney disease (ckd) one of these six cats was the ragdoll cat with iris stage 2 ckd. in one ragdoll cat, pkd could not be excluded on ultrasonography because one cyst was detected in one kidney. however, none of the ragdoll cats was genetically positive for pkd.based on this study, pkd and ckd appear to be uncommon in ragdoll cats residing in belgium and the netherlands. however, renal infarcts were seen more commonly in ragdoll cats compared to an age-matched control group spain urine markers are advocated to early detect kidney damage in the clinical practice, nevertheless histology remains the gold standard. the aim of this study was to evaluate quali-quantitative proteinuria and possible renal damage using different non-invasive tests in dogs affected by leishmaniasis.based on clinical signs and serology/cytology, 26 affected dogs (leish) were included. fifteen healthy, non-proteinuric dogs were selected as control. upon admission, all dogs underwent to physical examination, systolic blood pressure (sbp) measurement, clinicopathological evaluation urine high resolution agarose and silver staining sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (hre; sds-page) were performed. a cut-off of 66 kda was selected to classify bands in high or low molecular weight (hmw; lmw). data were analyzed with non-parametric statistics and roc curve analysis (roc). a difference was considered significant for p<0.05 leish dogs presented significantly higher upc (mean 3, median 1.29) and uac (mean 1.62, median 0.29). rri values were significantly higher in leish (mean 0.72) than control (mean 0.64). rri was significantly correlated to wbc (r=0.51), hemoglobin (r=0.52) and albumin concentrations (r=0.61), usg (r=0.60) and upc (r=0.48). hre and sds-page protein patterns allow to distinguish p from np and control dogs. sds-page revealed a significantly higher number of bands in leish dogs (35-40) than in control (25-30). np and blp dogs presented a significantly lower number of lmw bands than p. number of bands was significantly correlated to upc (r=0.66) and uac (r=0.64) in humans, diabetes mellitus (dm) is an important cause of renal damage. main lesions include thickening of the glomerular basement membrane and mesangial expansion, whereas tubular atrophy and vascular hypertrophy are less frequent. in cats, although diabetes is a common endocrinopathy, it is yet unknown whether dm causes renal damage. the aim of the present study was to compare renal histopathological features and clinical parameters of kidney function in diabetic cats against a well-matched control population.formalin-fixed, paraffin-embedded kidney samples were retrieved from diabetic and control cats that died between 1997 and 2009 due to any disease at the clinic for small animal internal medicine, university of zurich (switzerland), and in which a post-mortem examination was performed. control cats were selected to be matched for age, sex, breed and body weight. serum creatinine and urea levels were analyzed if they had been measured within 10 days before death. kidney sections were stained with haematoxilin-eosin, periodic acid-schiff (pas), masson's trichrome, acid fuchsine orange-g (afog), and periodic acid methenamine silver (pams). with optical microscopy glomerular, tubulointerstitial and vascular parameters were identified and scored using a grading scale. data were analyzed with contingency tables and t-tests.thirty-two diabetic cats and 20 matched controls were included. with optical microscopy, scores of glomerular lesions (i.e., sclerotic glomeruli, mesangial or endocapillary hypercellularity, increased mesangial matrix, immunodeposits, glomerular basement membrane thickening, mesangial interposition), tubulointerstitial lesions (i.e., inflammation, fibrosis, tubular atrophy, necrosis and lipidosis, intratubular mineralizations) and vascular lesions (i.e., small or large artery hypertrophy) did not differ between the 2 groups. overall, glomerular, tubulointestitial and vascular lesions were observed in 43.8%, 57.9% and 6.3% of diabetic cats and in 57.9%, 78.9% and 15.7% of the controls. similarly, serum creatinine and urea levels were not different between groups (creatinine: 197 ± 42 vs. 199 ± 46 lmol/l, reference: 98-163 lmol/l; urea: 18.2 ± 2.5 vs. 18.4 ± 4.6 mmol/l, reference: 7.4-12.6 mmol/ l).the results suggest that dm in cats does not lead to microscopically detectable renal lesions or clinically relevant renal dysfunction when compared to a well-matched control group. we hypothesize that the short life expectancy of diabetic cats and the low prevalence of hypertension are main reasons for the difference to human diabetics.early diagnosis of aki and differentiation from non-renal disease or ckd remains challenging in veterinary medicine. in human medicine ngal is used as a real time indicator of aki but few data exist in veterinary medicine. in this study plasma and urine ngal was measured in 18 healthy dogs with normal gfr (plasma inulin clearance) and 83 dogs with renal azotemia (creatinine > 1.4mg/dl and/or urea > 59mg/dl persisting at least 24 hours after correction of prerenal factors). based on history, clinical course, laboratory and ultrasonographic findings, azotemic dogs were diagnosed with aki (n=53) or ckd (n=30). urine and plasma ngal was measured with a dog ngal elisa kit (bioporto ® diagnostics a/s, gentofte, denmark). intra-assay variability for plasma and urine ngal was 3.1% and 4.8%, respectively. azotemic dogs had significantly higher plasma ngal concentrations and urine ngal-creatinine ratios compared to healthy dogs (p< 0.001, mann-whitney u-test). median (min-max) plasma ngal concentration in healthy dogs, dogs with aki and ckd was 10.7 (2.5 -21.2) ng/ml, 49.1 (5.7 -469.0) ng/ml and 35.3 (7.7 -97.9) ng/ml, respectively. using a multiple linear regression model in the azotemic dogs with ngal as dependent and age, weight, sex, aki vs. ckd, dialysis and survival as independent variables revealed a significant differ-ence only for aki vs. ckd (p = 0.005). in conclusion, ngal can be measured successfully in plasma and urine of healthy dogs and dogs with kidney disease. dogs with aki had significantly higher plasma ngal concentration compared to dogs with ckd. xanthine urolithiasis is a rare condition accounting for 0.1% of all canine urolithiasis in one study. this pathology has been reported as a primary disorder in dogs, most notably in cavalier king charles spaniels (ckcs). xanthine is an intermediate product of purine metabolism, which is converted from hypoxanthine by xanthine oxidase. xanthine is only slightly soluble in urine and therefore hyperxanthinuria may lead to urolith formation. it has been speculated that some ckcs have an inherited mutation in the xanthine oxidase gene. in humans, isolated deficiency of xanthine oxidase occurs rarely and approximately 50% of individuals are asymptomatic, despite having significant xanthinuria. therefore we hypothesised that asymptomatic xanthinuria may be commonplace in the uk population of ckcs. in support of this, a previous case report of a symptomatic ckcs reported significant xanthinuria occurring in an asymptomatic sibling. in order to examine the prevalence of xanthinuria in ckcs, urine concentrations of hypoxanthine and xanthine metabolites as well as creatinine were measured in 35 clientowned cavalier king charles spaniel dogs and 24 dogs of other breeds from three first-opinion veterinary practices in the uk. urine samples were collected by free catch and purine metabolites were measured by high-performance liquid chromatography. ratios of xanthine/creatinine and hypoxanthine/creatinine from the two populations were compared by mann whitney u test and were found not to be significantly different (p=0.41 and p=0.59 respectively). in the control population, the xanthine/creatinine ratio ranged from 0.00018 to 0.01611 (median 0.00069), while in the ckcs population it ranged from 0.000154 to 0.005794 (median 0.000435). these results are markedly lower than the previously reported case of xanthine urolithiasis in a uk ckcs dog, which utilised the same reference laboratory (xanthine/creatinine ratio 0.406). these data suggest that asymptomatic xanthinuria is not prevalent in the uk ckcs population. key: cord-293151-g3758oes authors: nemzek, jean a.; lester, patrick a.; wolfe, a. marissa; dysko, robert c.; myers, daniel d. title: biology and diseases of dogs date: 2015-07-10 journal: laboratory animal medicine doi: 10.1016/b978-0-12-409527-4.00012-2 sha: doc_id: 293151 cord_uid: g3758oes historically, the dog played an important role as a laboratory animal in biomedical research. although numbers are declining, the use of dogs continues to be common in pharmacokinetics and cardiovascular studies. the normal biology of the dog as both a laboratory and a companion animal has been well studied and reference values are presented here as a clinical and experimental resource. this provides the necessary background to discuss the spontaneous diseases, including infectious and neoplastic conditions, prevalent in purpose bred as well as random source dogs used in biomedical research. in addition, diseases and conditions that arise secondary to the housing and experimental manipulation of dogs is discussed with emphasis on treatment and prevention. laboratory animal medicine mellitus. a comprehensive but concise review of the use of the dog as a research subject is available in gay (1984) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger dog breeds for use in surgical research studies. some specific breeds with congenital or spontaneous disorders have also been maintained by research institutions (see examples below). random-source dogs used in research are most frequently mongrels or larger dog breeds (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for "beagle" for the years 2012-2013, a significant portion of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. other common areas of research using beagles were dental and periodontal disease and surgery, orthopedic surgery, skeletal physiology, and imaging studies. other research areas that utilized beagles included canine infectious disease, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established and the organs of larger dog breeds are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies had been maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of the blood, most notably the neutrophil population. these dogs can be used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., 1995) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by the absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., 1994) . other genetic disorders studied in dog colonies include hereditary canine spinal muscle atrophy (cork, 1991) and narcoplepsy in doberman pinschers (ripley et al., 2001) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease) and the development of spontaneous diabetes mellitus and hypothyroidism has been studied in several breeds of dogs for comparisons with the human conditions. although historically the dog has been a common laboratory animal, their use in research has waned over the past 30 years. according to the u.s. department of agriculture (usda), animal and plant health inspection service (1998, 2011) , the number of dogs used in research has declined from 211,104 in 1979 to 75,429 in 1997 (prior to the previous edition of this text) and 64,930 in 2010. this decrease was caused by a variety of factors, including (but not limited to) decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), increased cost, and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purposebred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against rabies virus, canine distemper virus, parvovirus, adenovirus type 2, parainfluenza virus, leptospira serovars canicola, icterohaemorrhagiae, grippotyphosa, and pomona, and bordetella bronchiseptica (jasmin, personal communication). purpose-bred dogs are also usually treated prophylactically for intestinal helminths and ectoparasites, and possibly given a heartworm preventative. random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting and racing) or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. random-source dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations and tissue/organ harvest. options for procurement of dogs for biomedical research typically include purchase from a usdadesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title 9, chapter 1 (1-1-92 edition), subchapter a, animal welfare, 1.1 definitions, and 2.1 requirements and application (office of the federal register, 2002) . briefly, class a licensees are breeders who raise all animals on their premises from a closed colony. class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and record-keeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. in december 2013, the national institutes of health (nih) issued notice not-od-14-034 entitled notice regarding nih plan to transition from use of usda class b dogs to other legal sources (national institutes of health, 2013) . this nih policy begins in the fiscal year 2015 and prohibits the procurement of dogs from class b dealers using nih grant funds. from that point forward, dogs on nihfunded studies will have to be obtained from class a vendors, privately owned colonies (such as institutional breeding colonies), or client-owned animals (e.g., animals participating in veterinary clinical trials). the best resource for identification of possible vendors are online 'buyer's guide' sites or 'buyer's guide' issues of trade periodicals. online sites include the buyer's guide of the american association of laboratory animal science (http://laboratoryanimalbuyersguide.com), and the trade journals lab animal (http://guide.labanimal. com) and animal lab news (http://www.alnmag.com/ content/buyers-guide). a 'buyer's guide' typically lists sources for both purpose-bred and random-source dogs, and denotes such features as pathogen-free status, health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within issues of the journals. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal welfare act (7 cfr 2.17, 2.51, and 371.2[g] ) are described in 9 cfr chapter 1 (1-1-92 edition), subchapter a, animal welfare (office of the federal register, 2002) . regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats of part 3 (standards) of subchapter a. particular attention should be paid to section 3.6c (primary enclosures-additional requirements for dogs) because the space required for housing dogs is calculated using body length rather than weight (a parameter used for other species and also for dogs in the national research council (nrc) guidelines). section 3.8 (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute for laboratory animal research (ilar) has written the guide for the care and use of laboratory animals (national research council, 2011) . the 'guide' is the primary document used by institutional animal research units to develop their programs and by animal care evaluation groups, such as the association for assessment and accreditation of laboratory animal care international (aaalac international), to facilitate site visits and inspections. the primary difference between the 7th and 8th editions of the 'guide' (national research council, 1996 regarding the care of dogs is the notation that "enclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs, or kennels) are preferable." the ilar committee on dogs authored dogs: laboratory animal management (national research council, 1994) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. the information presented in the tables represents a range of normal values that can vary depending on the analytical method, as well as the age, breed, and sex of the animal. cohen, covance laboratories, inc., cumberland, va (2013) . physiological data for a mixed population of dogs of both sexes. fig. 12 .1 demonstrates the normal weights and corresponding ages for both male and female beagle and hound dogs. tables 12.2 and 12.3 feature hematology data from beagles of both sexes from two commercial facilities. tables 12.4 and 12.5 list serum chemical data for beagles of both sexes from two commercial facilities. representative blood gas, coagulation data, and normal urinalysis parameters can be found in tables 12.6-12.8, respectively. finally, the reviews in arterial and venous blood gas anaylses (rieser, 2013) and the manual of canine and feline cardiology (tilley et al., 2007) are excellent resources. good nutrition and a balanced diet are essential to the health, performance, and well-being of the animal. the nrc of the united states national academy of sciences is the leading provider of nutrient recommendations for dogs and provides average requirements needed to maintain growth and prevent deficiencies (subcommittee on dog and cat nutrition, 2006) . the nrc publications form the basis for the association of american feed control officials (aafco) nutrient profiles, which are updated periodically (baldwin et al., 2010) . the aafco is an advisory body comprising state representatives from across the united states. it provides a mechanism for developing and implementing uniform and equitable laws, regulations, standards, and enforcement policies, and establishes nutrient profiles for cat and dog foods (dzanis, 1994; thatcher et al., 2010) . additional resources should be consulted for details on the nutritional requirements for dogs of all ages (dzanis, 1994; subcommittee on dog and cat nutrition, 2006; baldwin et al., 2010; thatcher et al., 2010; hand et al., 2010) . recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment. the mer for most breeds may be calculated using the following equation: mer (metabolizable kcal/day) = bw × 0.75 × 550 kj de, where bw = body weight (kg), kj = kilojoules, de = digestable energy (kienzle and rainbird, 1991) . in-depth overviews of diets used in biomedical research are available in diet-specific literature. open-formula diets have defined concentrations of all ingredients and the information is publicly available. this allows researchers to control for this important environmental variable and enables retrospective analysis of possible diet composition effects on research results (barnard et al., 2009) . open-formula diets occasionally may require changes in formulation to maintain nutrient composition or meet changing nutrient requirements. these changes in quantitative ingredient formulation are made public when open-formula diets are modified. in contrast, closed-formula diets are commercially available, balanced diets that meet and label the minimum requirements for protein and fat and the maximum values for ash and fiber; however, the exact composition of ingredients may vary from batch to batch. ingredient composition varies as the manufacturer applies a leastcost strategy, referring to formulating diets to maximize profit by using the least-expensive ingredients. although the ingredients are listed, the quantitative ingredient formulation is not publicly available and can vary without public disclosure, due to proprietary nature of commercial diets produced and marketed under vendor trade names. closed-formula diets have also been referred to as as 'fixed formula' or 'constant nutrition' (labdiets, pmi nutrition international, st. louis, missouri) by manufacturers (barnard et al., 2009) . in fixed-formula diets, the quantitative ingredient formulation does not change; however, this information is proprietary and therefore laboratory animal medicine not disclosed publically (barnard et al., 2009) . semipurified and purified diets provide the strictest control of ingredients and are formulated from purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. although purified and semipurified diets do differ in the types of ingredients used, the terms are generally used to mean the same thing. purified-ingredient diets are generally 'open' formulas, meaning that they are published and available to the scientific community. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are safe for consumption up to 6 months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf-life, but the best strategy is to feed only fresh diets and use each lot based on the date of manufacture. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. from dr. asheley wathen, covance laboratories, inc., madison, wi, and dr. kimberley cohen, covance laboratories, inc., cumberland, va (2013) . overall health, body condition, nutrition, and age greatly influence reproductive efficiency (gavrilovic et al., 2008; johnson, 2008) . therefore, only normal, healthy animals in excellent body condition should be used in breeding programs. beagles between 2 and 3.5 years of age have the best conception rates and litter size with the lowest neonatal mortality. after 5 years of age, conception rates and litter size decline and neonatal mortality increases (johnson, 2008) . the vagina is a long, musculomembranous canal that extends from the uterus to the vulva. during physical examination, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva, avoiding the deep ventral clitoral fossa. examination should proceed at an angle of approximately 60° until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. female dogs are monoestrous, typically nonseasonal, spontaneous ovulators that have a spontaneous luteal phase approximately 5 days longer than the 65 ± 1 days of pregnancy followed by obligate anestrus. puberty (beginning of the first estrus) occurs between 6 and 14 months in most breeds. the time of onset positively correlates with the body size (concannon, 2011) . the canine cycle is divided into four phases: proestrus, estrus, diestrus, and anestrus. the duration of laboratory animal medicine proestrus is 5-20 days with an average of 9 days and reflects the follicular phase rise in estrogen. during this stage, the vulva is enlarged and turgid, and a serosanguinous vaginal discharge is present (concannon, 2011) . estrus may be from 5 to 15 days in duration but generally lasts 9 days. the endocrine feature of estrus is the first abrupt increase in progesterone (>5 ng/ml), which occurs concomitantly with the luteinizing hormone (lh) surge 95% of the time, followed by ovulation within 24-72 h. the vulva is softer and smaller than in proestrus. the vaginal discharge persists and may remain serosanquinous or become straw colored. diestrus begins approximately 9 days after the onset of standing heat. the end of this stage is 60 days later, which would be coincident with whelping if the bitch had become pregnant. defined behaviorally as starting when estrous behavior ceases (concannon, 2011) , diestrus represents the peak of serum progesterone. anestrous may last from 80 to 240 days and is the stage of reproductive quiescence. it is characterized by an absence of ovarian activity and serum progesterone levels of less than 1 ng/ml. the onset of puberty in the male ranges from 5 to 12 months of age and is affected by breed, season, nutrition, and disease status. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial or leydig's cells. at this time, the testicular growth is rapid, the seminiferous tubules begin to differentiate, and sertoli cells form the blood-testis barrier. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including, inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, whereas inhibin and estrogen provide negative feedback to the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in 45 days, with subsequent maturation of sperm occurring in the epididymis for approximately 15 days. thus, the entire process from the initiation of spermatogonial mitosis to the delivery of mature sperm to the ejaculate is 60 days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality semen production. supression of sexual behavior and problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities. animals with poor hind limb conformation or trauma to the back may be unable to properly mount the female. there is a positive correlation between scrotal circumference and the number of sperm produced. finally, the quality of sperm is assessed by motility, morphology, volume, and concentration. an ejaculate (5 ml) that contains approximately 500 million progressively motile sperm without significant morphological abnormalities is a good indicator of normal male fertility. complete anatomy of the bitch and dog can be found in miller's anatomy of the dog (evans and de lahunta, 2012) . cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. cornification occurs approximately 2 days prior to the estrogen peak and 4 days prior to standing heat. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear/ cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear 'anuclear' and are classified as 'cornified' or 'anuclear squames.' the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified 6 days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear/cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, observation of behavioral estrus is the best criterion to use in breeding management. during proestrus, the male is attracted to the bitch and will investigate her hindquarters, but she laboratory animal medicine will not accept breeding. estrus is characterized by proactive receptivity to mounting by males and increased male-seeking behavior (concannon, 2011) . during this stage, the bitch will exhibit 'flagging,' or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate and litter size, it is recommended to breed the bitch on days 1, 3, and 5 of the standing heat. due to the long life span of canine sperm, fertilization occurs in the oviduct up to 8 days after coitus. the ovulated oocyte is a primary oocyte that must undergo two meiotic divisions before fertilization can occur. this overall maturation process takes approximately 2 days. after maturation, the oocyte remains viable for 4-5 days. optimal conception rates tend to occur when the bitch is bred from 4 days before to 3 days after ovulation; best litter size is achieved when the bitch is bred 2 days after ovulation. implantation is evident by areas of local endometrial edema 17-18 days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary, indicating the placental villi are arranged in a belt, and deciduate, reflecting that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is 59-63 days. luteal progesterone is responsible for maintaining pregnancy and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than 1 ng/ml in late proestrus to a peak of 30-60 ng/ml during gestation, and then declines to 4-5 ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility (johnson, 2008; verstegen-onclin and verstegen, 2008) . pregnancy detection can be performed by several methods. abdominal palpation of the uterus may be most informative at approximately 28 days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings and are approximately 2 inches in length at 28-30 days. by day 35, the uterus begins to enlarge diffusely and the vesicles become difficult to identify by palpation. radiology can be used to confirm pregnancy and facilitate determination of gestational age, beginning 45 days after the lh surge (lopate, 2008) . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. ultrasonography can be used to confirm pregnancy beginning on days 18-22, at which point the gestational sacs will be approximately 1 cm in diameter, and until parturition (shille and gontarek, 1985; lopate, 2008) . ultrasonography can assess fetal viability by visualizing fetal heartbeats and fetal movement beginning on gestational days 23-25 and 35, respectively (lopate, 2008) . it can also predict gestational age using the inner diameter of the chorionic cavity in early pregnancy and the biparietal diameter in late pregnancy luvoni and beccaglia, 2006) . however, ultrasonography for determination of gestational age is most accurate at day 30 of pregnancy when using correction factors for small (< 9 kg) and large (> 40 kg) body weight dogs (kutzler et al., 2003) . thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged because they may adhere to the umbilical cord and predispose to ascending infections. heat lamps may be placed 24 h prior to parturition and remain until all neonates demonstrate vigorous suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. monitoring of parturition is important, but human intervention should be minimal in order to prevent stressinduced cannibalism. an abrupt drop in body temperature to less than 100°f indicates impending parturition within 18-24 h. the process of parturition has been divided into three stages. stage 1 of labor lasts 6-12 h and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include panting and increased pulse rate (johnson, 2008) . fetal expulsion occurs during stage 2, which lasts approximately 3-6 h. as the fetus engages the cervix, there is release of oxytocin, referred to as the ferguson reflex, which strengthens the uterine contractions and may elicit abdominal contractions as well. the bitch is able to inhibit this stage of labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between deliveries of each pup is irregular, but the average is less than 1 h between pups. veterinary assistance laboratory animal medicine is necessary if the bitch remains in stage 2 for more than 5 h without delivering the first pup, or for more than 2 h before delivering subsequent pups. during stage 3 of labor, the placentas are expelled either immediately or within 15 min of delivery of each pup. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. the peripartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. oxytocin should not be used in the event of systemic illness or abnormalities precluding vaginal delivery. indications for its use include lack of delivery 24 h after onset of stage 1 labor, greater than 1 h of unproductive stage 2 labor, inadequate contractions, or abnormal vaginal discharge. in these cases, radiographs are recommended to assess fetal size in relation to the birth canal and any possible obstructions, followed by 0.25-2.00 iu of oxytocin intramuscularly or subcutaneously. the oxytocin can be repeated 30-60 min after the first dose for a total of two doses (plunkett, 1993) . in some cases, treatment with 0.5-1.5 ml/kg of 10% calcium gluconate, delivered slowly iv while monitoring closely for bradycardia, and 25% dextrose iv may be indicated. uterine involution occurs during anestrus within 4-5 weeks of parturition. during this time, a greenish to red-brown vaginal discharge, or lochia, is considered normal. the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the 6th postpartum week, with complete repair by 3 months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies, the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately 12 days, and ears are patent at approximately 12-20 days. solid food can be introduced between 4.5 and 6 weeks of age, and puppies can be weaned at 6-8 weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as a vaginal-vestibular stricture, narrow vagina, vaginal septum, and vaginal hyperplasia, or if there is a behavioral incompatibility between the male and female dogs (kutzler, 2005) . semen is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the scent of an estrous bitch and manually stimulated. the first two fractions are collected followed by a sufficient amount of the third fraction (predominantly of prostatic fluid) to bring the total semen volume to 4-6 ml. the semen can be introduced into the cranial vagina or directly into the uterus either through trans-cervical catheterization with a norwegian ai catheter or utilizing fiberoptic endoscopy. use of the norwegian ai catheter for intrauterine insemination of frozen-thawed, fresh, and chilled-extended semen results in significantly higher whelping rates than intravaginal insemination (linde-forsberg et al., 1999; thomassen and farstad, 2009) . for trans-cervical insemination, the bitch is either standing on all four legs or standing with hindquarters raised. the ai catheter and guiding tube are inserted into the vestibulum as far as the pseudocervix. firm abdominal palpation is then used to locate and fix the cervix in the other hand, at which point the catheter is further inserted along the dorsal vaginal fold until the cervical opening is located and semen is deposited into the uterus lumen (thomassen and farstad, 2009) . surgical and laparoscopic ai has been used successfully for intrauterine and intratubal insemination; however, these techniques are invasive and require anesthesia. therefore, the nonsurgical techniques mentioned above are recommended, as these approaches are less invasive and can be completed without anesthesia in nonsedated or sedated dogs depending on the experience of the personnel and personality of the dogs. ai with freshly collected sperm can be done on days 1, 3, and 5 of standing heat or on days of maximal vaginal cornification. the viability of frozen-thawed sperm is significantly reduced compared to fresh or chilled sperm that may live up to 5 or 6 days in the reproductive tract of the bitch; frozen-thawed sperm live only a few hours. therefore, the ova must be mature and insemination with frozen-thawed semen must be done 2-3 days after ovulation in the bitch as determined by serum progesterone concentrations (thomassen et al., 2006) . false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. a comprehensive review of canine pseudocyesis exploring its cause, clinical features, and treatments is covered by c. gobello (gobello et al., 2001) . reproductive performance in the bitch is optimal prior to 4 years of age. cycling does not completely cease; however, after 5-8 years of age, bitches demonstrate significant decreases in conception rate and the number of live pups whelped. by 8-9 years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia, are extremely common. dogs prefer living in a social environment. dogs have well developed olfactory glands, vision, and auditory and tactile senses that allow them to gain environmental cues and information from other dogs and humans (field and jackson, 2006; joint working group on refinement, 2004) . much of their instinctive behavior is dependent on learning to interact with other members of their species. beagles have been a popular animal model because of their docile nature. they are easily handled and, for the most part, respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiograms (ecgs), oral gavage, and venipuncture. although sexually mature by 6-9 months of age, dogs are not socially mature until 18-36 months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from 3 to 8 weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks 5 and 12, puppies are most capable of learning how to interact with people. by 10-12 weeks of age, dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the canine behavior section of the manual of clinical behavioral medicine for dogs and cats (overall, 2013) . by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of its disorders. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting, especially infectious diseases associated with the use of random-source dogs and conditions seen frequently in the beagle. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks. etiology canine infectious respiratory disease (cird) is a highly contagious illness and several organisms have been incriminated including bordetella bronchiseptica; streptococcus equi subsp. zooepidemicus; canine parainfluenza virus (cpiv); canine influenza virus (civ); canine respiratory coronavirus; canine adenovirus type 2 (cav-2); canine herpesvirus; canine reovirus types 1, 2, and 3; and mycoplasma and ureaplasma. naturally occuring infection can result in coinfection by two or more organisms (garnett et al., 1982; ford, 2012) . clinical signs cird can be subdivided into mild or severe forms. the mild form is more common and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough may be elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise, dogs are typically asymptomatic. mild tracheobronchitis usually lasts 7-14 days, even when untreated. the severe form results from poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and can be the determinant of severity (sherding, 1994) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. epizootiology and transmission the natural reservoir for b. bronchiseptica is the respiratory tract (bemis, 1992) , and it is very easily spread by aerosol and direct contact. transmission is heightened by confined housing of multiple animals. bordetella bronchiseptica is highly infectious with an incubation period of 3-10 days. pathogenesis the most common clinical isolates are cpiv and b. bronchiseptica (mochizuki et al., 2008) . however, b. bronchiseptica is often recovered from clinically healthy animals (chalker et al., 2003) . during clinical infection, b. bronchiseptica attaches to the cilia of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv laboratory animal medicine or cav-2 alone are usually subclinical but can cause necrotizing tracheobronchiolitis (dungworth, 1985) . diagnosis and differential diagnosis diagnosis is often based on clinical signs and known history; however, cough elicited by tracheal palpation may be inconsistent and should not be used for definitive diagnosis. presumptive diagnosis can be made by isolation of b. bronchiseptica or mycoplasma by nasal swabs. viral isolation or paired serology is often impractical and expensive. if cough persists for more than 14 days, other disease conditions should be considered. differential diagnoses include civ, canine distemper virus, pneumonia, heartworm disease, tracheal collapse, mycotic infections, and diseases resulting in tracheal compression (johnson, 2000) . prevention prevention is best achieved by avoiding exposure to infected animals. dogs should be vaccinated prior to or upon admission to the animal facility. intranasal vaccines protect against infection and disease and can be given to dogs as young as 3 weeks of age (greene and levy, 2012) . combination vaccines for b. bronchiseptica, cav-2, and cpiv are preferred. vaccinations should be boostered every 6 months when multiple animals are housed in a confined area. control staff must practice proper hygiene to prevent transmission by fomites. sanitation, proper ventilation, and proper humidity are critical for control. symptomatic animals should be isolated and kennels should be disinfected with agents such as bleach, chlorhexidine, or quaternary ammonium chloride. treatment bordetella bronchiseptica is sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur and should be continued for 14 days. for severe or unresponsive infection, treatment should be based on bacterial culture/sensitivity patterns. nebulized gentamicin or kanamycin may be helpful in severe cases. antitussives should be avoided if the cough is productive; however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction. research complications due to the altered respiratory tract physiology, infected animals should not be used for pulmonary studies. etiology β-hemolytic lancefield's group c streptococcus (s. equi ssp. zooepidemicus) is a gram-positive, non-spore-forming coccus that causes pneumonia and sepsis in dogs. clinical signs clinical signs vary based on the organ system affected. pneumonic disease is typically associated with sudden onset of clinical signs including coughing, weakness, fever, dyspnea, and hematemesis. the rapid progression of disease is similar to that seen in humans with toxic shock syndrome (tss) caused by streptococcus pyogenes. peracute death has been reported in research and shelter dogs (bergdall et al., 1996; pesavento et al., 2008) . epizootiology and transmission streptococcus equi ssp. zooepidemicus is not considered a commensal of healthy dogs as most of the β-hemolytic commensal organisms belong to group g, specifically streptococcus canis. asymptomatic carriers are suspected to be the route by which infection enters populations. streptococcus equi ssp. zooepidemicus is considered an opportunistic pathogen and stressful factors such as transport can predispose to disease (priestnall et al., 2010) . pathologic findings in peracute cases, hemorrhage from the mouth and nose and within the pleural cavity can be the most striking lesion. ecchymotic and petechial hemorrhages can be noted on several organs ( fig. 12 .2). 'bull's-eye' lesions may be observed on the pleural surface of affected lung lobes. histologic lesions can include fibrino-suppurative, necrotizing, and hemorrhagic pneumonia. gram-positive cocci can be found in intracellular clusters throughout the lung ( fig. 12. 3), tonsils, and spleen of affected animals (bergdall et al., 1996; priestnall and erles, 2011) . pathogenesis predisposing factors such as transport stress and viral coinfection have been shown to contribute to the virulence of s. zooepidemicus (priestnall and erles, 2011) . due to the similarities with the clinical signs seen in human cases of tss, superantigens are thought to contribute to the virulence of s. zooepidemicus in cases of acute hemorrhagic pneumonia. these superantigens work by bypassing the conventional mechanisms of antigen presentation and binding to major histocompatibilitity complex class ii receptors. as a result, there is a hyperactive proinflammatory response and an 'avalanche' of cytokines including interleukin 1β (il-1β), interleukin 6 (il-6), and tumor necrosis factor alpha (tnf-α). three novel superantigen-encoding genes have been identified from a case of acute fatal hemorrhagic pneumonia, szef, szen, and szep. however, it is currently unclear what effect these superantigens have in vivo priestnall et al., 2010) . while superantigens have been detected in some isolates, there is not enough data to determine if superantigens play a role in the pathogenesis (byun et al., 2009; kim et al., 2007) . diagnosis and differential diagnosis definitive diagnosis is based on bacterial culture of nasal swabs or transtracheal lavage. polymerase chain reaction (pcr) can be done on post-mortem lung tissue. bacterial pneumonias or bacteremias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and b. bronchiseptica. nonbacterial causes of respiratory disease include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control there is no vaccine for prevention of s. zooepidemicus. the organism has been isolated from the environment during active outbreaks (pesavento et al., 2008) , so dogs diagnosed with s. zooepidemicus should be quarantined and any potential fomites (e.g. food bowls, enrichment) should be properly disinfected. treatment antibiotic therapy should be based on culture and sensitivity. resistance to doxycycline and tetracycline has been demonstrated (garnett et al., 1982; pesavento et al., 2008) . research complications dogs with severe hemorrhagic pneumonia or systemic disease are not appropriate for research study. the association between epizootics of this disease and transportation supports operational policies that require adequate acclimation periods for animals upon arrival. etiology serovars canicola, bratislava, and grippotyphosa result in renal or hepatic disease, whereas serovars icterohaemorrhagiae and pomona predominantly result in hepatic disease . clinical signs canine leptospirosis can present as subclinical, acute, or chronic disease. clinical signs in acute infection can be nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, vomiting, muscle tenderness, and pyrexia. clinical signs can be related to renal failure including polyuria and polydipsia, with or without azotemia, oliguria, or anuria. leptospirosis can also lead to hepatic failure with signs such as icterus or bleeding abnormalities. peracute leptospirosis is characterized by shock, vascular collapse, and rapid death. uveitis, abortions, stillbirths, and pulmonary hemorrhage have also been associated with leptospirosis (klopfleisch et al., 2010; van de maele et al., 2008) . bivalent vaccines against the most common canine serovars, icterohaemorrhagiae and canicola, have resulted in the increased prevalence of other serovars including grippotyphosa, pomona, bratislava, and autumnalis. increased movement of wild animal reservoirs (rats, raccoons, skunks, opossums) into urban/suburban areas have also contributed to the greater prevalence of previously uncommon serovars (sykes et al., 2011) . transmission occurs primarily through environmental contact, although direct transmisson between hosts may also occur. leptospires passing from urine into water is the most common route of contamination (goldstein, 2010) . leptospirosis is a zoonotic disease. pathologic findings the kidneys consistently have gross and microscopic lesions. in the acute phase, the kidneys are swollen with subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and necrotic foci (searcy, 1995) . in chronic stages of leptospirosis, the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis the severity and course of leptospirosis depend on the causative serovar as well as the age and immune status of the dog. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly, reaching the renal tubular epithelium several days postinfection. acute or progressive renal failure leading to oliguria or anuria may occur. nephritis may or may not be accompanied by hepatitis, uveitis, pulmonary hemorrhage, and meningitis. disseminated intravascular coagulation is often a secondary complication. diagnosis and differential diagnosis paired serology for the microscopic agglutination test is the most reliable means of definitive diagnosis, and successive serum sampling should be done 7-14 days after the first sample. pcr can be used to identify active infection early in the disease when serologic testing is negative or in previously vaccinated animals (sykes et al., 2011) . differential diagnoses include other causes of acute renal failure and hepatitis. prevention and control according to the american animal hospital association's 2011 vaccination guidelines, vaccination for leptospirosis is recommended based on geographic location and exposure risk (welborn et al., 2011) . both quadrivalent and bivalent inactivated bacterins are available. quadrivalent bacterins protect against canicola, icterohaemorrhagiae, grippotyphosa, and pomona serovars, whereas bivalent bacterins cover only canicola and icterohaemorrhagiae. immunization does not prevent the development of the carrier state or protect against other serovars. control requires preventing contact with wildlife reservoirs as well as identification of carrier animals. treatment doxycycline is the drug of choice as it can eliminate renal colonization. if vomiting or allergic reactions prohibit treatment with doxycycline, ampicillin or other penicillins should be utilized. aggressive fluid therapy and supportive care may also be needed. research complications due to the zoonotic potential, dogs with clinical leptospirosis should not be used in research studies. etiology campylobacter spp. are thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rods. many species of campylobacter have been isolated from normal and diarrheic animals; however, the most common pathogenic species include campylobacter jejuni ssp. jejuni and c. coli (marks et al., 2011) . clinical signs most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than 6 months of age (greene, 2000; burnens et al., 1992) . in cases of clinical illness, mild and intermittent mucoid or watery diarrhea, with or without frank blood, is most commonly noted. signs typically last 5-21 days but can persist for several months. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea (marks et al., 2011) . bacteremia and cholecystitis secondary to c. jejuni have also been documented in dogs (fox, 2012) . epizootiology and transmission the role of campylobacter spp. as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, 1994) . stress or immunosuppression may make animals more susceptible. transmission is via the fecal-oral route, mostly through contaminated food or water. campylobacter jejuni can be zoonotic with immunocompromised individuals at greatest risk. pathologic findings lesions depend on the mechanism of the enteropathy (van kruiningen, 1995) . enterotoxin production results in dilated, fluidfilled bowel loops, with little or no histopathologic alteration. cytotoxin-mediated disease results in a friable, hemorrhagic mucosal surface. histologically, the mucosa is ulcerated with lymphoplasmacytic infiltration. translocation can result in edema and congestion of the lamina propria with focal accumulation of granulocytes. epithelial hyperplasia and decreased goblet cell numbers are also noted. campylobacter jejuni may be visualized between enterocytes with warthin-starry silver-stained sections. pathogenesis clinical disease may be produced by several different mechanisms as campylobacter spp. have a variety of virulence factors including enterotoxins, cytotoxins, and adherence or invasion properties. campylobacter jejuni can cause an erosive enterocolitis by invasion of epithelium and production of the cytolethal distending toxin (cdt) (fox, 2012; van kruiningen, 1995) . in addition, c. jejuni can produce illness via translocation to regional lymph nodes causing a mesenteric lymphadenitis. diagnosis and differential diagnosis fresh feces (per rectum) can be used for presumptive diagnosis by demonstration of highly motile, curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and johnson, 1994) . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of 37°c. a pcr multiplex assay for differentiation of c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus ssp. fetus has been developed (wang et al., 2002) . any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis. prevention and control proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. in enzootic situations, group housing should be avoided. outbreaks are controlled by isolation and treatment of affected individuals. treatment antibacterial treatment should be considered in severely ill dogs. erythromycin, neomycin, enrofloxacin, clindamycin, and doxycycline are all effective. resistance to quinolones and ciprofloxacin has been documented (acke et al., 2009) . treatment should be a minimum of 10-14 days with bacterial cultures repeating 1 and 4 weeks after treatment. research complications dogs with clinical campylobacteriosis have temporary derangements to digestive and absorptive functions. etiology helicobacters are gram-negative, microaerophilic, spiral bacteria that infect the gastrointestinal tract. helicobacter spp. can be separated into gastric and enterohepatic groups. the gastric helicobacters commonly identified in dogs are referred to as non(haesebrouck et al., 2011; joosten et al., 2013) . the most common enterohepatic species found in dogs include h. bilis, h. canis, and h. cinaedi (castiglioni et al., 2012; dewhirst et al., 2005; fox, 2012 (haesebrouck et al., 2009; fox, 2012) . clinical signs most infections are subclinical in the dog. gastric infections may present with vomiting, diarrhea, and fever, accompanied by anorexia, pica, or polyphagia. enterohepatic helicobacters have been linked with inflammatory bowel disease in experimental animal models. heavy infections in dogs have been associated with inflammatory lesions of the large intestine (castiglioni et al., 2012; nguyen et al., 2013) . epizootiology and transmission the epizootiology and transmission of helicobacter spp. in the dog remain unknown. both oral-oral and fecal-oral routes for transmission have been suggested in humans, but transmission via canine saliva is a less reliable source of infection (craven et al., 2011) . enterohepatic infections of pet dogs are as high as 52% (castiglioni et al., 2012) . prevalence of gastric helicobacter infections in colony or shelter dogs can be as high as 82-100% (fox, 1995; hermanns et al., 1995) . pathologic findings gastritis is usually mild and characterized by reduced mucus content of the surface epithelium with vacuolation, swelling, karyolysis, and karyorrhexis of parietal cells. multifocal infiltrates of plasma cells and neutrophils occur around blood vessels and between gastric pits (hermanns et al., 1995) . intestinal lesions include mild to moderate lymphoplasmacytic infiltration as well as crypt dilation and crypt hyperplasia (castiglioni et al., 2012) . pathogenesis gastric helicobacters are urease positive, which assists with survival in the acidic environment of the stomach (kusters et al., 2006; uberti et al., 2013) . enterohepatic helicobacters are urease negative and typically reside in the lower intestine. the mechanism by which enterohepatic helicobacters colonize the liver is thought to be through portal circulation after uptake by enterocytes or through retrograde movement from the intestine into the bile duct (fox, 2012) . diagnosis and differential diagnosis organisms may be demonstrated with histopathology on endoscopic or surgical biopsy tissue samples. warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. culture may be difficult depending on the helicobacter spp. for species that produce urease, a positive urease test on a gastric biopsy specimen may give a presumptive diagnosis. the urea breath test has been successfully used to diagnose helicobacter spp. in laboratory beagles with a sensitivity and specificity of 89% (kubota et al., 2013) . western blot has been used to detect serum antibodies to enterohepatic species and pcr can be used to detect helicobacter spp. in fecal samples (oyama et al., 2012; wadström et al., 2009) . any causes of acute or chronic vomiting and diarrhea in the dog are differential diagnoses. prevention and control until more is known about the epizootiology and transmission of helicobacter spp., specific recommendations cannot be made for prevention and control. treatment for gastric species, combination therapy of amoxicillin (10 mg/kg q12 h), metronidazole (30 mg/kg q24 h), and sucralfate (0.25-0.5 mg/kg q8 h) has proven to be most effective (hall and simpson, 2000) . replacing sucralfate with famotidine, omeprazole, or bismuth subsalicylate may also be effective (marks, 1997; jenkins and bassett, 1997; denovo and magne, 1995) . recurrence rates within 60 days of treatment can be as high as 80% (anacleto et al., 2011) . treatment of enterohepatic helicobacters may depend on species susceptibility. aminoglycosides have been successful in treating h. cinaedi, but resistance to fluoroquinolones has been documented (tomida et al., 2013) . combination therapy of amoxicillin, clarithromycin, metronidazole, and omeprazole in medicated chow has been successful in eliminating various enterohepatic helicobacters from mice (del carmen martino-cardona et al., 2010) . long-term antibiotic treatment at a minimum of 21 days is suggested for enterohepatic and gastric helicobacters. research complication dogs used in gastrointestinal physiology or oral pharmacology studies should be free from helicobacteriosis. etiology parvoviral enteritis in dogs is caused by canine parvovirus strain 2 (cpv-2) of the family parvoviridae, genus protoparvovirus, species carnivore protoparvovirus 1. currently, there are three antigenic variants, 2a, 2b, and 2c. parvoviruses are nonenveloped, single-stranded dna viruses. clinical signs while parvoviral infection can affect the gastrointestinal tract, bone marrow, myocardium, and nervous tissues, the most common manifestation of disease is acute enteritis. clinical signs usually appear 5 days after fecal-oral inoculation and include anorexia, fever, depression, vomiting, and profuse intractable diarrhea which may become hemorrhagic. excessive fluid and protein losses through the gastrointestinal tract result in rapid and severe dehydration. dogs can develop severe leukopenia with a total leukocyte count of 1000 cells/μl or less. repeated hemograms may provide prognostic value, as rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. epizootiology and transmission parvovirus can infect dogs of any age, but puppies between 6 and 20 weeks of age are particularly susceptible. puppies less than 6 weeks of age are protected by passive maternal antibody. strain cpv-2c has been associated with severe disease in adult vaccinated dogs (calderon et al., 2009) . pathogenesis canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes acute enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus, leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis parvovirus can be detected with a commercially available fecal enzyme-linked immunosorbent assay (elisa). due to intermittent and brief shedding of the virus, fecal elisas can have false-negative results. pcr can be used to confirm an elisa result and to differentiate the viral strain. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. prevention and control parvoviral-positive animals should be quarantined for at least 10 days as the infectious virus is shed for several days after onset of clinical signs. although pcr has been used to detect viral dna in feces for up to 6 weeks (decaro et al., 2005) , it is currently unknown if the material being shed at this time is still infectious. disinfection of exposed areas with dilute bleach (1:30) or a commercial disinfectant is essential for elimination of the virus. six-week-old puppies should be vaccinated every 2-4 weeks with a modified live vaccine until at least 16 weeks of age. treatment treatment is largely supportive and aimed at restoring fluid and electrolyte balance. antimicrobial therapy is recommended due to intestinal compromise and risk of sepsis. early nutritional support continued throughout the disease has been shown to decrease recovery times (mohr et al., 2003) . research complications infection with parvovirus precludes the use of a particular dog in an experimental protocol. due to the significant discomfort of the animal, as well as the intensive therapy required, humane euthanasia is usually chosen in a research setting. etiology rabies virus is a lyssavirus belonging to the family rhabdoviridae. clinical signs clinical progression of neurologic disease occurs in three stages. the first, prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, furious, stage, animals are easily excited or hyperreactive to external stimuli and will readily bite at inanimate objects. the third, paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death due to respiratory failure usually occurs after onset of the third stage. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact with infected saliva, usually via bite wounds. pathogenesis the incubation period for rabies is 3-8 weeks to the onset of clinical signs but can range from 1 week to 1 year. bites to the head and neck result in shorter incubation periods due to the close proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to neurons within the brain, resulting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis definitive diagnosis is based on immunofluorescence of the virus in negri bodies of hippocampal cells. submission of the whole, unfixed brain, including the cerebellum and proximal brain stem, should be done within 48 h of collection. the tissue should be kept refrigerated as freezing can cause delays in testing. differential diagnoses laboratory animal medicine include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. prevention and treatment puppies should be vaccinated by 16 weeks of age, again at 1 year, and then annually or triennially, depending on state and local laws. research complications immuno-prophylaxis is recommended for animal care and research personnel who may have work-related risks of exposure. due to risk of human exposure, animals with suspected infection should be humanely euthanized and brain tissue should be submitted for confirmation. giardiasis giardia lamblia, also known as g. duodenalis and g. intestinalis, is a binucleate flagellate protozoan that usually causes subclinical infestation of the small intestine. clinical disease is usually seen in young dogs and the characteristic sign is voluminous, light-colored, foul-smelling, soft to watery diarrhea, which is the result of malabsorption and hypersecretion. giardia has a direct life cycle with infection resulting after consuming cyst-contaminated food or water. the change in ph between the stomach and duodenum activates excystation and trophozoites then attach to the enterocytes. for diagnosis, direct fecal smears are considered best for observing trophozoites and zinc sulfate centrifugation is preferred for detection of cysts. a commercial elisa kit is licensed for use in dogs, but the positive predictive value is poor and zinc sulfate centrifugation techniques should be used in conjunction with elisa (rishniw et al., 2010) . pcr assays are also available for diagnosing giardiasis. differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. metronidazole at 25-30 mg/kg po q12 h for 5-10 days is effective at treating giardiasis as well as other enteric protozoans, which may be potential differential diagnoses or coinfections. albendazole, fenbendazole, pyrantel, and praziquantel are also effective. coccidiosis intestinal coccidia associated with enteropathy in dogs include isospora canis, i. ohioensis, i. neorivolta, i. burrowsi, and hammondia heydorni (dubey and greene, 2012) . coccidian oocysts can be found in feces of clinically healthy dogs, as well as animals with diarrhea. clinically affected animals are young or immunosuppressed and develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. coccidia oocysts are typically spread by fecal-oral transmission, but dogs can ingest monozoic cysts in intermediate host tissues. the coccidian life cycle is both sexual and asexual, and results in the release of unsporulated eggs, which sporulate under appropriate environmental conditions. other causes for diarrhea should be excluded before a coccidial etiology is implicated. treatment may not be necessary, as infections are typically self-limiting and clinically insignificant. treatment may help to limit the number of oocysts shed in a kennel-housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine (50-60 mg/kg po q24 h for 10-20 days) or trimethoprim sulfa (30 mg/kg po q8 h for 10 days). ascarids roundworms of dogs are most often toxocara canis; however, toxascaris leonina can also affect dogs. clinical illness is usually only seen in young animals with large worm burdens. diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization can be seen. puppies may have a classical 'potbellied' appearance. heavy infestations can cause intussusception and/or intestinal obstruction. puppies that experience lung migrations of larval worms can develop fatal pneumonia. toxascaris canis can infect dogs by transplacental migration, transmammary migration, or ingestion of infective eggs. the infective stage of t. canis is the third-stage larva (l3). in transplacental infections, puppies may be born with l3 larvae in their lungs (sherding, 1989) . for diagnosis, large (70-85 μm in diameter) and relatively round ascarid eggs can be seen by standard fecal flotation methods. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention (hall and simpson, 2000) . most anthelmintics are effective for treatment. puppies should be treated early and often (every other week until 16 weeks of age) because of the possibility of prenatal or neonatal infection. pregnant bitches can be treated with extended fenbendazole therapy (50 mg/kg po once a day from day 40 of gestation through day 14 of lactation). hookworms the most common and most pathogenic hookworm of dogs is ancylostoma caninum. ancylostoma braziliense can also be found in dogs, but only a. caninum infestation typically results in clinical illness. puppies with hookworm infections can present as anemic with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. these signs are a direct result of the worms' consumption of blood and body fluids. infective larvae (l3) are ingested from the environment and develop directly in the intestinal tract. infestation can also be transmammary, from ingestion of a paratenic host, and, less often, by transplacental migration. on histological sections, embedded worms with mouthparts may be identified. diagnosis is made by identification of eggs or larvae by either fecal flotation or direct smear. a differential diagnosis of parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in young dogs with anemia. pyrantel pamoate is the anthelmintic of choice because it is safest in young ill animals. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention and control (hall and simpson, 2000) . due to transplacental or milkborne infection, puppies should be treated q2 weeks from 2 to 16 weeks of age. whipworms trichuris vulpis, the canine whipworm, can cause acute or chronic large intestinal diarrhea. the adult worm resides in the cecum or ascending colon. most infections are subclinical, but in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss may also be seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. trichuris vulpis has a direct life cycle with eggs passed in the feces. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to diarrhea. factors that influence development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. whipworm infestation is diagnosed by the presence of barrel-shaped, thickwalled eggs with bipolar plugs on fecal flotation. adult worms intermittently release eggs; therefore, negative results do not exclude infection. differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for 3 months (jergens and willard, 2000) . several species of cestodes parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. most cestode infestations are subclinical, but severe infestations with dipylidium can cause diarrhea, weight loss, and poor growth. the cestode requires an intermediate host, which for d. caninum are fleas and lice. ingestion of these arthropods results in transmission of the tapeworm. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. the most significant means to limit cestode infestation is to control flea and/or louse exposure. praziquantel at 5-12.5 mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against d. caninum (hall and simpson, 2000) . demodicosis canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles and is passed from dams to nursing pups. localized demodicosis is typically asymptomatic, but disease can present with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and face, and around the ears (demanuelle, 2000a). generalized demodicosis can develop in juvenile or adult populations and is indicative of an underlying immunosuppressive disorder. demodex has a characteristic 'cigar shape' and can be identified from deep skin scrapings mounted on mineral oil (campbell, 2000; noli, 2000) . differential diagnoses include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma, which is also a common secondary complication of generalized demodicosis. ivermectin at 200-600 μg/kg and oral milbemycin at 1-2 mg/kg/day are effective treatments. treatment duration can be extensive and must be accompanied by repeated skin scrapings. sarcoptic mange canine sarcoptic mange is caused by sarcoptes scabiei var. canis, which is zoonotic. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas of the ear pinnae, elbows, ventral thorax, and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. adult mites, mite eggs, or mite feces may be observed on superficial skin scrapings, but diagnosis may be difficult because multiple skin scrapings may yield negative results. even if scrapings are negative, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige either in the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, 2000) . histologic examination is nondiagnostic; however, suggestive lesions include small foci of edema, exocytosis, degeneration, and necrosis . an important differential diagnosis is flea allergy dermatitis (fad). unless antiparasitic therapy would interfere with research objectives, all dogs with sarcoptic mange should be treated. in addition, their kennel mates should also be treated due to the contagious nature of the disease and its zoonotic potential. the usual means of treatment is either ivermectin at 200-400 μg/kg q14 days or milbemycin at 2 mg/kg q7 days for three oral doses . ticks ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, amblyomma, and ixodes. the primary significance of tick infestation is vector-borne infectious diseases, including rocky mountain spotted fever (rickettsia rickettsii), lyme disease (borrelia burgdorferi sensu stricto), thrombocytic anaplasmosis (anaplasma platys), and canine monocytic ehrlichiosis (ehrlichia canis). ticks alone cause minimal signs unless the dog develops a hypersensitivity reaction leading to a more granulomatous response at the bite location (merchant and taboada, 1991) . some species (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that causes an ascending flaccid paralysis (malik and farrow, 1991) . uncomplicated tick bites and tick-bite paralysis are diagnosed by identification of the tick and clinical signs of paralysis. dogs with tick-bite paralysis usually show improvement within 24 h of tick removal, with complete recovery within 72 h (malik and farrow, 1991) . formamidines (amitraz), pyrethroids, and phenylpyrazoles (fipronil) are available as spot-ons, collars, sprays, and foggers to treat tick infestations in both the animal and the environment (halos et al., 2014; beugnet and franc, 2012) . differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, 1991) . fleas the most common flea to infest dogs is ctenocephalides felis felis, the cat flea (sousa, 2010) . flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop severe fad, which features papules, crusting, and excoriations over the lumbosacral region, flanks, thighs and abdomen. these animals may require oral corticosteroids to relieve clinical signs (muller et al., 1983) . secondary bacterial and fungal infections can also develop. fleas can also transmit other parasitic diseases, such as dipylidium tapeworms. flea infestations and fad are definitively diagnosed by observing the fleas on the host's skin; however, the presence of flea excrement can support a presumptive diagnosis (demanuelle, 2000b) . treatment of flea infestations should use an integrated pest management (ipm) approach that targets adult fleas, immature stages, and environmental contamination in order to limit the risk of chemoresistance. combining ovicidal treatments, such as lufenuron and selamectin, with adulticidal treatments, such as fipronil, spinosad, selamectin, and imidacloprid, is recommended (halos et al., 2014; beugnet and franc, 2012; dryden et al., 2012) . certain chemicals (i.e., imidacloprid and selamectin) have both adulticidal and larvacidal abilities, but the principles of ipm preclude the use of one product solely for both adulticidal and larvacidal properties (schwassman and logas, 2009 ). differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs. canine dermatophytoses are commonly caused by microsporum spp., trichophyton spp., and epidermophyton spp. (moriello and deboer, 2012) . uncomplicated infections are characterized by circular areas of alopecia and crusting with or without follicular papules, usually around the face, neck, and forelimbs. dermatophytes infect the hair shaft and follicle, as well as the surrounding skin. infected hairs become brittle and broken shafts remain infective in the environment for months. dermatophytoses are zoonotic and easily transmitted to other animals through the environment or by direct contact. definitive diagnosis is made using dermatophyte test medium for culture. hair and crust material from infected sites can be plucked and placed on culture; however, the 'toothbrush' method is more effective for sampling multiple sites. the brush is used to comb hairs and scales from several infected sites and then pressed into the culture media. media plates should be visually inspected daily for 14 days. positive cultures will become red at the same time as growth of a fluffy white colony. microscopic examination of hairs and scales to visualize fungal elements can be done using skin scrapings in 20% koh or mineral oil; however, this method is not very sensitive. topical and systemic therapy should be initiated together after all suspected areas are clipped to reduce spreading of contaminated fragile hairs. wholebody topical therapies with antifungal shampoos, rinses, and creams are recommended rather than spot treatment. systemic therapy can be achieved with griseofulvin, ketoconazole, itraconazole, or fluconazole. due to the highly infective nature of this disease, animals should be isolated and the environment thoroughly disinfected. chlorhexidine and virkon ® s are ineffective at clearing environmental spores, but lime sulfur (1:33), enilconazole (0.2%), and bleach (1:10) are effective across many strains of microsporum canis (moriello and deboer, 2002) . although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, 1994) , deficiency in thyroid hormone can significantly laboratory animal medicine affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology primary hypothyroidism affects the thyroid gland directly, whereas secondary hypothyroidism has indirect effects through dysfunction of the pituitary gland (seguin and brownlee 2012) . both of these causes result in a gradual loss of functional thyroid tissue (avgeris et al., 1990; kemppainen and clark, 1994) . the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, 1962; beierwaltes and nishiyama, 1968; manning 1979) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, 1989; kemppainen and clark, 1994) . clinical signs because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including nonpruritic, bilaterally symmetrical alopecia, hyperpigmentation, seborrhea, and pyoderma (avgeris et al., 1990; peterson and ferguson, 1989; panciera, 1994) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, 1989; panciera, 1994) . normocytic, normochromic, nonregenerative anemia may be seen in approximately one-half of the cases (avgeris et al., 1990) . increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, 1989; panciera 1994) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, 1989) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, 1989) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bichsel et al., 1988; panciera, 1994) , and a true causal relationship with hypothyroidism has not been completely defined (panciera, 1994) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, 1989) . hypothyroidism can also cause abnormalities of the cardiovascular system including bradycardia, hypocontractility, increased vascular volume, and atherosclerosis (seguin and brownlee 2012) . abnormalities that may be detected by ecg include a decrease in p-and r-wave amplitude (peterson and ferguson, 1989 ) and inverted t waves (panciera, 1994) . these ecg abnormalities are caused by lowered activity of atpases and calcium channel function. an association between hypothyroidism and von willebrand disease has been suggested. however, the relationship is probably one of shared breed predilection and not a true correlation. contradictory studies have shown either deficient (avgeris et al., 1990) or normal (panciera and johnson 1994, 1996; avgeris et al., 1990) von willebrand factor antigen and bleeding times in hypothyroid dogs. most importantly, hypothyroidism does not appear to cause overt, clinical von willebrand disease. however, it may exacerbate existing subclinical von willebrand disease (seguin and brownlee, 2012) . epizootiology the prevalence of hypothyroidism in the general canine population is reportedly less than 1% (panciera, 1994) . the disorder occurs most often in middle-aged, larger breed dogs (avgeris et al., 1990) , and reports suggest a higher incidence of hypothyroidism in spayed, female dogs (panciera, 1994; peterson and ferguson, 1989) . doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism compared with other breeds (panciera, 1994; peterson and ferguson, 1989; scarlett, 1994) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, 1979; tucker, 1962; beierwaltes and nishiyama, 1968) . diagnosis and differential diagnosis because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, 1989; ferguson, 1994) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t 4 (thyroxine) and free t 4 (peterson and ferguson, 1989; ferguson, 1994) . t 4 serves primarily as a precursor for t 3 and is heavily protein bound. free t 4 represents the laboratory animal medicine unbound fraction that is available to the tissues (peterson and ferguson, 1989) . the measurement of total t 4 carries a sensitivity of around 95% and can be used as a good screening tool. with the measurement of both serum total t 4 and free t 4 , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial may be in order (peterson and ferguson, 1989) . however, nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, 1989; ferguson, 1994) . therefore, low values do not always indicate that hypothyroidism is present and animals should not be treated solely on the basis of serum hormone levels if clinical signs are not present. if the clinical signs are equivocal or only total t 4 or free t 4 is decreased, further diagnostic testing is warranted (peterson and ferguson, 1989) . although t 3 is the most biologically active form of thyroid hormone, the measurement of serum t 3 levels is an unreliable indicator of hypothyroidism (peterson and ferguson, 1989; ferguson, 1994) . serum t 3 can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t 3 may be preferentially released and conversion of t 4 to t 3 may be enhanced by the failing thyroid (peterson and ferguson, 1989; ferguson, 1994) , particularly early in the disease. in one study, t 3 was within normal limits in 15% of the hypothyroid dogs (panciera, 1994) . autoantibodies can be responsible for false elevations in the concentrations of t 3 and t 4 found in these respective assays. it has been recommended that free t 4 , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t 3 and t 4 . autoantibodies have been found in less than 1% of the samples submitted to one laboratory (kemppainen and behrend, 2000) . other means of diagnosing hypothyroidism have been described. in humans, endogenous thyroid-stimulating hormone (tsh) levels provide reliable information on thyroid status, and an assay is available for dogs. however, endogenous tsh levels can be normal in some dogs with hypothyroidism and high tsh levels have been noted in normal dogs and sick animals that are actually euthyroid. it is therefore recommended that tsh levels be considered along with other information (clinical signs, t 4 ) prior to diagnosis and treatment (kemppainen and behrend, 2000) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, 1989; ferguson, 1994) . another drawback of tsh testing is that the test must be postponed for 4 weeks if thyroid supplementation has been given (peterson and ferguson, 1989) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is 0.045 u of tsh per pound of body weight (up to a maximum of 5 u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and 6 h after. a normal response to the administration of tsh should create an increase of t 4 levels at least 2 μg/dl above the baseline levels or an absolute level that exceeds 3 μg/dl (peterson and ferguson, 1989; wheeler et al., 1985) . treatment the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is 0.01-0.02 mg/kg once a day (avgeris et al. 1990 ). if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in 6-8 weeks, and blood samples should be drawn 4-8 h after the morning pill. a clinical response is usually seen in 6-8 weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, 1994) . ecg abnormalities also return to normal (peterson and ferguson, 1989) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, 1994) . weight gain and eventual obesity are frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must address obesity and its potential effects on animal welfare and research results. etiology obesity is defined as a body weight 20-25% over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy, the result of overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, 1992) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household because access to food is more restricted, and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and limitation to exercise reduces energy expenditure. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy laboratory animal medicine expenditure are followed (butterwick and hawthorne, 1998) . as in humans, genetics plays an important role in the development of obesity in dogs, and certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, 1986) . several metabolic or hormonal changes are also associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese compared with intact females (macewen, 1992) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, 1986 ). in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, 1992) . differential diagnosis the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict the total body fat (wilkinson and mcewan, 1991) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see above), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to 60% of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to 50% produces no adverse health effects. however, t 3 levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of 1-2% of body weight per week (laflamme et al., 1997) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to have rebound weight gain after restrictions are relaxed. there has been a great deal of attention in humans as to the correct diet to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, 1992) . there has been much concern about the addition of fiber to the diet as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorie-restricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick et al., 1994; butterwick and markwell, 1997) . research complications it is important to control weight gain in research animals because of the association of obesity with metabolism. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, 1992) , this relationship is not consistently apparent (edney and smith, 1986) . joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, 1992; kealy et al., 1997) . in addition, diabetes mellitus has been linked to obesity and obesity-induced hyperinsulinism in several experimental models (macewen, 1992) . a recent study demonstrated metabolic disease, typified by hyperinsulinemia and hypoadiponectinemia, in approximately 20% of obese dogs (tvarijonaviciute et al., 2012) . research that requires anesthesia may be complicated by a greater risk of cardiovascular diseases (edney and smith, 1986) including hypertension and compromise to the respiratory tract. etiology in the laboratory setting, the majority of traumatic wounds will be small in size and quickly observed. occasionally, dogs may sustain minor trauma during transport or have a small, previously undetected, chronic wound upon arrival at the facility. when dogs are group housed, they may sustain bite wounds during early socialization periods. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or when the basic principles of wound management are not followed. clinical signs the signs and appearance of a traumatic wound will vary with the cause and the duration of time since wounding. abrasions, sustained by shear forces, are partial thickness skin wounds characterized laboratory animal medicine by minimal bleeding or tissue disruption. puncture wounds have a small surface opening but penetrate into deep tissues with the potential for contamination. lacerations are wounds caused by sharp separation of skin that may extend to deeper tissues. acute wounds are characterized by bleeding tissue, sharp edges and no obvious devitalization. they have variable degrees of contamination. chronic wounds generally do not exhibit active bleeding and will have curled or rounded edges. these wounds often have necrotic tissue and are considered contaminated. treatment to aid decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a 'golden period.' it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the competence of the host's defense systems (swaim, 1980; waldron and trevor, 1993) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as clean, clean-contaminated, contaminated, or dirty (see table 12 .9). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds require more aggressive therapy. postsurgical infections or complications of initial therapy would be considered dirty wounds. when in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of its classification. when first recognized, the wound should be covered with a sterile dressing until definitive treatment can be rendered. bleeding should be controlled with direct pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, 1980) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, 1980) . anesthesia or analgesia may be necessary and the choice of agent will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed. then a water-soluble lubricant gel may be applied directly to the wound to prevent it from further contamination during the hair removal process. a wide margin of hair should be clipped and a surgical scrub performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., 1990a, b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. several irrigation solutions have been recommended (lozier et al., 1992; waldron and trevor, 1993; sanchez et al., 1988) , but type may not be as important as the volume and pressure of delivery. it has been suggested that 8 psi is required to obtain adequate tissue irrigation, and this may be achieved by using a 35-ml syringe with an 18-or 19-gauge needle (waldron and trevor, 1993) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, 1993) . if one is unsure about tissue viability in areas that are devoid of waldron and trevor (1993) . extra skin, the tissue may be left (swaim, 1980; waldron and trevor, 1993) , and nonviable areas will demarcate within 2-3 days (waldron and trevor, 1993) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, 1980) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be placed. subcutaneous closure should be performed with absorbable suture such as polydioxanone, polyglactin 910, or polyglycolic acid. it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon (3-0 or 4-0). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. the wound may be covered by a nonadherent dressing such as vaseline-impregnated gauze (swaim, 1980) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within 3-5 days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after 5 days, this is considered secondary closure (waldron and trevor, 1993) . second-intention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, 1980) . it is important to note that second-intention healing will take longer than with surgical repair, and, in the case of large wounds, it will be more expensive because of the cost of bandaging materials. several factors must be weighed when considering the use of antibiotics in traumatic wound care, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. topical application of bacitracin, neomycin sulfate, and polymixin b combinations may be used in wounds with minor contamination. in skin wounds with more extensive contamination, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. when systemic antibiotics are necessary, cephalosporins, amoxicillinclavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, 1993) . prevention in facilities with good husbandry practices and a diligent staff, potentially injurious equipment or surfaces are identified quickly. appropriate attention to surgical technique and to initial wound care will generally reduce the occurrence of postprocedure wound infection. etiology pressure sores (decubital ulcers) can be a problem in long-term studies and housing situations that require chronic skin contact with hard surfaces. decubital ulcers often develop over a bony prominence such as the elbow, tuber ischii, tarsus, or carpus. the compression of soft tissues between hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, 1990) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores, including poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting coaptation devices (swaim and angarano, 1990) . clinical signs initially, the skin will appear red and irritated. over time, constant trauma can result in full-thickness skin defects and can progress to necrosis of underlying tissues. the severity of the sores may be graded from i to iv according to the depth of the wound and the tissues involved, from superficial skin irritation to involvement of underlying bone (waldron and trevor, 1993) . epizootiology the problem usually occurs in large dog breeds, but any type of dog can be affected. prevention and control minimizing or eliminating predisposing factors is important to both the prevention and treatment of this condition. if a dog will experience long periods of recumbency, adequate bedding or padding must be provided. recumbent animals should be moved frequently, ideally every 2 h, to prevent continuous compression on a specific laboratory animal medicine area (waldron and trevor, 1993) . skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain body weight will minimize compressive forces experienced over areas susceptible to ulceration (swaim and angarano, 1990) . treatment the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive diagnostics and therapy must be performed. the affected area should be radiographed to assess bone involvement and the wound should be cultured. all of the damaged tissue should be debrided and basic wound management guidelines should be followed (see above). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, 1990) . with extensive lesions, reconstruction with skin flaps may be necessary (waldron and trevor 1993) . bandaging should be performed on all full-thickness wounds; however, it is important to remember that illfitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound, in order to displace pressure over a larger area and onto healthier tissue. the doughnut is then incorporated into a padded bandage. if a cast has been applied to the area for treatment or research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, 1990) . bandages should be removed at least once or twice a day to allow wound care. etiology an acral lick granuloma is a skin lesion caused by self-trauma. in a few cases, the self-trauma is due to initial irritation caused by an identifiable neurologic or orthopedic condition (tarvin and prata, 1980) . allergy may also be a source of irritation that leads to self-trauma. however, the majority of cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, 1990) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., 1988) . the laboratory environment could promote the abnormal behavior and lead to acral lick granuloma. epizootiology the lesions associated with acral lick granuloma are seen most often in large dog breeds, particularly dobermans. however, any type of dog can be affected (walton, 1986) . clinical signs early lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, 1990) . the predilection for the limbs may be due to accessibility or possibly a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated and the wound develops a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of the aforementioned problems can be ruled out by the history of the animal. however, a complete history may be unavailable in the laboratory setting. fungal cultures and allergy testing may aid in diagnosis. biopsy of the affected area would rule out neoplasia. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, 1986) . prevention and control behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. environmental enrichment including exercise, co-housing and various toys is already a basic requirement and may be increased to combat self injurious behaviors. treatment several treatments have been reported for acral lick granuloma and the selection of a treatment should be based on the underlying cause. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, 1990; walton, 1986) . opioid antagonists have been applied as treatments for acral lick granulomas and self-injurious behaviors with the theory that this will block the effects of endogenous opioids. naltrexone and nalmefene have been used successfully to reduce excessive licking behaviors and resolve associated lesions. however, lesions did recur after the drugs were discontinued (dodman et al., 1988; white, 1990) . the topical administration of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide has also been shown to be effective (walton, 1986) . in addition, psychoactive drugs have been suggested to relief of laboratory animal medicine boredom or anxiety. these have included phenobarbital, megestrol acetate, and progestins. however, side effects have been reported (swaim and angarano, 1990) . other behavior-modifying medications such as clomipramine may be effective in the treatment of compulsive anxiety disorder. the potential for effects that could interfere with experimental results must be determined prior to initiation of treatment. it is important to note that none of the above-mentioned treatments have been successful in all cases. the overall prognosis for acral lick granuloma should be considered guarded since the lesions often recur when treatment is discontinued. etiology hygromas are fluid-filled sacs that develop as a result of repeated trauma or pressure over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., 1974) . epizootiology elbow hygromas are most frequently reported in large and giant breeds of dogs, less than 2 years of age (johnston, 1975; white, 2003; cannap et al., 2012) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment of research dogs, especially cage bottoms and cement runs, may predispose them to hygromas. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs the clinical presentation will depend upon the chronicity of the problem. a dog with an elbow hygroma usually presents with a painless, fluctuant swelling over the point of the elbow without signs of lameness. the condition may be unilateral or bilateral. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma becomes secondarily infected, the animal may exhibit pain and fever (johnston, 1975; white, 2003) . pathology the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is a yellow or red serous transudate. this fluid is less viscous than joint fluid and elbow hygromas do not communicate with the joint (johnston, 1975) . treatment the treatment of elbow hygromas should be conservative whenever possible. conservative management of the elbow hygroma is aimed at relieving the source of pressure at the point of the elbow. in early and mild cases, simply providing padding to cover hard surfaces will result in resolution of the hygroma. a soft padded bandage or doughnut bandage around the affected site may also be of benefit. neoprene/polyester sleeves that cover the elbows and fit over the shoulders are also available as an option for either prevention or treatment of hygromas (cannap et al., 2012) . more aggressive therapies, including needle drainage and injection of corticosteroids into the hygroma, have been described but are not recommended due to the risk of infection (johnston, 1975) . surgical options should be reserved for complicated or refractory cases. even simple excision can be associated with complications such as wound dehiscence and ulceration (johnston, 1975) due to the location of the bony prominence at the surgical site. this issue may be avoided by using a skin advancement flap (white, 2003) that allows intact, healthy skin to cover the boney prominence. a muscle advancement flap has also been described (green et al., 2008) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology in the research environment, corneal ulcers are most often associated with direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these would be rare in the laboratory setting. clinical signs the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may appear normal however, in cases of deeper ulceration, the cornea may appear roughened or have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. diagnosis a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers is made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies, abnormal eyelids, or abberant cilia. treatment the treatment of corneal ulcers will depend on the depth and size of the affected area, as well as the underlying cause. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given three times a day for 2-3 days usually provides adequate treatment. simple corneal ulcers are restained with fluorescein after 3 days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. deep ulcers may require further debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. ulcers caused by entropion, ectropion, or dystichiasis will not resolve until the condition is repaired, and descriptions for this can be found elsewhere. prevention the proper application of lubricant eye ointment at the time of anesthesia will prevent drying due to exposure and may also protect the eye from scrub solutions applied near the eye. early treatment of superficial ulcers should prevent self-trauma and progression of the wound. etiology research protocols often require the placement of chronic implants. implants such as cardiac or other biomedical devices may be the primary focus of the research study. implants may also be used as chronic monitoring devices, for delivery of compounds, or to collect serial samples. infection may occur at the time of implant. alternatively, the implant may serve as a nidus after hematogenous spread from other sources. one of the most common sources of infection is from colonization of the device from an external component, which is a frequent complication with indwelling catheters. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. one study (hysell and abrams, 1967 ) examined the lesions found at necropsy in animals with chronic indwelling catheters, which included traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. these lesions were primarily associated with catheter-induced trauma or secondary to embolization of fibrin. in a veterinary clinical setting, infections in peripheral catheters were more likely when the catheters were used for blood collection immediately after placement and when a 't' connector rather than a 'y' connector was used. . intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., 1993) . the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., 1993; kwei et al., 1995) . clinical signs dogs with implant infections may not exhibit signs initially . localized swelling around the implant may occur. in the case of indwelling catheters, signs may include redness and swelling of the skin around the external port or discharge from the skin wound. vascular access ports may develop fluctuant subcutaneous abscesses. in more severe cases, systemic signs may be noted (bach et al., 1998; hysell and abrams, 1967) . the systemic signs of infection are covered elsewhere in this chapter. treatment the treatment of catheter infections almost invariably requires removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, 1984; darif and rush, 1983 ). superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes, and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. localized abscesses or sinus tracts may be managed by establishing drainage and copious flushing. aerobic and anaerobic cultures of blood and locally infected sites should be performed prior to initial treatment (ringler and peter, 1984) . systemic antibiotic therapy should be initiated for a 10-day period. the choice of drug will ultimately be based on previous experience and culture results. if retention of a catheter is important, the catheter lumen may be safely disinfected with chlorine dioxide solution (dennis et al., 1989) . the solution is removed after 15 min and replaced with heparinized saline. all of the extension lines and fluids used with an infected catheter should be discarded. the blood cultures should be repeated 3 days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. prevention it is highly desirable to prevent complications that may result in loss of an implanted device. catheters and other implants should be made of nonthrombogenic material and be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. intravenous catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. ideally, catheters are secured to reduce movement and irritation of the skin, which may predispose to infection around external ports. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that long extension tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, 1984) . for intestinal access ports, catheter security may be improved with a synthetic cuff added to the end of the catheter allowing better attachment to the intestine (meunier et al., 1993) . after any catheter placement, animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide. in addition, a solution of the antibiotic ceftazidime used on alternate days with the heparin locking solution has been shown to effectively reduce infections in indwelling vascular catheters (bach et al., 1998) . throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. periodically, the placement of an indwelling catheter may be verified by radiography. when placed and managed correctly, catheters and ports of any kind may remain in place for months without complications. etiology sepsis is defined as the systemic response to infection caused by bacteria (gram negative and/or gram positive), fungi, or viruses. in laboratory animals, sepsis is most often seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course, dogs may present with signs of a hyperdynamic sepsis, including increased heart rate, increased respiratory rate, red mucous membranes, and a normal-to-increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals may show classic signs of septic shock including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, 1993) . pathogenesis the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell (wbc) count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of band neutrophils than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., 1997) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several areas should be evaluated for infection, including the urinary, reproductive, respiratory, alimentary, and cardiovascular systems, as well as the abdominal cavity (kirby, 1995) . treatment the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is infected, it should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or thirdgeneration cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. this category of sepsis treatment is the focus of much research. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no 'magic bullet' for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology in research animals, aspiration may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. pathogenesis aspirated compounds can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response, probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after 24-48 h. clinical signs the severity and clinical manifestation of aspiration lung injury are dependent upon the ph, osmolality, and volume of the aspirate. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on witness of aspiration, history consistent with aspiration, and/or the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. treatment the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. if the aspiration is witnessed, the mouth and, idealy, the upper airway should be cleared of residual material. when small amounts of a relatively innocuous substance (e.g., barium) have been aspirated, treatment may not be necessary. when severe inflammation is present, systemic as well as localized therapy may be necessary. oxygen therapy may be instituted; however, the concentration and time frame are controversial, because lung injury may be exacerbated by long-term administration of oxygen at high concentrations (nader-djahal et al., 1997) . fluid therapy may also be necessary in severe cases; however, cardiovascular support should be performed judiciously as fluid overload could lead to an increase in pulmonary edema. the use of colloids is also controversial because of the increase in vascular permeability that occurs in the lungs. several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. corticosteroids are contraindicated (raghavendran et al., 2011) . in humans, antibiotics are reserved for cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be immediately treated with antibiotics when the aspirated material is not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, 2000) . the presence of pneumonia should be verified by tracheal wash and cultures. prevention aspiration of drugs and other compounds may be avoided through careful administration of oral medications by experienced individuals. likewise, gavage or orogastric administration of liquids should be performed by experienced individuals, and the procedure should be aborted if coughing or other respiratory signs occur. the aspiration of stomach contents can largely be avoided by appropriate fasting prior to anesthesia for at least 12 h for food and 2 h for water. if appropriate fasting times are not observed, anesthesia should be postponed whenever possible, particularly if intended procedures require manipulation of the viscera or head-down positioning of the dog. if anesthesia cannot be avoided, it should be rapidly induced and the dog should be intubated. during recovery from anesthesia, the endotracheal tube should be removed with the cuff partially inflated and with the dog in a head-up position (haskins, 1993) . based on the source of energy, burn wounds may be categorized into four groups: thermal, chemical, radiation, and electrical. in laboratory animals, accidental burns are usually the result of thermal injury (heating pads, water bottles), chemicals (strong alkalis, acids, disinfectants, drugs), or experimental irradiation protocols. etiology inappropriate use of external heating devices is the most common cause of burns in laboratory animal medicine. the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs the clinical signs vary with the depth, location, and surface area of burn injury. classification systems for thermal burns are generally based on the depth of the injury, varying from superficial involvement of only epidermis to complete destruction of skin and subcutaneous tissues (bohling, 2012) . superficial burns appear erythematous and inflamed. in some cases, matting of the overlaying hair with exudate may be the first sign of a previously undetected skin lesion. progressive hair and skin loss may be evident over the first few days after injury (johnston, 1993) . although blistering is a characteristic of partial thickness burns in humans, this is rarely seen in dogs (bohling, 2012) . uncomplicated, superficial burn wounds heal by reepithelialization within 3-5 days. deeper burn wounds are characterized by a central area of nonviable tissue surrounded by edematous, inflamed tissues. a thick eschar, composed of the coagulated proteins and desiccated tissue fluid, develops over deep burn wounds. these wounds heal by granulation under the eschar, which will eventually slough. the amount of pain associated with burns depends upon several factors including the depth and area of the wound, procedural manipulations, and movement at the affected site (bohling, 2012) . pain associated with superficial burn wounds usually subsides in 2-3 days. theoretically, deep burns destroy nerve endings and result in less pain than superficial burns. however, inflammatory pain may still be present due to the tissue reaction around the necrotic site. in addition, sharp procedural pain and breakthrough pain have been described in humans during the healing phases of burn injuries and should be considered as potential complications in dogs as well (bohling, 2012) . severe and widespread accidental burn injury can result in clinical signs associated with multiple organs including the pulmonary, gastrointestinal, hematopoietic, and immune systems. in addition, extensive burn injury can predispose to infection and even sepsis. this type of injury with the associated complications would be extremely rare in the laboratory setting. treatment appropriate and timely treatment of a burn wound will reduce the extent of tissue damage and associated pain. thermal injuries should be immediately exposed to cool water (15°c) to reduce edema and pain. exposure to very cold water and ice does not improve outcomes (bohling, 2012) . topical wound dressings are recommended in the early stages of treatment for both partial-and full-thickness burns that are of small size. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, a thin film of a water-soluble, broad-spectrum antibiotic ointment should be applied to the wound surface. silver sulfadiazine has a broad spectrum, penetrates eschar, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it has been associated with pain upon application (demling and lalonde, 1989) . once a topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds (demling and lalonde, 1989; bohling, 2012) . after the initial treatment, burn wounds should be gently cleansed two to three times a day, followed by reapplication of the topical antibiotic and rebandaging (demling and lalonde, 1989) . systemic antibiotics are indicated in cases where local or systemic infection is present and their ultimate selection should be based on culture results. burn wounds can be extremely painful, and analgesia should be instituted immediately and adjusted accordingly throughout the treatment period. surgical intervention may be necessary in some cases. with small or moderately sized wounds, the eschar over the burn wound may actually impede wound contraction and reepithelialization. in such cases, once the eschar has become fully defined, a complete resection may improve wound healing. with large and severe burn wounds, repeated debridement by surgery or other means might be necessary. in the laboratory setting, a decision to pursue extensive surgical intervention would be dependent upon full consideration of the effects on animal welfare and research results. prevention thermal burns can be prevented in the research setting. electric heating pads and heat lamps should be avoided if possible. only heated water blankets or circulating warm air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore, these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. basic fire prevention precautions should be taken particularly around oxygen sources and flammable agents etiology chemical injury may be due to skin contact with concentrated solutions such as disinfectants or inadvertent exposure to laboratory chemicals. in addition, perivascular injection of certain drugs (pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin) have been associated with extensive tissue damage (swaim and angarano, 1990; waldron and trevor, 1993) . the mechanism of action will vary depending upon the ph, osmolality, and chemical composition of the agent and may include oxidation, reduction, disruption of lipid membranes, or other reactions (bohling, 2012; swaim, 1990; waldron and trevor, 1993) . clinical signs surface contact with chemicals may result in mild irritation and redness of superficial layers of the skin. however, many agents may cause progressive injury until the chemical reaction has been neutralized. this may result in tissue necrosis and secondary infection. the immediate signs of perivascular injection are withdrawal of the limb or other signs of discomfort and swelling at the injection site. the area may appear red, swollen, and painful as inflammation progresses. there may eventually be necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a 1-to 4-month period. this is because the drug is released over time from the dying cells (swaim and angarano, 1990) . treatment in cases of skin contact with chemical agents, the affected area should be thoroughly and repeatedly lavaged with warm water to dilute or remove the substance. the material safety data sheet for the substance should be consulted for any possible neutralization protocol. additional treatment will depend upon the severity of the tissue damage and will follow the same guidelines as for the thermal injury described earlier. for the treatment of perivascular injections, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions. the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, 1989) . the local infiltration of hyaluronidase accompanied by warm compresses has been suggested for perivascular vinblastine (waldron and trevor, 1993) and for doxirubicin. the use of dmso or another free radical scavenger, dexrazoxane, infused at the site has also been suggested for doxorubicin toxicities. despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, 1990) . in all cases, the condition can be painful and analgesia should be addressed. prevention prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. the material safety data sheets should be available for all compounds and storage recommendations followed closely. for intravenous administration of toxic compounds, insertion of an indwelling catheter is extremely important. prior to the injection, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. etiology radiation burns are generally a complication of therapeutic administration and are a result of free oxygen radical formation (waldron, 1993) . the severity of radiation burns and their treatment will depend upon the dose, frequency, total surface area, and location of the radiation. damage to epithelial layers of the skin can lead to desquamation. direct injury to fibroblasts results in decreased collagen production and poor wound healing. in addition, there may be fibrosis of blood vessels (pavletic, 2010) and subsequent hypoxia causing necrosis of deeper tissues. clinical signs the tissues most often affected are the skin and mucus membranes. with superficial injury, affected skin may exhibit hair loss and erythema, and produce a clear exudate. the intensity of the inflammation may increase for 1-2 weeks after the completion of radiation treatment. deeper and more serious injury manifests with subcutaneous fibrosis and can lead to disfigurement . the skin and underlying deep structures including the bone may become necrotic over several weeks (pavletic, 2010) . these deeper injuries are prone to infection due to their lack of blood supply. systemic signs such as vomiting are rare in dogs unless there has been direct radiation treatment to organs . treatment with superficial skin burns, the wound should be kept clean and should be covered if possible. in cases of oral mucous membrane damage, there may be special feeding requirements. when wounds are ulcerated, avascular tissues should be excised. treatments with silver sulfadiazine, mafenide acetate, or other topical agents are recommended to control infection. in addition, infection is avoided by closure of the wound as soon as possible. the goal of surgery is to cover the wound with healthy tissue to promote vascularization of the area. in some cases, this may require muscle and/ or skin grafts. prevention radiation burns can be limited by selection of appropriate, fractionated therapy and application of shielding to reduce exposure. prompt treatment of the injuries can reduce the occurrence of infection. since radiation is associated with poor wound healing, complications may arise when additional procedures are required. it is recommended to wait at least 1 week (pavletic, 2010) or even longer (laing, 1990) prior to administering radiation to a surgical site. after radiation, routine surgeries should be avoided for 1-2 months (pavletic, 2010 ). the prevalence of cancer in the general canine population has increased over the years (dorn, 1976) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, 1982) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. (1994) found death rates similar to the death rate of the at-large dog population (bronson, 1982) . approximately 22% of the male beagles died of cancer. the majority of the tumors were lymphomas (32%) and sarcomas (29%), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer (26% of the population studied), three-quarters had mammary cancer (40%), lymphomas (18%), or sarcomas (15%). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, 1975; benjamin et al., 1996) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. a complete review of clinical oncology in the dog is beyond the scope of this chapter but can be found elsewhere (withrow et al., 2013) . fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients with local anesthesia. an instrument such as a tru-cut ® needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a 1-mm × 1-1.5-cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies aid in histopathological examination and are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least 1 cm around the tumor and 3 cm if mast cell tumors are suspected (morrison et al., 1993) . incisional biopsies are performed when large softtissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. canine lymphoma represents 5-7% of canine tumors and a majority (85%) of canine hematopoetic disease (ettinger, 2003; vail and young, 2013) . whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide 2,4-dichlorophenoxyacetic acid as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, 1991) . in addition, tobacco smoke, environmental chemicals, and waste emissions are considered possible risk factors (marconato et al., 2009; gavazza et al., 2001) clinical signs multicentric high-grade lymphoma (mhgl) accounts for the majority of reported cases of canine lymphoma. depending upon grade, immunophenotype, and location involved, dogs with mhgl usually present with painless, enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, fever, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. less commonly, dogs develop alimentary, mediastinal, cutaneous, and extranodal lymphomas. alimentary lymphoma is associated with vomiting and diarrhea, in addition to clinical signs associated with mhgl. dogs with mediastinal lymphoma often present with respiratory signs (dyspnea and exercise intolerance) secondary to pleural effusion or cranial vena caval syndrome. hypercalcemia is most frequently associated with this form of lymphoma and may result in polyuria, polydypsia, and weakness. cutaneous lymphoma is an uncommon epitheliotrophic form of lymphoma. it is often referred to as mycosis fungoides and is typically of a cd8+ t-cell immunophenotype. it varies in presentation from solitary to generalized and may mimic any of a number of other inflammatory skin disorders including oral mucosal lesions. the lesions may occur as erythema, plaques, erosions, scales, nodules, crusts, hypopigmentation, and alopecia (fontaine et al., 2009) . approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement (e.g., nervous system: seizures, paresis, paralysis). epizootiology the incidence of lymphoma is highest in dogs 5-11 years old, accounting for 80% of cases. although the neoplasm generally affects dogs older than 1 year, cases in puppies as young as 4 months have been reported (dorn et al., 1967) . no gender predilection has been reported. diagnosis and pathologic findings a fine-needle aspirate is initially performed on accessible lymph nodes. thoracic radiographs and abdominal ultrasound ± fine needle aspiration of the liver or spleen can be used if mediastinal or abdominal involvement is suspected. additional staging can be determined through complete blood counts, serum biochemistry, flow cytometry for immunotyping, bone marrow aspiration, or surgical lymphadenectomy and histology. enlarged neoplastic lymph nodes vary in diameter from 1 to 9 cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. flow cytometry and lymphoblastic markers (cd34) can aid in diagnosis and subtyping of tumors. in addition, positron emission tomography is being explored for detection of extranodal and metastatic lymphoma (leblanc et al., 2009; marconato, 2011; elstrom et al., 2003) . classification of lymphoma types is based upon cytological, morphological, and immunological characteristics using the kiel classification criteria (vail and young, 2013) . histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. lymphoma subtypes can be further characterized based upon genetic, molecular, and immunological criteria (ponce et al., 2004) . pathogenesis all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is 4-6 weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, 1991) . median survival time with aggressive therapy is generally less than 12 months. hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. differential diagnosis differential diagnoses for multicentric lymphoma include systemic mycosis; salmon-poisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections for histopathologic examination may be needed. treatment therapy for lymphoma primarily consists of one or a combination of several chemotherapeutic agents. in addition, radiation therapy and bone marrow transplantation have been utilized. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. although treatment may induce clinical remission and prolong short-term survival, most treatment is palliative and aimed at improving quality of life. a thorough discussion of therapeutic options for the treatment of lymphomas in the dog can be found elsewhere (chun, 2009; marconato, 2011) . future directions include development of molecular and cellular targeted therapies to enhance traditional chemotherapy treatment, prolong remission, and treat immunologic subtypes of lymphoma (e.g., t-cell lymphoma). research complications given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with significant clinical illness. etiology mast cells are derived from cd34+ bone marrow progenitor cells. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog and may account for up to 21% of canine skin laboratory animal medicine tumors (bostock, 1986; welle et al., 2008) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin, lung, liver, and gastrointestinal tract. current research has linked mast cell tumor development to multifactorial causes including breed predisposition and a genetic component, chronic inflammation, and mutations in the surface growth factor, c-kit (ma et al., 1999; reguera et al., 2000; webster et al., 2006) . clinical signs well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, 1-to 10-cm nodules in the dermis and subcutaneous tissue. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema to surrounding tissues. mast cell tumors can be found on any portion of the dog's skin but frequently affect the trunk and hind limb extremeties along with perineal and preputial areas. the tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. abdominal organs are rarely involved but may be associated with anorexia, vomiting, melena, abdominal pain, and gastrointestinal ulceration. mast cell tumors have also been reported in extracutaneous areas such as the salivary glands, larynx, nasopharynx (london and thamm, 2013) and conjunctiva (fife et al., 2011) . mast cell tumors within the perineal, preputial, or inguinal areas are associated with a greater predilection for recurrence or metastasis (misdorp, 2004) . epizootiology these tumors tend to affect middleaged dogs but have been observed in dogs ranging from 4 months to 18 years (pulley and stannard, 1990) . pathologic findings because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. (1986) has become widely accepted. in this system, grade i has the best prognosis and are well differentiated, with round to ovoid, uniform cells with distinct cell borders. the nuclei are round and regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have the worst prognosis. the cells contain large, irregular nuclei with multiple prominent nucleoli and few cytoplasmic granules. mitotic figures are much more frequent. cells are pleomorphic with indistinct borders. in addition to associated skin lesions (e.g., ulceration, collagenolysis, necrosis, and infection), mast cell tumors have been associated with gastric ulcers in the fundus, pylorus, and/or proximal duodenum, most likely secondary to tumor production of histamine. histamine stimulates the h 2 receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (>75%) of dogs with mast cell tumors (howard et al., 1969) . pathogenesis although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver, and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis using fineneedle aspiration, mast cell tumors can be distinguished cytologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue to metachromatically stain the cytoplasmic granules red or purple. mast cell granules can also be stained with wright's, giemsa, and romanowsky stains. in addition, mast cells may contain tryptase, chymase, or both (fernandez et al., 2005) . histological evaluation is generally required for grading. examination of regional lymph nodes may be warranted if metastatic or systemic disease is suspected. in addition, radiographs and ultrasound with guided aspirates of the liver, spleen, or sublumbar lymph nodes can be used to determine metastatic disease. treatment depending upon the grade, initial treatment for mast cell tumors is generally wide surgical excision (3-cm margins), which may be followed by radiation, chemotherapy, or glucocorticoid therapy. aspiration or surgical removal of regional lymph nodes is recommended if lymphatic tumor drainage is suspected. if the tumor is not completely resectable or is grade ii or iii (moderately to undifferentiated), then debulking surgery and adjunct therapy may be used. treatment algorithms are outlined elsewhere (withrow et al., 2013) research complications because of the potential for systemic release of substances such as histamine, vasoactive substances, heparin, eosiniphilic chemotactic factor, and proteolytic enzymes, along with the possibility of delayed wound healing and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed monthly. grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every 3-6 months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the canine transmissible venereal tumor (ctvt) is transmitted horizontally to the genitals by coitus (nielsen and kennedy, 1990) . ctvt is a 'parasitic-like' tumor that appears to have originated from dogs or wolves thousands of years ago and despite immense mutation, ctvt adapted, survived, and spread across multiple continents making it the oldest known continuously passaged somatic cell line (rebbeck et al., 2009; murchison et al., 2014; murgia et al., 2006) . it has been described as a round cell tumor of histiocytic origin. although this tumor has been reported in most parts of the world, it is most prevalent in tropical or temperate climates (macewen, 1991) . clinical signs the tumors are usually cauliflowerlike masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to 10 cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis. less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission ctvts are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for exfoliation and transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, 1990) . extragenital lesions may be the result of oral contact with previously traumatized areas. pathologic findings histologically, cells are arranged in compact masses or sheets. the cells are round, ovoid, or polyhedral, and have large, round nuclei with coarse chromatin. the cytoplasm is eosinophilic with small vacuoles arranged in a 'string of pearls' pattern. pathogenesis tumor growth occurs within 2-6 months after mating or implantation, and then growth generally slows. metastasis is rare (<5-17% of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. spontaneous regression may occur within 6-9 months of tumor development. diagnosis and differential diagnosis transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. however, cytological examination of impression smears, swabs, and fine-needle aspirates generally provide a definitive diagnosis. although not usually required, histopathology of a biopsy from the mass can aid in diagnosis. prevention thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control removing affected individuals from a breeding program should stop further spread through the colony. treatment surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine (0.5-0.7 mg/m 2 ) iv once weekly for four to six treatments will induce remission and cure in greater than 90% of the cases (macewen, 1991) . research complications experimental implantation of ctvts has been shown to elicit formation of tumor-specific igg (cohen, 1972) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs single nodules are found in approximately 75% of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger, invasive, and coalescent with adjacent tissues. inflammatory mammary carcinomas may mimic mastitis or severe dermatitis and must be ruled out to prevent misdiagnosis. epizootiology mammary tumors are uncommon in dogs under 5 years of age with the incidence rising sharply after that. the median age at diagnosis is 10-11 years. a longitudinal study of a large beagle colony showed that significant risk for development of mammary tumors begins at approximately 8 years of age (taylor et al., 1976) . mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings the t (tumor size), n (lymph node involvement), and m (metastasis) system is commonly used to stage mammary tumors. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, 1977) . histopathologic grades are scored based upon tubule formation, nuclear pleomorphism, and mitosis (elston and ellis, 2002) . extensive discussions of classification, staging, and histopathologic correlations can be found elsewhere (moulton, 1990; sorenmo et al., 2011) . pathogenesis mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in 60-70% of tumors. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs and extraskeleton. diagnosis and differential diagnosis both benign and malignant mammary tumors must be distinguished from mammary hyperplasia, mastitis, and severe dermatitis. cytological evaluation from fine-needle aspirates correlates well with histological examination of benign and malignant tumors (simon et al., 2009) . radiographs and possibly ultrasound should be performed to rule out metastatic disease prior to surgery. prevention the lifetime risk of developing mammary tumors can effectively be reduced to 0.5% by spaying bitches prior to the first estrus (schneider et al., 1969) . this is commonly done in the general pet population at 6 months of age. the protective effects of early spay rapidly decrease after several estrus cycles. dogs spayed prior to the first estrus had a risk of 0.8%, whereas dogs spayed after the first and second estrus had risks of 8% and 26%, respectively. treatment surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. single mammary tumors should be surgically removed with 2-cm lateral margins or margins wide enough for complete resection. deep margins may include removing sections of abdominal fascia or musculature en bloc with mammary tumor. multiple mammary tumors should be removed via regional or unilateral chain mastectomies. bilateral, staged mastectomies are reserved for more aggressive tumors. there is insufficient evidence at this time to recommend routine complete unilateral or bilateral chain mastectomies. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. sorenmo et al. (2013) provide a thorough review of canine mammary gland neoplasia. research complications treatment of early-stage or low-grade mammary tumors may be rewarding, allowing dogs to continue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. beagles are subject to many of the inherited and/ or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, 2000) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm 1 gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). other defects observed include cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than 1.0% incidence. etiology benign prostatic hyperplasia (bph) is an age-related condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs. epizootiology and transmission bph typically affects older dogs (>4 years), although glandular hyperplasia begins as early as 3 years of age. approximately 95% of inact male dogs will develop bph by 9 years of age (smith, 2008) . pathologic findings in the early stages of bph, there is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular with a honeycomb appearance (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to 5α-dihydrotestosterone (kustritz and klausner, 2000) mediating bph. diagnosis and differential diagnosis bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis that may be characterized by hemorrhage. a prostatic biopsy can be performed to confirm diagnosis. differential diagnoses include squamous metaplasia of the prostate, para-prostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve with castration. prevention castration is the primary means for prevention of benign prostatic hyperplasia. treatment the first and foremost treatment for bph is castration. in pure cases of bph, castration results in involution of the prostate gland detectable by rectal palpation within 7-10 days. for most dogs in research studies, this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases where semen collection is necessary from a valuable breeding male (e.g., genetic diseases). if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. finasteride, a synthetic 5α-reductase inhibitor, has been used in dogs to limit the metabolism of testosterone to 5α-dihydrotestosterone. treatment at daily doses of 0.1-0.5 mg/kg orally for 16 weeks was shown to reduce prostatic diameter and volume without affecting testicular spermatogenesis (sirinarumitr et al., 2001) . upon discontinuation of finasteride, the prostate generally returns to its pretreatment size within several months (smith, 2008) . gonadotropin-releasing hormone analogs such as desorelin inhibit production of testosterone and estrogen via negative feedback on the hypothalamus-pituitary axis. this is available in a sustained release subcutaneous implant, which has demonstrated efficacy in reducing prostatic size in dogs (junaidi et al., 2009) . however, medical therapy has not shown to be as advantageous as castration. research complications bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. research model older dogs with benign protastic hypertrophy are used in research to evaluate the use of ultrasonic histotripsy as a precise nonsurgical urethralsparing alternative to prostate surgery (lake et al., 2008; hall et al., 2009; schade et al., 2012) . etiology juvenile polyarteritis syndrome (jps), also known as steroid-responsive meningitis-arteritis, is a painful disorder seen in young beagles (occasionally reported in other breeds) caused by a systemic necrotizing vasculitis. the cause of the vasculitis has not been established but appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to have a hunched posture and/ or an extended head and neck. most dogs seem to be in pain when touched, especially in the neck region. neurological examination may reveal proprioceptive deficits, paresis, or paralysis. the syndrome typically has a course of remissions and relapses characterized by 3-7 days of illness and 2-4 weeks of remission (scott-moncrieff et al., 1992) . there may be a component of this condition that is subclinical, given that vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission jps typically affects young beagles (6-40 months), with no sex predilection. jps has been reported in other breeds including sibling welsh springer spaniels (caswell and nykamp, 2003) . pathologic findings on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., 1995) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to mediumsized arteries are seen. these lesions are most noticeable where gross lesions are observed, but they may be seen in other visceral locations. arterial fibrinoid necrosis leading to thyroid gland hemorrhage and inflammation was also reported (peace et al., 2001) . the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries. the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., 1995) . fibrinous thrombosis of the affected arteries is also seen. a subclinical vasculitis has also been diagnosed in beagles post mortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclic nature and respond to treatment with corticosteroids, and the affected dogs have elevated α 2 -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resulted in one in seven affected pups (scott-moncrieff et al., 1992) . diagnosis and differential diagnosis differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). prevention and control no prevention and control measures are known at this time. treatment clinical signs can be abated by administration of corticosteroids. prednisone administered orally at 1.1 mg/kg, q12 h, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of 0.25-0.5 mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. research complications because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes. the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym 'sterile pyogranuloma complex'). clinical signs dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, 1993) . a retrospective study of beagles housed in a research industry setting, linked development of interdigital cysts to body codition score, age, location of cyst, and type of caging, and may result from chronic interdigital dermatitis (kovacs et al., 2005) . pathologic findings histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at 1 mg/kg q12 h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require surgical removal. excision includes the removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, 1993) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weight-bearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, 1993) . research complications research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. etiology 'cherry eye' is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating membrane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit. excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. pathologic findings typically, the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, 1989) . prevention hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, 1989 ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland or its removal might compromise ophthalmologic studies. antimicrobial resistance profiles and mechanisms of resistance in campylobacter jejuni isolates from pets life span and cancer mortality in the beagle dog and human studies of distribution and recurrence of helicobacter spp. gastric mucosa of dogs after triple therapy plasma von willebrand factor concentration and thyroid function in dogs catheter-related infections in long-term catheterized dogs. observations on pathogenesis, diagnostic methods, and antibiotic lock technique aaha nutritional assessment guidelines for dogs and cats open-and closed-formula laboratory animal diets and their importance to research comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma respiratory infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog insecticide and acaricide molecules and/ or combinations to prevent pet infestation by ectoparasites neurologic manifestations associated with hypothyroidism in four dogs veterinary surgery: small animal neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis variation in age at death of dogs of different sexes and breeds comparison of campylobacter carriage rates in diarrheic and healthy pet animals advances in dietary management of obesity in dogs and cats effect of amount and type of dietary fiber on food intake in energy-restricted dogs effect of level and source of dietary fiber on food intake in the dog an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv2c in vaccinated dogs external parasites: identification and control orthopedic coaptation devices and small-animal prosthetics enterohepatic helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations intradural vasculitis and hemorrhage in full sibling welsh springer spaniels respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide laboratory animal medicine do not correlate with bacterial isolation or clinical respiratory symptoms lymphoma: which chemotherapy protocol and why? detection of humoral antibody to the transmissible venereal tumor of the dog laboratory animal management: dogs reproductive cycles of the domestic bitch hereditary canine spinal muscular atrophy: an animal model of motor neuron disease. can evaluation of the helicobacteraceae in the oral cavity of dogs management of septicemia in rhesus monkeys with chronic indwelling catheters clinical and virological findings in pups naturally infected by canine parvovirus type 2 glu-426 mutant eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound burn trauma chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis discordant 16s and 23s rrna gene phylogenies for the genus helicobacter: implications for phylogenetic inference and systematics use of narcotic antagonists to modify stereotypic selflicking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma evaluation of the ovicidal cctivity of lufenuron and spinosad on fleas' eggs from treated dogs enteric coccidiosis the respiratory system the association of american feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the united states study of obesity in dogs visiting veterinary practices in the united kingdom pathological prognostic factors in breast cancer. i. the value of histological grade in breast cancer: experience from a large study with long-term follow-up utility of fdg-pet scanning in lymphoma by who classification principles of treatment for canine lymphoma miller's anatomy of the dog update on diagnosis of canine hypothyroidism immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors the laboratory canine canine conjunctival mast cell tumors: a retrospective study canine cutaneous epitheliotropic t-cell lymphoma: a review canine infectious respiratory disease helicobacter-associated gastric disease in ferrets, dogs, and cats enteric bacterial infections hemorrhagic streptococcal pneumonia in newly procured research dogs association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners reproductive patterns in the domestic dog-a retrospective study of the drever breed the dog as a research subject a review of canine pseudocyesis leptospirosis surgical treatment of an elbow hygroma utilizing microvascular free muscle transfer in a newfoundland bacterial diseases immunoprophylaxis leptospirosis gastric helicobacters in domestic animals and nonhuman primates and their significance for human health non-helicobacter pylori helicobacter species in the human gastric mucosa: a proposal to introduce the terms h. heilmannii sensu lato and sensu stricto diseases of the small intestine histotripsy of the prostate: dose effects in a chronic canine model flea control failure? myths and realities small animal clinical nutrition operating room emergencies shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs pulmonary parenchymal disease canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats current veterinary therapy x: small animal practice congenital defects of the dog gastric and duodenal ulcers in dogs with mastocytoma complications in the use of indwelling vascular catheters in laboratory animals helicobacter infection diseases of the large intestine pregnancy management in the bitch tracheal collapse. diagnosis and medical and surgical treatment hygroma of the elbow in dogs thermal injuries joint working group on refinement factors contributing to the contamination of peripheral intravenous catheters in dogs and cats diversity in bacterium-host interactions within the species helicobacter heilmannii sensu stricto morphological study of the effects of the gnrh superagonist deslorelin on the canine testis and prostate gland five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism maintenance energy requirement of dogs: what is the correct value for the calculation of metabolic body weight in dogs? outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs kirk's current veterinary therapy xii: small animal practice an emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome? tarsal joint contracture in dogs with golden retriever muscular dystrophy an epidemiological study of interdigital cysts in a research beagle colony value of the (13)c-urea breath test for detection of gastric helicobacter spp. infection in dogs undergoing endoscopic examination pathogenesis of helicobacter pylori infection prostatic diseases semen collection in the dog accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs problems in wound healing associated with chemotherapy and radiation therapy histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model 18fdg-pet imaging in canine lymphoma and cutaneous mast cell tumor comparison of fertility data from vaginal vs intrauterine insemination of frozenthawed dog semen: a retrospective study withrow & macewen's small animal clinical oncology estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review effects of four preparations of .05% chlorhexidine diacetate on wound healing in dogs the prediction of parturition date in canine pregnancy clustering of activating mutations in c-kit's juxtamembrane coding region in canine mast cell neoplasms transmissible venereal tumors kirk's current veterinary therapy 11: small animal practice canine lymphoma and lymphoid leukemias the staging and treatment of multicentric highgrade lymphoma in dogs: a review of recent developments and future prospects association between waste management and cancer in companion animals tick paralysis in north america and australia thyroid gland and arterial lesions of beagles with familial hypothyroidism and hyperlipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs mast cells and canine mast cell tumours: a review etiologic study of upper respiratory infections of household dogs effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis determination of strain variability of microsporum canis to disinfectants cutaneous fungal infections diagnosis of neoplasia tumors of the mammary gland transmissible dog cancer genome reveals the origin and history of an ancient cell lineage clonal origin and evolution of a transmissible cancer notice regarding nih plan to transition from use of usda class b dogs to other legal sources (not-od-14-034) guide for the care and use of laboratory animals surgical closure of elbow hygroma in the dog colitis and colon cancer in waspdeficient mice require helicobacter species tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases animals and animal products, subchapter a, parts 1, 2, and 3 comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part 2: clinical trial in 100 dogs manual of clinical behavioral medicine for dogs and cats. mosby-year book identification of and screening for human helicobacter cinaedi infections and carriers via nested pcr identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep hypothyroidism in dogs: 66 cases (1987-1992) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and buccal bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs atlas of small animal wound management and reconstructive surgery what's your diagnosis? fever and leukocytosis in a young beagle. canine juvenile polyarteritis syndrome (beagle pain syndrome) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus thyroid diseases urogenital emergencies emergency procedures for the small animal veterinarian prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy streptococcus zooepidemicus: an emerging canine pathogen characterization of pneumonia due to streptococcus equi subsp. zooepidemicus in dogs tumors of the skin and soft tissue aspiration-induced lung injury origins and evolution of a transmissible cancer canine mast cell tumors express stem cell factor receptor arterial and venous blood gas analyses dogs and cats as laboratory animals hypocretin levels in sporadic and familial cases of canine narcolepsy comparison of 4 giardia diagnostic tests in diagnosis of naturally acquired canine chronic subclinical giardiasis effects of chlorhexidine diacetate and povidone-iodine on wound healing in dogs genetic evidence for an east asian origin of domestic dogs urethral-sparing histotripsy of the prostate in a canine model factors influencing canine mammary cancer development and postsurgical survival how to treat common parasites safely muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology, second ed. mosby-year book thyroid and parathyroid glands diseases of the small bowel canine infectious tracheobronchitis (kennel cough complex) diseases of the intestines the use of ultrasonography for pregnancy diagnosis in the bitch cytologic examination of fine-needle aspirates from mammary gland tumors in the dog: diagnostic accuracy with comparison to histopathology and association with postoperative outcome effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment pathologic features of naturally occurring juvenile polyarteritis in beagle dogs development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms withrow & macewen's small animal clinical oncology nutrient requirements of dogs and cats (nutrient requirements of domestic animals) trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention lumbosacral stenosis in dogs mammary neoplasia in a closed beagle colony complete mitochondrial genomes of ancient canids suggest a european origin of deomestic dogs artificial insemination in canids: a useful tool in breeding and conservation artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach manual of canine and feline cardiology comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of helicobacter cinaedi thyroiditis in a group of laboratory dogs: a study of 167 beagles obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome pro-inflammatory properties and neutrophil activation by helicobacter pylori urease of agriculture, animal and plant health inspection service annual report animal usage by fiscal year. available from: <www leptospirosis in dogs: a review with emphasis on clinical aspects thomson's special veterinary pathology, second ed. mosby-year book endocrinology of pregnancy in the dog: a review immunoblot analysis as an alternative method to diagnose enterohepatic helicobacter infections management of superficial skin wounds psychodermatosis colony multiplex pcr assay for identification and differentiation of campylobacter jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus subsp. fetus the role of c-kit in tumorigenesis: evaluation in canine cutaneous mast cell tumors aaha canine vaccination guidelines canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment serum concentrations of thyroxine and 3,5,3′-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs surgical treatment of specific skin disorders naltrexone for treatment of acral lick dermatitits in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs mammal species of the world: a taxonomic and geographic reference withrow & macewen's small animal clinical oncology the authors thank dr. bambi jasmin of marshall farms for contributions on preventive health practices for class a dogs. key: cord-021453-vf8xbaug authors: dysko, robert c.; nemzek, jean a.; levin, stephen i.; demarco, george j.; moalli, maria r. title: biology and diseases of dogs date: 2007-09-02 journal: laboratory animal medicine doi: 10.1016/b978-012263951-7/50014-4 sha: doc_id: 21453 cord_uid: vf8xbaug nan status as a cooperative companion animal of reasonable size. dogs were used in the mid-1600s by william harvey to study cardiac movement, by marcello malpighi to understand basic lung anatomy and function, and by sir christopher wren to demonstrate the feasibility of intravenous delivery of medications (gay, 1984) . the use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by pavlov to observe and document the conditioned reflex response and by banting and best to identify the role of insulin in diabetes mellitus. for a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by gay (1984) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for beagle for the years 1998-1999, approximately 40% of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. the next most common areas of research using beagles were dental and periodontal disease and surgery (12% of publications), orthopedic surgery and skeletal physiology (7%), and radiation oncology (4%). other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies is maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. these dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., 1995) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., 1994) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions. although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. according to the u.s. department of agriculture (1998) , the number of dogs used in research has declined from a high use of 211,104 in 1979 211,104 in to only 75,429 in 1997 . this decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against canine distemper virus, parvovirus, adenovirus type 2, parainfluenza virus, leptospira serovars canicola and icterohemorrhagiae, and bordetella bronchiseptica. rabies virus vaccination may also be included. purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (r. scipioni and j. ball, personal communication, 1999) . random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. options for procurement of dogs for biomedical research typically include purchase from a u.s. department of agriculturedesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title 9, chapter 1 (1-1-92 edition), subchapter a, animal welfare, 1.1, definitions, and 2.1, requirements and application. briefly, class a licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically class a dealers). class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. regulations regarding the sale of pound dogs to research facilities or class b dealers vary from state to state and include some bans on this practice. the best resource for identification of possible vendors is the "buyer's guide" issue of the periodical lab animal. typically the last issue of each year, the "buyer's guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within that issue of the journal. welfare act (7 cfr 2.17, 2.51, and 371.2[g] ) are described in 9 cfr chapter 1 (1-1-92 edition), subchapter a, animal welfare. regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats, of part 3 (standards) of subchapter a. particular attention should be paid to section 3.6c (primary enclosures--additional requirements for dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to national research council (nrc) guidelines). section 3.8 (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute of laboratory animal research (ilar) has written the "guide for the care and use of laboratory animals" (seventh edition, 1996) . the "guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the association for assessment and accreditation of laboratory animal care international (aaalac international) and other animal care evaluation groups to facilitate site visits and inspections. the ilar committee on dogs has also written "dogs: laboratory animal management " (1994) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. growth data for beagles from a purpose-bred dog breeding facility are provided in table i . table ii features hematology data from beagles from the same commercial facility. table iii lists serum and urine chemical data for beagles. normal physiologic data for dogs (no breed specified) are provided in table iv . the information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. the basic nutrient requirements for dogs have been compiled by the nrc and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (national research council, 1985) . the reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. these diets do not necessarily provide the identical composition of ingredients from batch to batch. ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. an "openformula" (or "fixed-formula") diet provides more precise dietary control. in these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are generally safe for consumption up to 6 months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the basal metabolic rate, or basal energy requirement (ber), refers to the amount of energy expended following sleep, 12-18 hours after food consumption, and during thermoneutral conditions (kleiber, 1975; lewis et al., 1987) . the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the ber (lewis et al., 1987) . for dogs weighing greater than 2 kg, the mer may be calculated using this simplified linear equation: mer (metabolizable kcal/day) = 2(30 weightkg + 70) (national research council, 1985; lewis et al., 1987) . the quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the mer by the energy density of the diet. fat provides three major dietary functions, including absorption of fat-soluble vitamins (a, d, e, and k), enhancement of palatability, and provision of essential (unsaturated) fatty acids. dietary fat is an excellent, highly digestible energy source, providing 2.25 times more energy on a per weight basis than either soluble carbohydrates or proteins (lewis et al., 1987) . however, fats are not needed for this purpose when adequate carbohydrate and protein are present. consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. however, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." dogs require linoleic (f2-6) acid, an essential fatty acid (national research council, 1985) , and more recently it has been demonstrated that the f2-3 fatty acids may play a role in maintaining healthy skin (logas and kunkle, 1994) . supplementation with a balanced essential fatty acid product (e.g., derm caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (logas and kunkle, 1994; miller, 1989) . essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (lewis et al., 1987) . there are 22 a-amino acids, 10 of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. the essential amino acids and the minimal requirements for growth are listed elsewhere (lewis et al., 1987) . chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (lewis et al., 1987) . conversely, inadequate protein intake results in retardation of growth and adata graciously provided by r. scipioni and j. ball of marshall farms usa, inc., north rose, new york. beagles tested for period 2/28/99-9/01/99. b s.d., standard deviation; wbc, white blood cells; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; rdw, red cell distribution width; hdw, hemoglobin distribution width; plt, platelets; mpv, mean platelet volume; neut, neutrophils; lymp, lymphocytes; mono, monocytes; eos, eosinophils; baso, basophils; luc, large unstained cells; li, lobularity index; mpxi, mean peroxidase activity index reduction in production and/or performance. protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. as a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. thus, caloric needs must be met before protein needs (lewis et al., 1987) , an important concept to bear in mind in the event of research experiments that may predispose to anorexia. in general, providing a good quality commercial diet that supplies the required amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. appropriate mineral balance in the diet is very important. the best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. the recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (lewis, 1987) . determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/ 2.0 (basal) 12.6 (anestrus) 2.0 c <0.5 c 20-100 c 4-73 r (continues) deficiencies are similar and nonspecific. a definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. vitamins function as enzymes that regulate a wide variety of physiologic processes. they are divided into two groups based on their solubility. the fat-soluble vitamins include a, d, e, and k, whereas the rest are water-soluble. a list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (lewis et al., 1987) . cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (b complex and c) (lewis et al., 1987) . however, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. this section is largely based on information assimilated from texts such as "miller's anatomy of the dog" (evans and christensen, 1993) , "veterinary reproduction and obstetrics" (arthur et al., 1989) , and an issue of veterinary clinics of north america: small animal practice devoted to pediatrics of puppies and kittens (hoskins, 1999) . the ovaries of the bitch are attached to the dorsolateral walls of the abdominal cavity caudal to the kidneys by the broad ligaments and are not palpable abdominally. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the birchard and sherding (1994) . ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. thus, semen is deposited at the external cervical os during natural or artificial insemination. the vagina is a long musculomembranous canal that extends from the uterus to the vulva. when the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. examination should proceed at an angle of approximately 60 ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the bitch has a monoestrous cycle, with clinical estrus occurring predominantly in january or february and again in july or august (although it can occur at any time of year). the estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. the average duration of proestrus is 9 days. during this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. estrus generally lasts 9 days, and the vulva is softer and smaller than in proestrus. a vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. the endocrine feature of estrus is the luteinizing hormone (lh) surge, followed by ovulation within 24-72 hours. diestrus begins approximately 9 days after the onset of standing heat. the end of this stage is 60 days later, which would be coincident with whelping if the bitch had become pregnant. serum progesterone levels peak during diestrus. the duration of anestrus is approximately 4 months. anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than 1 ng/ml. components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. the cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at 2~176 lower than basal body temperature. sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. the penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. it is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. the accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. the dog does not have seminal vesicles or bulbourethral glands. the onset of puberty ranges from 5 to 12 months of age and is affected by breed, season, and nutritional and disease status. testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. the sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or leydig's, cells. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in 45 days, with subsequent maturation of sperm occurring in the epididymis for approximately 15 days. thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is 60 days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. there is a positive correlation with the size of the testicles as measured by scrotal circumference and the number of sperm produced. finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. an ejaculate (5 ml) that contains approximately 500 million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. in general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. on mounting, the initial thrusting and ejaculation of semen last about 1 minute. the bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. the male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." ejaculation of the accessory gland fluid continues for 5-30 minutes. the continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. fertilization occurs in the oviduct and may occur as late as 8 days after coitus, because of the long life span of sperm in the dog. however, once ovulated, oocytes generally remain viable for only 12-24 hours. therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear "anuclear" and are classified as "cornified" or "anuclear squames." cornification occurs approximately 2 days prior to the estrogen peak and 4 days prior to standing heat. the percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified 6 days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear-cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. during proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. the behavioral hallmark of estrus is standing receptivity toward the male. during this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days 1, 3, and 5 of the standing heat. fertilization is completed in the mid-to distal oviduct. implantation is evident by areas of local endometrial edema 17-18 days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is 59-63 days. luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than 1 ng/ml in late proestrus to a peak of 30-60 ng/ml during gestation, then declines to 4-5 ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. pregnancy detection can be performed by abdominal palpation of the uterus 28 days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings in the early gravid uterus. they are approximately 2 inches in length at 28-30 days, the time at which pregnancy is most easily and accurately diagnosed. by day 35 the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. fetal skeletons become calcified and are radiographically evident by day 42. bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. real-time ultrasound can be utilized for pregnancy detection of vesicles as early as 25-28 days. an abrupt drop in body temperature to less than 100~ indicates impending parturition within 18-24 hours. the process of parturition has been divided into three stages, stage 1 of labor lasts 6-12 hours and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include hard panting and increased pulse and respiration rates. fetal expulsion occurs during stage 2, which lasts approximately 3-6 hours. as the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the ferguson reflex. oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. the bitch is usually recumbent during stage 2 but is able to inhibit this stage if labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between delivery of each pup is irregular, but the average time lapse is less than 1 hour between pups until parturition is complete. veterinary assistance is necessary if the bitch remains in stage 2 for more than 5 hours without delivering the first pup, or for more than 2 hours before delivering subsequent pups. the placentas are expelled during stage 3 of labor, immediately following delivery of a pup, or up to 15 minutes thereafter. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. heat lamps may be placed 24 hours prior to parturition and remain until all neonates dem-onstrate vigorous and successful suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. the postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. in these cases, 5-20 units of oxytocin may be administered intramuscularly. uterine involution occurs during anestrus within 4-5 weeks of parturition. during this time a greenish to red-brown vaginal discharge, or lochia, may be noted. although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the sixth postpartum week, with complete repair by 3 months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately 12 days, and ears are patent at approximately 12-20 days. solid food can be introduced between 4.5 and 6 weeks of age, and puppies can be weaned at 6-8 weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. semen for ai is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the bitch's scent and manually stimulated. after collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to 4-6 ml. the semen is then drawn into a sterile 10 or 12 ml syringe attached to a sterile disposable insemination pipette. the bitch is inseminated either standing or with raised hindquarters. a gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. the tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. the semen is injected, and 2-3 ml of air are then flushed through the syringe and pipette. the pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. the bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. as with natural breeding, ai should be performed on days 1, 3, and 5 of standing heat, or on the days of maximal vaginal cornification. the bitch should be palpated for pregnancy approximately 4 weeks after the first insemination. false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. however, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (cheque drops) at an oral dose of 16 ~tg/kg q24 hr for 3-5 days (brown, 1984) . reproductive performance in the bitch is optimal prior to 4 years of age. although normal cycle lengths are reported to occur up to the ages of 5-7 years, the interestrous interval tends to increase by 4 years of age. cycling does not completely cease; however, after 8 years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. by 8-9 years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common. beagles have been a popular animal model because of their docile nature. they are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ecg) recordings, oral gavage, and venipuncture. dogs are sexually mature by 6-9 months of age, but they are not socially mature until 18-36 months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from 3-8 weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks 5 and 12, puppies are most capable of learning how to interact with people. by 10-12 weeks of age dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. the extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. in general, breed-specific patterns do tend to emerge. for example, it appears that beagle pups are very motivated by food reward (overall, 1997 ). this is not surprising, because the breed was selected to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "clinical behavioral medicine for small animals" (overall, 1997). by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "current veterinary therapy" series (j. d. bonagura and r. w. kirk, eds.), "veterinary internal medicine" (s. j. ettinger and e. c. feldman, eds.), and "infectious diseases of the dog and cat" (c. e. greene, ed.) . full citations of some chapters from these texts are listed in the references (w. b. saunders co. of philadelphia publishes all three texts.) canine infectious tracheobronchitis (kennel cough complex) etiology. infectious tracheobronchitis (itb) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. several organisms have been incriminated as causative for this condition: bordetella bronchiseptica; canine parainfluenza virus (cpiv); canine adenovirus types 1 and 2 (cav-1, cav-2); canine herpesvirus; canine reovirus types 1, 2, and 3; and mycoplasms and ureaplasms. clinical signs. clinical infectious tracheobronchitis can be subdivided into mild or severe forms. the mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. mild tracheobronchitis usually lasts 7-14 days, even if left untreated. the severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and may be the determinant of severity (sherding, 1994) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. these cases require more aggressive treatment and may be fatal. bordetella bronchiseptica is considered to be the respiratory tract of infected animals (bemis, 1992) . this bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (bemis, 1992) . transmission is favored by confined housing of multiple animals. in experimental studies, b. bronchiseptica transmission to susceptible individuals was 100% (thompson et al., 1976; mccandlish et al., 1978) . the incubation period is 3-10 days. cpiv and cav-2 are also spread by aerosols. of these two viruses, cav-2 is the most persistent, lasting for up to several months in the environment, whereas cpiv is fairly labile (hoskins, 2000a) . both viruses can be destroyed by quaternary ammonium disenfectants. pathogenesis. the most common clinical isolates are cpiv and bordetella bronchiseptica. however, b. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. in cases of clinical infection, b. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv or cav-2 alone are usually subclinical; coinfections with b. bronchiseptica or other microbes may result in clinical itb (keil and fenwick, 1998; wagener et al., 1994) . the characteristic lesion from cpiv or cav-2 infection is necrotizing tracheobronchiolitis (dungworth, 1985) . pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction. diagnosis and differential diagnosis. diagnosis of infectious tracheobronchitis is often based on clinical signs. isolation of bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. viral isolation or paired serology can be done but is often impractical and expensive. if cough persists for more than 14 days, other disease conditions should be considered. canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (johnson, 2000) and should be considered as a differential for itb. prevention. prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. intranasal vaccine combinations for bordetella bronchiseptica and cpiv are preferred. intranasal vaccines protect against both infection and disease, can be given to dogs as young as 2 weeks of age, and can produce immunity within 4 days. control. sanitation and ventilation are critical for control. the animal care staff must practice proper hygiene to prevent fomite transmission. symptomatic animals should be isolated, and animal-to-animal contact avoided. kennels should be disinfected with agents such as bleach, chlorhexidine (nolvasan) or quaternary ammonium chloride (roccal-d). proper ventilation and humidity are important in controlling spread of these infectious agents; 15-20 air changes per hour at 50% relative humidity are recommended (sherding, 1994) . no specific treatment is available for viral infections. bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for 14 days. use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. for severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. tis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. animals with clinical disease would also be poor surgical candidates. etiology. [3-hemolytic lancefield's group c streptococcus (streptococcus zooepidemicus) is a gram-positive non-spore-forming coccus and an etiologic agent for pneumonia and septicemia in dogs. clinical signs. clinical signs vary based on the organ system affected. pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. peracute death without clinical signs has been reported in a previously healthy research dog (bergdall et al., 1996) , and conjunctivitis can also be caused by this organism (murphy et al., 1978) . epizootiology and transmission. lancefield's group c streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (olson et al., 1973) . epizootics have been reported in both racing greyhounds and research colonies (sundberg et al., 1981; garnett et al., 1982) . in these epizootics, and in the reported case of peracute death (bergdall et al., 1996) , recent transportation (within 7 days) was associated with the disease. as such, lancefield's group c streptococcus may be an opportunistic pathogen in dogs. pathologic findings. in the peracute case reported (bergdall et al., 1996) , hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. ecchymotic and petechial hemorrhages were seen on other organ surfaces. the lungs were heavy and wet, and blood oozed from cut surfaces. "bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (fig. 2) . histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. gram-positive cocci were seen in both the lung and the tonsils. pathogenesis. the pathogenesis for disease caused by lancefield's group c streptococcus is unclear. strain variation with respect to virulence and host immune factors is probably significant. diagnosis and differential diagnosis. definitive diagnosis is made based on bacterial culture and identification. any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. bacterial pneumonias or septicemias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and bordetella bronchiseptica. nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control. too little is known about the pathogenesis of lancefield's group c streptococcus to make any recommendations about prevention and control. treatment. antibiotic therapy should be provided, based on culture and sensitivity. intravenous fluids are indicated for febrile or systemically ill patients. for dyspneic patients, oxygen therapy and strict activity restriction are required. research complications. clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. the association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. etiology. serovars of the spirochete leptospira interrogans sensu lato cause canine leptospirosis. disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. clinical signs. leptospirosis may present as either an acute or a chronic problem. clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. icterus, congested mucous membranes, or signs referable to disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. uveitis, abortions, and stillbirths have also been associated with leptospirosis. epizootiology and transmission. vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past 30 years. wild animals, cattle, and rodents are reservoirs for leptospira. the epidemiology of the disease is not static, and recent changes have been observed. serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. this may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (rentko et al., 1992; brown et al., 1996; kalin et al., 1999) . transmission occurs primarily by environmental contact, and not directly from animal to animal. infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. recovered animals may shed organisms in their urine for months to years. the organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. leptospirosis is a zoonotic disease. pathologic findings. the kidneys consistently have gross and microscopic lesions. in the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (searcy, 1995) . in chronic stages of leptospirosis the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly. several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. the hematogenous phase lasts 4-14 days. acute renal failure or progressive renal failure leading to oliguria or anuria may occur. the most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (greene, 2000) . the nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. icterus, if it develops, is most common in the acute phase. the combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. disseminated intravascular coagulation is often a secondary complication. the severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient. diagnosis and differential diagnosis. zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection. prevention and control. vaccination for leptospirosis is standard veterinary practice. bivalent inactivated bacterins for serovars of l. interrogans canicola and serovars of l. interrogans icterohemorrhagiae are commercially available. however, immunization does not prevent development of the carrier state or protect against other serovars. for outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. control requires identification and either treatment or elimination of carrier animals. treatment. penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. aggressive fluid therapy and supportive care may also be needed. elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. should not be used in research studies because of the effects of the disease on renal and hepatic function. etiology. campylobacteriosis in dogs is caused by campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. clinical signs. most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than 6 months of age (greene, 2000; burnens et al., 1992) . in cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. these signs are usually mild, may be intermittent, and typically last 5-21 days. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. epizootiology and transmission. the role of c. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, 1994) . clinical signs of disease most often occur in dogs less than 6 months of age, although any age may be affected. stress or immunosuppression may make animals more susceptible to clinical disease. pound and shelter populations have the highest rates of fecal excretion of c. jejuni (sherding and johnson, 1994) . transmission is via the fecal-oral route, mostly through fecally contaminated food or water. unpasteurized milk, poultry, and meat are other sources of infection. campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk. pathologic findings. the actual lesions observed depend upon the mechanism of the enteropathy (van kruiningen, 1995) . enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. in cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. on histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. when translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. with warthin-starry silver staining, c. jejuni may be seen between enterocytes but only rarely inside them. pathogenesis. clinical disease may be produced by several different mechanisms after the campylobacter has populated the intestinal tract (van kruiningen, 1995) . after colonization of the enterocyte surface, c. jejuni can produce an enterotoxin that causes a secretory diarrhea. campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. in addition, c. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. this causes mesenteric lymphadenitis. diagnosis and differential diagnosis. fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of 42~ any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, giardia, and salmonella infections; helminth infestations; and hemorrhagic gastroenteritis. clinical signs. based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (c. g. couto, personal communication, 1993; waddle and littman, 1988; woody and mcdonald, 1985) . a history of tick exposure may be noted prior to onset of signs. in the acute phase, clinical signs range from mild to severe and may last 1-2 weeks. they include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. central nervous system (cns) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (kuehn and gaunt, 1985) . clinical signs are generally absent during the subclinical phase. mild thrombocytopenia, anemia, or leukopenia may be seen. the chronic phase develops 1-4 months after the initial infection, and signs may be subclinical to severe. an extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. the following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. on bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in 50-100% of e. canis seropositive or infected dogs (kuehn and gaunt, 1985; breitschwerdt et al., 1987; shimon et al., 1996) . proteinuria and/or hypoalbuminemia have also been seen. epizootiology and transmission. ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. the definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. the primary vector and reservoir is the brown dog tick, rhipicephalus sanguineus. ehrlichia canis is found worldwide and follows the distribution of the vector. infection in dogs is most prevalent in tropical and subtropical areas (greene, 1991) . in the united states, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (breitschwerdt, 2000) . transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as 5 years. ticks become infected by feeding on an infected dog that is in the first 10-15 days of an acute infection (lewis et al., 1977) , and ticks can shed the organisms for up to 5 months. within the tick population, e. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (groves et al., 1977) . pathogenesis. in experimental infections, the incubation period prior to the onset of the acute phase is 7-21 days. during the acute phase, which can last from 2-4 weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. the thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. the subclinical phase of the disease occurs 6-9 weeks after initial infection. during this stage, dogs that can mount an effective immune response clear the infection. those that cannot mount such a response progress to the chronic stage. infection does not confer protective immunity in dogs that recover. german shepherds and doberman pinschers seem to be more severely affected than other breeds. pathologic findings. gross lesions are varied and change, depending on the phase of the disease. the most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (woody and hoskins, 2000) . the most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. diagnosis and differential diagnosis. the most sensitive, specific, and commonly employed method for diagnosing e. canis infections is the indirect fluorescent antibody (ifa) test. antibodies can be detected as early as 7 days postinfection, although some dogs may not seroconvert until 28 days postinfection (buhles et al., 1974) . cross-reaction may occur between e. canis, e. chaffeensis, and e. ewingii. titers greater than 1:10 are considered positive and indicative of infection and may persist for up to 1 year. effective treatment typically produces seronegative results in 6-9 months. in some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (bartsch and greene, 1996) . the exact mechanism for this finding has not been elucidated. ehrlichia canis morulae can be demonstrated in circulating monocytes of giemsa-stained blood smears. however, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. using buffy coat smears from capillary blood may increase the diagnostic yield. polymerase chain reaction (pcr) assays are also available to identify e. canis. differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. prevention. preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. avoid exercising dogs in areas infested with ticks. use topical acaricides to prevent tick infestations. keep kennel areas tick-free. dogs used as blood donors and dogs from unproven sources should be tested for e. canis. treatment. doxycycline is the drug of choice for treating monocytic ehrlichiosis. oral doses of either 2.5-5 mg/kg q12 hr or 10 mg/kg q24 hr for 21 days are very effective at eliminating the organism. tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. in chronic cases, antibiotic treatment should be extended for an additional 1-2 weeks. research complications. the most significant research complication is the thrombocytopenia that persists for all stages of the disease. additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. for these reasons, dogs positive for antibodies to e. canis should not be used in research. etiology. this disease, caused by ehrlichia platys, was first described as cyclic thrombocytopenia by harvey et al. in 1978 . clinical signs. in most cases, infection with e. platys results in subclinical disease. a generalized lymphadenopathy may be noted. epizootiology and transmission. the vector for e. platys is assumed to be a tick; however, this mode of transmission has not been established. experimental studies by simpson et al. ( 1991) failed to demonstrate rhipicephalus sanguineus as a vector for e. platys. coinfection with e. canis has been reported, which suggests a common vector for both organisms (french and harvey, 1983; kordick et al., 1999) . dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (harvey et al., 1978; gaunt et al., 1990) . the geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other ehrlichia organisms. the highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as michigan and as far west as oklahoma (wilson, 1992; mathew et al, 1997) . the prevalence of seropositive dogs can be high in some parts of the country. a study by bradfield et al. (1996) reported that 74% of the dogs entering a research institute's quarantine facility from sources in eastern north carolina were seropositive for e. platys. hoskins et al. (1988) reported a 54.2% seropositive prevalence in healthy dogs from kennels in louisiana. pathologic findings. gross and histopathologic findings during experimental e. platys infection in dogs have been described by baker et al. (1987) . generalized lymphadenopathy was the only gross lesion noted. follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. all dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. multifocal kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. pathogenesis. the pathogenesis of e. platys in dogs has primarily been determined through experimental infection (harvey et al., 1978) . after inoculation the organism directly infects platelets. thrombocytopenia occurs by day 10-14 and fluctu-ates, along with parasitemia, at 10 to 14 day intervals. in some cases the rebound may be within the normal range for thrombocyte counts. the nadir can be lower than 20,000 platelets/~d. concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of e. platys infection. the mechanism responsible for the cyclic nature of the infection has not been elucidated. diagnosis and differential diagnosis. ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. however, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. available ifa assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other ehrlichia species. dogs usually develop detectable titers 2-3 weeks postinfection. pcr assays for e. platys have now been developed as well (chang and pan, 1996; mathew et al., 1997) . differential diagnoses for thrombocytic ehrlichiosis include e. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (dic). platys is the same as described for e. canis, above. research complications. ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. coinfection with e. platys may potentiate the pathogenicity of other infectious agents, in particular e. canis (breitschwerdt, 2000) . etiology. lyme disease is caused by borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. clinical signs. clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. the onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. over the course of the disease, signs may wax and wane over a period of weeks to months. dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (greene, 1991; manley, 1994) . hematologic and biochemical profiles are generally unremarkable. lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the united states. it affects humans and dogs worldwide. the geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. three major endemic foci that have been identified in the united states account for 94% of reported human cases (appel and jacobson, 1995). the distribution of these cases is as follows: northeast/mid-atlantic focus, 85%; midwestern focus (michigan, wisconsin, minnesota, iowa, illinois, and missouri), 10%; and california and oregon, 4%. for the most part, dogs in the remainder of the country are not at risk for contracting lyme disease. borrelia burgdorferi is transmitted exclusively by ixodes ticks. other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. ixodes scapularis, a three-host tick with a 2 to 3 year life cycle, is the prototypical vector for north america. the spirochetes are spread by tick bites from both nymphs and adults. ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). in endemic areas, 50-80% of adult ticks may be infected (appel and jacobson, 1995) . the primary reservoir for the organism is the whitefooted deer mouse, peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. evidence also indicates that the eastern chipmunk, tamias striatus, is an important reservoir (slajchert et al., 1997) , and birds may also be a significant reservoir. deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. pathogenesis. the pathogenesis of lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. infection can be induced experimentally by the bite of a single infected tick. clinical signs develop 60-90 days postinfection. some evidence points to the host's inflammatory response to the organism as etiologic for disease (pershing et al, 1994; greene, 1991) . seroconversion in dogs occurs 4-6 weeks after infection with b. burgdorferi. antibody titers may remain extremely elevated for at least 18 months. igm titers also remain elevated for several months and are indicative of neither acute nor active infection (appel and jacobson, 1995) . because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for 30 days with antibiotics) can be reactivated by steroid treatment up to 420 days postinfection (straubinger et al., 1998) . diagnosis and differential diagnosis. appel and jacobson (1995) recommend that three of the following four criteria be met to establish a diagnosis of lyme disease in dogs: (1) history of exposure to ixodes ticks in an endemic area, (2) characteristic clinical signs, (3) positive serology, and (4) rapid resolution of clinical signs with antibiotic therapy. ifa or elisa tests for borrelia antibodies are the assays of choice. it should be re-membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. responses to vaccine versus infection may be distinguished by western blot. culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents. prevention and control. prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, section iii,a,l,e above). a vaccine against b. burgdorferi is available but should not be necessary in a research setting. treatment. doxycycline is the drug of choice for treating lyme borelliosis. a typical dosing regimen is 10 mg/kg q12 hr for 3-4 weeks. amoxicillin, tetracycline, and the quinolones are also effective. of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. (fox and lee, 1997) . "helicobacter heilmannii" and h. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. this species, as well as h. rappini and h. canis, is considered to be zoonotic (fox and lee, 1997) . clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. epizootiology and transmission. the epizootiology and transmission of helicobacter spp. in the dog remains to be elucidated. the prevalence of canine helicobacter infections in colony or shelter situations has been reported to range from 82% to almost 100% (fox, 1995; hermanns et al., 1995) . both oral-oral and fecal-oral routes for transmission have been suggested. pathologic findings. no gross lesions are noted; the primary lesion is that of histologic gastritis. this is typically characterized by reduced mucus content of the surface epithelium; vacu-olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (hermanns et al., 1995) . focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. pathogenesis. some helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. helicobacter felis and "h. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (fox and lee, 1997) . the mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the helicobacter's ability to produce urease. urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (marshall et al., 1990; shimoyama and crabtree, 1998 ). diagnosis and differential diagnosis. any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. definitive diagnosis for dogs requires either endoscopic or surgical biopsy. confirmation of infection with helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by pcr. a positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. the use of warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. prevention and control. until more is known about the epizootiology and transmission of helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. treatment. combination therapy has proven to be the most effective method for treating helicobacter spp. infections in dogs. combination therapy of amoxicillin (10 mg/kg q12 hr), metronidazole (30 mg/kg q24 hr), and sucralfate (0.25-0.5 mg/kg q8 hr) for 21 days has been suggested for dogs (hall and simpson, 2000) . replacing the sucralfate with famotidine (0.5 mg/kg q24 hr), omeprazole (0.3 mg/kg q24 hr), or bismuth subsalicylate (0.2 ml/kg q4-6 hr) may also be effective (marks, 1997; jenkins and bassett, 1997; denovo and magne, 1995) . the benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. research complications. helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis. clinical signs. clinical signs of canine parvovirus usually appear 5 days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. profuse, intractable diarrhea ensues, which may become hemorrhagic. approximately 85% of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from 500-2000 wbc/~d or less. repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. parvovirus can infect dogs of any age, but puppies between 6 and 20 weeks of age appear to be particularly susceptible. puppies less than 6 weeks of age are generally protected from infection by passive maternal antibody. adult dogs probably incur mild or inapparent infections that result in seroconversion. pathogenesis. canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis. parvovirus can be detected in fecal samples with a commercially available elisa from cite. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. other lesions include myeloid degeneration and widespread lymphoid depletion. parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. prevention and control. prevention of transmission begins with isolation of affected animals and quarantine for 1 week after full recovery. disinfection of potentially infected kennel and diagnostic areas with diluted bleach (1:30) or commercially prepared disinfectant (such as kennesol, available from alphatech, lexington, massachusetts) is essential for elimination of the virus. six-week-old puppies should be vaccinated every 2-4 weeks with a commercially available modified live vaccine until 16-18 weeks of age. young rottweilers and doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every 3 weeks (5 times) from 6-18 weeks of age. treatment. treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance. research complications. infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. canine coronavirus infection is usually inapparent or causes minimal illness. this epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at 6 -8, 10 -12, and 12-14 weeks of age and then annually thereafter. the role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (hoskins, 1998) . etiology. canine distemper virus (cdv) belongs to the family paramyxoviridae, within the genus morbillivirus, which includes human measles virus and rinderpest virus of ruminants. although there is only one serotype of cdv, there is a wide difference in strain virulence and tissue tropism. some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. a transient subclinical fever and leukopenia occur 4-7 days after exposure, with a subsequent fever spike 7-14 days later, accompanied by conjunctivitis and rhinitis. other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. an immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (greene and appel, 1998) , because dogs that develop skin lesions often recover. neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete elimination by the immune system, possibly because of protective effects by the blood-brain barrier. classic neurologic signs that may occur in acute or chronic cdv infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. canine distemper is the most common cause of seizures in dogs less than 6 months of age. dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. cdv has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis." epizootiology and transmission. the virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of 3-6 months, coincident with the waning of maternal antibody. transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. pathologic findings. the predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may .. be accompanied by gliosis, necrosis, edema, and macrophage infiltration. acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. diagnosis and differential diagnosis. diagnosis of cdv is based on history of exposure and clinical signs. young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have cdv. ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. attenuated strains of cdv, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. because of the variety of clinical signs, there are many differential diagnoses for canine distemper. an important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. prevention and treatment. a series of three immunizations from 6 to 14 weeks of age, followed by yearly boosters, is a recommended preventative. treatment is largely supportive, but because of the profound immunologic effects and significant morbidity of cdv, humane euthanasia is usually undertaken in the research setting. etiology. canine herpesvirus (chv) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than 2 weeks of age. in adult dogs, chv causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress. clinical signs. clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. older puppies, aged 3-5 weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. infection in adult dogs may result in stillbirths, abortions, and infertility. lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. passage of puppies through the birth canal or venereally in adult dogs. puppies can also be horizontally infected by littermates. entire primiparous litters may be lost, with subsequent litters protected by colostral antibody. pathologic findings. pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings. diagnosis and differential diagnosis. diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. the diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (carmichael and greene, 1998) . differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia. there is no effective curative treatment. supportive therapy is unrewarding, and death usually ensues within 48 hours in in-fected neonates. in general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled. adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. because of the severity of clinical illness in puppies, such animals should be humanely euthanatized. etiology. rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals. clinical signs. clinical progression of neurologic disease occurs in three stages. the first, or prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. the third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death usually occurs within 2-7 days of the onset of clinical signs, due to respiratory failure. epizootiology and transmission. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds. pathogenesis. the incubation period for rabies is generally 3-8 weeks from the time of exposure to the onset of clinical signs but can range from 1 week to 1 year. bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis. diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. definitive diagnosis is based on fluorescent antibody demonstration of the virus in negri bodies of hippocampal cells. prevention and treatment. puppies should be vaccinated at 4-6 months of age, "boostered" in 1 year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. treatment of rabies is not recommended, because of the risk of human exposure. research complications. in a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. a healthy, purpose-bred dog that bites a human in a research facility should be quarantined for 10 days and observed for signs of rabies. this quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. a random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. the brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. a rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. a. protozoa i. giardiasis etiology. giardiasis is a small-intestinal disease of the dog caused by giardia duodenalis (lamblia), a binucleate flagellate protozoan. clinical signs. most giardia infections are subclinical. when dogs are clinically affected, diarrhea is the most prominent sign. the diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. weight loss has also been associated with clinical infection. clinical illness is more often seen in young animals. epizootiology and transmission. giardia has a direct life cycle. dogs (and people) typically become infected when they consume water (or food) contaminated with giardia cysts. the ph change from the stomach (acid) to duodenum (neutral) causes excystation. trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle. pathologic findings. giardiasis is rarely fatal. on histopathology of duodenal or jejunal specimens, giardia trophozoites can be seen attached to enterocytes. mucosal inflammation and ulceration, and villous atrophy, have been observed. pathogenesis. the exact pathogenesis of giardia-induced illness is unknown. it is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. it is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. diagnosis and differential diagnosis. definitive diagnosis requires observation of the organism in fecal or intestinal samples. direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. commercial elisa kits and direct immunofluorescent tests are available to detect fecal giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (barr, 1998) . differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. prevention. high-quality water sources will eliminate the possibility of infection developing within an animal research facility. use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. control. once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of giardia cysts. treatment. the most common treatment for giardiasis is metronidazole (flagyl) at 25-30 mg/kg per os twice per day for 5-10 days. quinacrine hydrochloride (atabrine) at 9 mg/kg per os once per day for 6 days, furazolidone (furoxone) at 4 mg/kg per os twice per day for 7-10 days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of giardia. a1bendazole is recommended at 25 mg/kg per os q12 hr for 2 days, and fenbendazole at 50 mg/kg per os q24 hr for 3 days. fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (barr, 1998) . research complications. typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies. clinical diarrhea would clearly need to be treated before a dog could be used as a research subject. ii. coccidiosis etiology. intestinal coccidia that have been associated with enteropathy in dogs include cystoisospora canis, c. ohioensis, c. burrowsi, and c. neorivolta. clinical signs. dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. epizootiology and transmission. cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. an indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. the gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. pathologic findings. dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. the mucosa may appear normal, raised, or ulcerated. histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. the oocysts are usually readily apparent within the epithelial cells (van kruiningen, 1995) . pathogenesis. intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. such factors include immunodeficiency, malnutrition, and/or concurrent disease. overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. diagnosis and differential diagnosis. diagnosis is somewhat difficult, as coccidian oocysts (of both cystoisospora and non-cystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. other causes for diarrhea (e.g., parvovirus, roundworms, giardia spp., campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. prevention. clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-crowding, and providing as stress-free an environment as possible. treatment. treatment for the presence of coccidial oocysts may often not be necessary, because cystoisospora infections are typically self-limiting and clinically insignificant. treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine (25-30 mg/lb per os for 10 days), trimethoprim sulfa (15 mg/lb per os for 10 days), or quinacrine (5 mg/lb per os for 5 days). amprolium, which is not labeled for dogs, can also be used as a coccidiostat. it can be given in gelatin capsules for 7-12 days at a daily dose of 100 mg for small-breed pups and 200 mg for larger breeds. research complications. as with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. dogs used in intestinal pharmacokinetic studies should be confirmed to be free of cystoisospora infections. b. nematodes i. ascarids etiology. the most common ascarid of dogs is toxocara canis. toxascaris leonina can also infect both dogs and cats. clinical signs. ascarid infestations are most commonly subclinical. however, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. puppies may have a classical "potbellied" appearance and dull hair coat. heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. visceral larval migrans caused by toxocara canis can cause pneumonia. epizootiology and transmission. toxocara canis typically infects puppies. in fact, a unique characteristic of t. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. pathologic findings. puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia. pathogenesis. the infective stage of t. canis is the third-stage larva (l3). infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. these larval migrations are often asymptomatic, and progression of the l3 larvae is arrested in the tissues. it is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. if the source of infection is transplacental, puppies may be born with l3 larvae in their lungs, because larval migration is already in progress (sherding, 1989 ). diagnosis and differential diagnosis. the characteristic large (70-85 ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. prevention and control. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, 2000) . treatment. most anthelmintics are effective for treatment of ascariasis. pyrantel pamoate (nemex) and fenbendazole (panacur) are commonly used. treatment should be started early in puppies (2, 4, 6, and 8 weeks) because of the possibility of prenatal or neonatal infection. pyrantel pamoate, dosed at 5 mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see section iii,a,3,b,ii). in breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. extended fenbendazole therapy (50 mg/kg per os twice per day for 14 days or once per day from day 40 of gestation through day 14 of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. research complications. puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. hookworms etiology. the most common and most pathogenic hookworm of dogs is ancylostoma caninum. other, less pathogenic canine hookworms found in north america are a. braziliense, which can be found in the american tropics and southern united states, and uncinaria stenocephala, which is distributed in the northern united states and canada. clinical signs. only a. caninum infestation typically results in clinical illness, because of the amount of blood that it con-sumes. puppies with a. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. epizootiology and transmission. infective larvae (l3) are typically ingested by puppies and develop directly in the intestinal tract. ingestion can be from the bitch's milk (transmammary migration occurs with a. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. transplacental migration does occur with a. caninum, but to a much lesser extent than is seen with toxocara canis. larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. the prepatent period is 3 weeks. pathologic findings. infected puppies often have severe anemia and eosinophilia. the anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. on gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (fig. 3a) . ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (fig. 3b) . pathogenesis. the severe pathogenicity of a. caninum is a direct result of its voracious consumption of blood and body fluids. each adult hookworm can consume 0.01-0.2 ml of blood; thus an extensive infection could deplete a puppy of 20 ml of blood per day, which is approximately 15% of the blood volume of a 2.0 kg animal. in contrast, a. braziliense and u. stenocephala consume 0.001 and 0.0003 ml per worm, respectively. diagnosis and differential diagnosis. diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. prevention and control. purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, 2000) . treatment. pyrantel pamoate (nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. because of the possibility of transplacental or milk-borne infection, puppies should be treated every 2 weeks from weeks 2-8. a follow-up treatment at 11 weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation. research complications. anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. iii. strongyloides etiology. strongyloides stercoralis is a small strongyle that can cause hemorrhagic enteritis in puppies. it is found in warm, humid climates such as the southeastern united states. fects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. the eggs of s. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. diagnosis and differential diagnosis. the baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva (280-310 ~tm x 30-80 ~tm). the larvae must be distinguished from larva of filaroides hirthi and hatched ancylostoma caninum. treatment. the usual treatment for s. stercoralis is fenbendazole (panacur) at 50 mg/kg per day for 5 days. iv. whipworms etiology. trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. the adult whipworm typically resides in the cecum or ascending colon. clinical signs. most whipworm infections are subclinical. in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss are also seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode. life cycle. adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. the eggs are very hardy and can persist for years. in optimal conditions, the eggs develop into an infective embryo within 10 days. after ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. the prepatent period is typically 2-3 months long. pathologic findings. dogs do not typically die from whipworm infestations. lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. pathogenesis. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. diagnosis and differential diagnosis. whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. these eggs are barrel-shaped, with thick walls and bipolar plugs. because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. adult worms can be seen on colonoscopy (jergens and willard, 2000) . differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. prevention and control. trichuris eggs are resistant to disinfection, making control difficult. dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. treatment. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for 3 months (jergens and willard, 2000) . treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. research complications. whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis. etiology. heartworm disease of dogs is caused by the filarial worm, dirofilaria immitis. adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them. clinical signs. most heartworm infestations are asymptomatic. the most common clinical signs observed are coughing and dyspnea. clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. epizootiology and transmission. successful heartworm transmission requires the presence of mosquitoes. for this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. the microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. this stage is then returned to the canine bloodstream during feeding. this stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. in the united states, transmission of heartworm by mosquitoes occurs over a 6month or shorter period, except for the southeastern and gulf coast states. here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (knight, 2000) . pathologic findings. on necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (fig. 4a ). there may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (fig. 4b) . endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis. pathogenesis. the physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. however, the host immunologic response to this infestation, coupled with secretion by the heart-worms of physiomodulative factors, contributes significantly to the complications seen with this disease. endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. these changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. the heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (dillon, 2000) . these factors are discussed in more detail below, under "research complications." diagnosis and differential diagnosis. for dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. a diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. false-negative results can occur during the prepatent period after initial infection (first 6-7 months), and when the adult worm burden is light or predominantly male. infections consisting of more than three mature female worms are usually detected by antigenic serology (knight, 2000) . a significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). if a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test 7 months later. examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified knott test and filter tests. tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of dipetalonema reconditum, a nonpathogenic filarial worm. other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. these same techniques can be used to diagnose clinical heartworm disease. additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis (complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure. prevention and control. for dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. purpose-bred dogs reared in such an environment are usually free from dirofilariasis. however, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. there are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). these could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. if a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. treatment. treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (hoskins, 1989) and immunologic reactions to dead worms lodged in the pulmonary vasculature. for this reason, medical treatment of heartworm disease is not usually attempted in research dogs. in a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (caparsolate) and ivermectin (ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). alternative choices include melarsomine (immiticide) as an adulticide and milbemycin (interceptor), levamisole (levasol), or fenthion (spotton) as microfilaricidal agents. dosing regimens for these agents are detailed in dillon (2000) . research complications. the physiomodulative properties of heartworm infection have been studied. such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (maksimowich et al., 1997; matsukura et al., 1997; mupanomunda et al., 1997) . the extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (kaiser et al., 1992) but refuted in others (tithof et al., 1994) . it is thought that the endothelium is perturbed by a factor released from the adult dirofilaria, possibly a cyclooxygenase product such as prostaglandin d2 (kaiser et al., 1990 (kaiser et al., , 1992 . these products have also been demonstrated to cause constriction in in vitro rat tracheal ring preparations (collins et al., 1994) , suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. platelet reactivity was also been found to be enhanced in dogs naturally infected with dirofilaria, when compared with uninfected controis (boudreaux and dillon, 1991) . based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements. etiology. several species of cestodes (tapeworms) parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. clinical signs. most cestode infestations are subclinical. severe infestations with dipylidium can be associated with diarrhea, weight loss, and poor growth. epizootiology and transmission. the cestode life cycle requires an intermediate host. for dipylidium caninum, the intermediate hosts are fleas and lice. thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. pathologic findings. adult cestodes in the small intestine are usually an incidental finding at necropsy. diagnosis and differential diagnosis. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. dipylidium egg packets are large (100 x 150 bm) and contain 1-63 eggs per packet (hall and simpson, 2000) . prevention and control. the most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. see the sections on these ectoparasites for effective means to treat infested dogs and kennels. treatment. praziquantel at 5-12.5 mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against dipylidium caninum (hall and simpson, 2000) . clinical signs. most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. epizootiology and transmission. the lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. on ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. pathologic findings. grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. pleural hemorrhages may also be caused by the migrating metacercariae (lopez, 1995) . pathogenesis. clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. diagnosis and differential diagnosis. definitive diagnosis of paragonimus infestation requires identification of the characteristic ovoid eggs (80-115 ~tm long) with a single operculum in either the feces or a transtracheal wash. identification from fecal samples requires sedimentation techniques. other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. prevention. use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal. treatment. praziquantel (at 25 mg/kg q8 hr x 3 days) or fenbendazole (25-50 mg/kg q12 hr x 10-14 days) are recommended for treatment of canine paragonimus infestation (hawkins, 2000) . effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. research complications. experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e. mites i. demodicosis etiology. canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles. it is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. clinical signs. demodex canis infestation is typically asymptomatic. clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and the face and around the ears (demanuelle, 2000a). secondary bacterial pyoderma is a common complication. epizootiology and transmission. demodex canis mites pass to nursing pups from the dam. they live their entire lives on one dog and are not considered contagious to other dogs or humans. certain breeds are predisposed to the generalized form of demodex dermatitis (see "pathogenesis," below). beagles are among the predisposed breeds, as are german shepherds, doberman pinschers, old english sheepdogs, collies, boxers, and shorthair brachycephalic breeds (muller et al., 1983) . pathologic findings. histologically, demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. cases with generalized demodicosis (see "pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression . pathogenesis. when clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one foot affected, or five or more small areas, or one large body area). localized demodicosis is typically seen in juvenile dogs (< 18 months) and usually resolves without treatment as natural immunological control develops. generalized demodicosis can develop in juvenile or adult populations. juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited t-lymphocyte dysfunction. adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration). diagnosis and differential diagnosis. demodex is readily identified from deep skin scrapings of lesioned areas (campbell, 2000; noli, 2000) . demodex canis has a characteristic "cigar shape," with short, stubby legs on a body 100-400 ~tm long. differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. prevention and control. dogs with generalized demodicosis should not be maintained in a breeding colony. treatment: ivermectin (ivomec) at 200-600 ~tg/kg and oral milbemycin (interceptor) at 1-2 mg/kg/day have been found to be effective treatments. these parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of demodex canis. amitraz (mitaban) dips (250 ppm every 14 days) can be used for more problematic cases. treatment duration can be extensive and must be accompanied by repeated skin scrapings. research complications. dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. sarcoptic mange etiology. canine sarcoptic mange is caused by sarcoptes scabiei var. canis. clinical signs. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. epizootiology and transmission. sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for 1-3 weeks away from the host, and it is this ability that enables them to spread from dog to dog. sarcoptes scabiei var. canis can also infect cats and humans. pathologic findings. histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis . pathogenesis. lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. the typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. diagnosis and differential diagnosis. sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige in either the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, 2000 ). an important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. prevention and control. use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. for random-source dogs, an ectoparasite control program should be in place to limit possible infestations. many institutions use ivermectin as a means to control both endoparasites and ectoparasites. treatment. unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. in research colonies, the usual means of treatment is either ivermectin (ivomec) at 200-400 ~tg/kg q14 days or milbemycin (interceptor) at 3 oral doses of 2 mg/kg q7 days . neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. research complications. the local skin inflammation and systemic immune response to sarcoptic mange probably make infected dogs poor subjects for dermatologic and immunologic studies. f lice and ticks i. lice etiology. dogs can be infested by one species of sucking louse (linognathus setosus) and two species of biting lice (trichodectes canis and heterodoxus spiniger). clinical signs. mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. more severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. these lesions lead to excoriation and secondary bacterial dermatitis. severe linognathus infestations could cause anemia, because this species feeds on blood. epizootiology and transmission. louse infestations are uncommon in both pet animal practice and the research setting. they would most likely be seen in random-source dogs that were obtained from a pound or shelter. transmission is usually by direct contact, for lice spend their entire lives on the host species. lice are host-specific and not zoonotic. pathogenesis. the biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. trichodectes canis can serve as vector for the canine tapeworm dipylidium caninum. the most severe complication of infestations by the sucking louse is the potential anemia. diagnosis and differential diagnosis. pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (muller et al., 1983) . differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea. prevention. use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony. treatment. most commercially available insecticide shampoos and dips readily treat louse infestations. treatment should be repeated in 10-14 days, because any nits that were not killed would have hatched by that time (muller et al., 1983) . there is probably minimal interference with research, unless severe linognathus infestations cause anemia. ii. ticks etiology. ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. except for the brown dog tick (rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, and ixodes. the primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including rocky mountain spotted fever (caused by rickettsia rickettsii), lyme disease (borrelia burgdorferi), and the canine forms of ehrlichiosis (ehrlichia canis and e. platys), babesiosis (babesia canis), haemobartonellosis (haemobartonella canis), and hepatozoonosis (hepatozoon canis). clinical signs. as an entity unto itself, tick infestation causes minimal clinical signs. most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (merchant and taboada, 1991) . some species of ticks (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (malik and farrow, 1991) . the paralysis develops within 5-9 days of tick attachment and can result from a single tick. this paralysis is fatal once the respiratory musculature is affected. epizootiology and transmission. in dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. the brown dog tick may reside within kennels (attics, bedding, wall insulation) (garris, 1991) . pathologic findings. under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death). pathogenesis. tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., dermacentor) while consuming a blood meal (malik and farrow, 1991) . interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. the primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including lyme borreliosis, ehrlichiosis, babesiosis, and rocky mountain spotted fever. diagnosis and differential diagnosis. for uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, 1991) . prevention. purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites. treatment. removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. dogs with tick-bite paralysis usually show improvement within 24 hr, with complete recovery within 72 hr (malik and farrow, 1991) to remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. the tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. the tick should be pulled away from the dog with steady pressure. many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. probably have minimal impact on research variables. the significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see sections iii,a,l,e-g). g. other i. flea infestation etiology. fleas are laterally flattened wingless insects that feed on animal blood. the most common flea to infest dogs is ctenocephalides felis, the cat flea. other fleas that can affect dogs are ctenocephalides canis, pulex irritans, and echidnophaga gallinacea. the fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (campbell, 2000) . clinical signs. flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as well as the flanks, thighs, and abdomen (muller et al., 1983) . the lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages. epizootiology and transmission. fleas are readily transmitted between animals and even between host species. they move readily between the host and the environment, making transmission easy and control difficult. because fleas require host blood for food, they can survive off of a host for only 1-2 months (muller et al., 1983) . pathologic findings. biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (muller et al., 1983) . pathogenesis. fleas are parasites that require animal blood for their meals. when they bite host animals, they inject some saliva into the host's skin. if the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. fleas can also transmit or serve as vectors for other pathogens (e.g., dipylidium tapeworms). flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (demanuelle, 2000b) . circulating eosinophilia is seen in some dogs with flea allergy dermatitis. differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity). prevention. most dogs obtained from high-quality purposebred facilities should be free from flea infestations. dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility. control. thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. treatment. treatment for fleas needs to address treatment of both the dog and the environment. many insecticide formulations such as shampoos, sprays, dips, powders, and oral systemics can be used for initial treatment of the individual dog. the active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. flea control in the kennel may need to include outdoor areas in warm climates. typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (demanuelle, 2000b) . in addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. oral prednisone or prednisolone at 0.5 mg/kg q12 hr for 5-7 days has been proposed as a starting therapy (muller et al., 1983) . the use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting. research complications. mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. in a research setting, the residual effects of insecticides may preclude their use in experimental animals. such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. the ability of fleas to transmit other parasitic diseases must also be considered. etiology. dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the united states are usually caused by either microsporum canis, m. gypseum, or trichophyton mentagrophytes (muller et al., 1983) . clinical signs. uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. these skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (muller et al., 1983) . ep&ootiology and transmission. the fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil. pathologic findings. on close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (muller et al., 1983) . stains that facilitate visualization of fungal elements include periodic acid-schiff (pas) or gomori methenamine-silver. the pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. pathogenesis. the dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. the hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. this causes short stubbly hair to be seen within the lesion. as the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. it is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (muller et al., 1983) . diagnosis and differential diagnosis. diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. these scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. fungal elements can also be seen on skin biopsy samples. for speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as sabouraud's medium or dermatophyte test medium (dtm). incubation should be at 30~ with 30% humidity for 14-30 days. lesions caused by m. canis may fluoresce when inspected using a wood's (253.7 nm ultraviolet) light. unfortunately, some strains of m. canis do not fluoresce, and neither does m. gypseum or t. mentagrophytes. differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (muller et al., 1983) . prevention. purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals. control. in cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. treatment, if acceptable, should be started immediately. treatment. topical antifungal therapy is most commonly used. shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (stannard et al., 2000) . severe cases may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. however, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). many of the newer agents are also expensive and not labeled for use in dogs. impact on most research applications for dogs. unfortunately, the zoonotic implications of dermatophytoses force the issue of aggressive treatment, and many antifungal agents may not be compatible with biomedical research studies. systemic fungal infections disseminate to multiple organ systems from a single mode of entry (usually through the respiratory tract). dogs are susceptible to several fungi that characteristically cause systemic mycosis, including blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans var. neoformans. these diseases are not typically seen in the research setting, because of the low overall incidence and noncontagious nature of these disorders and because of the use of purpose-bred animals. these conditions could, however, present in the rare random-source dog that was subclinical at its point of origin, especially if the animal becomes immunosuppressed (either naturally or by virtue of experimental manipulation). typical clinical signs include weight loss, fever, lymphadenopathy, and cough and dyspnea (if the lungs are affected). the reader is advised to read veterinary medical text chapters (e.g., taboada, 2000) for more complete information on these disorders and their possible treatments. although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, 1994) , deficiency in thyroid hormone can significantly affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology. the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. both of these causes result in a gradual loss of functional thyroid tissue (kemppainen and clark, 1994) . lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, 1962; beierwaltes and nishiyama, 1968; manning 1979) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, 1989; kemppainen and clark, 1994) . clinical signs. because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (peterson and ferguson, 1989; panciera, 1994) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, 1989; panciera, 1994) . normocytic normochromic nonregenerative anemia and increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, 1989; panciera 1994) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, 1989) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, 1989) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bischel et al., 1988; panciera, 1994) , and so the relationship between hypothyroidism and these problems has not been completely defined (panciera, 1994) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, 1989) . hypothyroidism can also cause bradycardia as a result of decreased myocardial conductivity. abnormalities that may be detected by ecg include a decrease in p and r wave amplitude (peterson and ferguson, 1989) and inverted t waves (panciera, 1994) . these electrocardiographic abnormalities are caused by lowered activity of atpases and calcium channel function. several reports have suggested that hypothyroidism is associated with von willebrand's disease and bleeding abnormalities. however, the relationship is probably one of shared breed predilection and not a true correlation. it has been demonstrated that dogs with hypothyroidism are not deficient in von willebrand's factor when compared with other dogs. in addition, the replacement of thyroid hormone in dogs did not increase the levels of vwf:ag in naturally occurring (panciera and johnson, 1994) or experimentally induced (panciera and johnson, 1996) hypothyroidism. epizootiology. the prevalence of hypothyroidism in the general canine population has been reported to be less than 1% (panciera, 1994) . the disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (panciera, 1994; peterson and ferguson, 1989; scarlett, 1994) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, 1979; tucker, 1962; beierwaltes and nishiyama, 1968) . in general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (panciera, 1994; peterson and ferguson, 1989 ). diagnosis and differential diagnosis. because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. the tests currently available and in popular use will be discussed further. however, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, 1989; ferguson, 1994) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t 4 (thyroxine) and free t 4 (peterson and ferguson, 1989; ferguson, 1994) . t 4 serves primarily as a precursor for t 3 in the body and is heavily proteinbound. free t4 represents the unbound fraction that is available to the tissues (peterson and ferguson, 1989) . using the measurement of serum total t 4 and free t4, hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (peterson and ferguson, 1989) . however, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, 1989; ferguson, 1994) . therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. if the clinical signs are equivocal or if only total t 4 or free t 4 is decreased, further diagnostic testing is warranted (peterson and ferguson, 1989) . although t 3 is the most biologically active form of thyroid hormone in the body, the measurement of serum t 3 levels is an unreliable indicator of hypothy-roidism (peterson and ferguson, 1989; ferguson, 1994) . like t4, serum t 3 can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t 3 may be preferentially released, and conversion of t 4 to t 3 may be enhanced in the hypothyroid dog (peterson and ferguson, 1989; ferguson, 1994) . t 3 was within normal limits in 15% of the hypothyroid dogs in one study (panciera, 1994) . autoantibodies can be responsible for false elevations in the concentrations of t 3 and t 4 found in these respective assays. it has been recommended that free t4, measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t 3 and t 4. autoantibodies have been found in less than 1% of the samples submitted to one laboratory (kemppainen and behrend, 2000) . other means of diagnosing hypothyroidism have been described. in humans, endogenous tsh (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous tsh is now available in dogs. however, tsh levels can be normal in some dogs with hypothyroidism, and high tsh levels have been noted in normal dogs. therefore, it is recommended that tsh levels be considered along with other information (clinical signs, t4) prior to diagnosis and treatment (kemppainen and behrend, 2000) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, 1989; ferguson, 1994) . another drawback of tsh testing is that the test must be postponed for 4 weeks if thyroid supplementation has been given (peterson and ferguson, 1989) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is 0.045 u of tsh per pound of body weight (up to a maximum of 5 u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and 6 hours after. a normal response to the administration of tsh should create an increase of t 4 levels at least 2 ktg/dl above the baseline levels or an absolute level that exceeds 3 ~tg/dl (peterson and ferguson, 1989; wheeler et al., 1985) . treatment. the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is 0.02 mg/kg once a day or 0.05 mg/m 2 (body surface area)/day for very small or very large dogs. if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in 6-8 weeks, and blood samples should be drawn 4-8 hours after the morning pill. a clinical response is usually seen in 6-8 weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, 1994) . ecg abnormalities also return to normal (peterson and ferguson, 1989) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, 1994) . weight gain and eventual obesity are also frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research. etiology. obesity is defined as a body weight 20-25% over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy. excessive caloric intake resuits from overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, 1992) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (butterwick and hawthorne,. 1998 ). as in humans, genetics plays an important role in the development of obesity in dogs. it has been established that certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, 1986) . in addition to genetics, several metabolic or hormonal changes are associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese when compared with intact females (macewen, 1992) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, 1986) . in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, 1992) . epizootiology. ewen, 1992) . obesity affects up to 40% of pet dogs (mac-diagnosis and differential diagnosis. the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (wilkinson and mcewan, 1991) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see section iii,b,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment. restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to 60% of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to 50% produces no adverse health effects. however, t 3 levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of 1-2% of body weight per week (laflamme et al., 1997) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. there has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, 1992) . there has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick and thorne, 1994; butterwick and thorne, 1997) . it is important to control weight gain in research animals, because of the association of obesity and several metabolic changes. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, 1992) , this relationship is not consistently apparent (edney and smith, 1986) . obesity in dogs over 10 years of age appears to be related to an increase in cardiovascular problems (edney and smith, 1986) , and obesity has been linked to hypertension. joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, 1992; kealy et al., 1997) . in addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (macewen, 1992) . in the laboratory setting, the majority of traumatic wounds will be small in size. in facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. to aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (swaim, 1980; waldron and trevor, 1993) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see table v ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. dirty wounds can occur as postsurgical infections or complications of initial wound therapy. when one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of the wound's classification. when first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. bleeding should be controlled with direct waldron and trevor (1993) . pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, 1980) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, 1980) . when the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. then a water-soluble lubricant gel may be applied directly to the wound. a wide margin of hair should then be clipped from around the wound, using a #40 blade. after the clipping, a surgical scrub is performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., 1990a,b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. two solutions, 0.05% chlorhexidine diacetate in water (lozier et al., 1992) and 1% povidone-iodine in saline, are most often recommended for wound lavage (waldron and trevor, 1993) . the chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (sanchez et al., 1988) . actually, the type of solution chosen may not be as important to wound care as the volume and pressure at which the solution is delivered. it has been suggested that 8 psi is required to obtain adequate tissue irrigation, and this may be achieved by using a 35 ml syringe and an 18-or 19gauge needle (waldron and trevor, 1993) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, 1993) . if one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (swaim, 1980; waldron and trevor, 1993) , and nonviable areas will demarcate within 2-3 days (waldron and trevor, 1993) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, 1980) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be place d. subcutaneous closure should be performed with absorbable suture such as polydioxanone (pds), polyglactin 910 (vicryl), or polyglycolic acid (dexon). it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon (3-0 or 4-0). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. when the sponges are later pulled from the wound, dried exudates will also be removed. when the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (swaim, 1980) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within 3-5 days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after 5 days, this is considered secondary closure (waldron and trevor, 1993) . secondintention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, 1980) . it is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. in skin wounds, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, 1993) . etiology. pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. the compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, 1990) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. these factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (swaim and angarano, 1990) . clinical signs. at first, the skin at the developing site will appear red and irritated. over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. the severity of the sores may be graded from i to iv, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. epizootiology. the problem usually occurs in large-breed dogs, but any type of dog can be affected. prevention and control. minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. if the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain good flesh and adequate healing is also important (swaim and angarano, 1990) . treatment. the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. the affected area should be radiographed to assess bone involvement, and the wound should be cultured. all of the damaged tissue should be debrided, and wound management guidelines should be followed (see section iii,c,1). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, 1990) . with extensive lesions, reconstruction with skin flaps may be necessary. bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. this will displace the forces acting on the wound over a larger area and over healthier tissue. then the doughnut is incorporated into the bandage. if a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, 1990 ). bandages should be removed at least once or twice a day to allow wound care. after wound care has been initiated the causative factors for the pressure sore must be addressed (see "prevention and control," above). recumbent animals should be moved frequently to prevent continuous compression on the wound. if the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. etiology. acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. in a few cases, self-trauma begins because of identifiable neurologic or orthopedic causes (tarvin and prata, 1988) . however, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, 1990) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., 1988) . the laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma. epizootiology. the lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (walton, 1986) . clinical signs. at first, lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, 1990 ). the predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis. acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of these problems can be ruled out by the history of the animal. when in doubt, a biopsy should be taken. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, 1986) . prevention and control. behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. the environment of a dog with this problem can be enriched with exercise and the introduction of toys. in addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. these drugs may produce side effects, however (swaim and angarano, 1990) , and may interfere with experimental results. treatment. several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in ah cases. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, 1990; walton, 1986) . opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. in one study, either naltrexone (1 mg/kg sq) or nalmefene (1-4 mg/kg sq) successfully reduced the excessive licking behavior in 7 of 11 dogs; however, lesions returned after the drug was discontinued (dodman et al., 1988) . the use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide (3 ml of banamine [schering] mixed with 8ml of synotic [diamond laboratories]) applied topically twice daily has also been shown to be effective (walton, 1986) . the prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. etiology. hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., 1974) . epizootiology. elbow hygromas are most frequently reported in large and giant breeds of dogs around 6-18 months of age (johnston, 1975; bellah, 1993) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs. a dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. the animals are not usually lame. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma is secondarily infected, the animal may exhibit pain and fever (johnston, 1975; bellah, 1993) . pathology. the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is yellow or red and is a serous transudate. this fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (johnston, 1975) . treatment. the treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see section iii,c,2). more aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this treatment (johnston, 1975) . likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (johnston, 1975) . a technique that has been used successfully involves placement of multiple penrose drains. the drains are kept in place for 2-3 weeks, and the limb remains bandaged for 4 weeks with this technique (bellah, 1993) . another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. the remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. the stents are removed in 5-7 days, and the wound is bandaged until suture removal in 10-14 days (newton et al., 1974) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology. in the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting. clinical signs. the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia. treatment. the treatment of corneal ulcers will depend on the depth and size of the affected area. deep ulcers may require debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given 3 times a day for 2-3 days usually provides adequate treatment. ointments are preferred over drops, because use of the former requires less frequent. simple corneal ulcers are restained with fluorescein after 3 days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. the catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. it is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. when appropriately maintained, indwelling catheters may remain functional for months without serious complication. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. this is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. one study (hysell and abrams, 1967 ) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact vascular locations not specified). the lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. the traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. the visceral infarcts were noted in the spleen, kidney (fig. 5) , and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. fatal hemorrhages were most often found in animals with experimentally induced hypertension. these animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm. etiology. the leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. clinical signs. the signs and treatment of systemic infection are covered in section iii,d,3. problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. the signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. prevention. because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. the catheter should be made of nonthrombogenic material. in addition, it is recommended that catheters be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, 1984) . the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. the placement of the catheter should be verified by radiography. catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. after catheter placement, the animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "treatment"). throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. treatment. the treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, 1984; darif and rush, 1983) . superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. systemic antibiotic therapy should be initiated for a 10-day period. the choice of drug will ultimately be based on previous experience and culture results. aerobic and anaerobic cultures of blood and locally infected sites should be performed (ringler and peter, 1984) . localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. again, the catheter should be removed. if retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. it has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (dennis et al., 1989) . the solution is removed after 15 min and replaced with heparinized saline. all of the extension lines and fluids used in the catheter should be discarded. the blood cultures should be repeated 3 days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., 1993) . when placed and managed correctly, these ports may remain in place for months without complications. the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., 1993 , kwei et al., 1995 . these infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. there have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. this complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (meunier et al., 1993) . the chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (figs. 6a,b) as complications of intestinal access ports. the procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. it is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. an omental patch placed over the site of entry may help form a firm adhesion. in addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. the placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (meunier et al., 1993) . etiology. sepsis is defined as the systemic response to infection. most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. in laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs. the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, 1993) . pathogenesis. the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~ as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis. in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., 1997) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (kirby, 1995) . treatment. the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is associated with the source of infection, the implant should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no "magic bullet" for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology. in research animals, aspiration into the lungs may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. clinical signs. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on a history consistent with aspiration and the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. pathogenesis. aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the ph, osmolality, and volume of the substance. the compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after 24-48 hr. treatment. the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. in cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. when severe inflammation is present, systemic fluid therapy should be instituted. support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. the use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (nader-djahal et al., 1997) . several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. in humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be treated with antibiotics immediately when the aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, 2000) . the presence of pneumonia should be verified by tracheal wash and cultures. etiology. in laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs. the clinical signs vary with the type and degree of burn injury. initially, the injury may not be noticed. the first signs may be oozing from the skin and matting of the overlying hair. within a couple of days, progressive hair and skin loss may be observed (johnston, 1993) . the wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. superficial wounds appear as red, inflamed skin similar to sunburn in humans. the pain associated with these injuries usually subsides in 2-3 days, and the wound reepithelializes without complications in 3-5 days. deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. the wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. within 2-3 days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. several days later, gram-negative organisms can appear in the burn wound (johnston, 1993) . at this point, signs of wound infection and sepsis may occur (see section iii,d,3). treatment. appropriate and timely treatment of a burn wound will reduce the extent of the injury. thermal injuries should be immediately cooled to reduce edema and pain (demling and lalonde, 1989) . chemical burns should be thoroughly lavaged for 60 min after wounding. the damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. after the initial treatment, all burn wounds should be gently cleansed 2-3 times a day (demling and lalonde, 1989) . burns involving the epidermis and part of the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, topical wound dressings are recommended in the early stages of treatment. a thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (demling and lalonde, 1989) . when signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. after the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. when the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected. prevention. obviously, prevention of burn wounds is preferable to a long course of treatment. care should be taken to prevent direct exposure to harsh chemicals. tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. etiology. research and/or anesthetic protocols may require the intravenous injection of various solutions. when these substances have a ph or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (swaim and angarano, 1990; waldron and trevor, 1993) . clinical signs. the immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. later, the area may appear red, swollen, and painful as inflammation progresses. often there will be eventual necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a 1 to 4 month period. this is because the drug is released over time from the dying cells (swaim and angarano, 1990) . prevention. because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. if a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. whenever possible, insertion of an indwelling catheter is extremely important. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. treatment. the treatment of perivascular injections will depend on the amount and type of substance injected. in most cases, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (swaim and angarano, 1990 ). the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, 1989) , and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (waldron and trevor, 1993) . despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, 1990) . in all cases, the condition can be painful, and analgesia should be addressed. etiology. hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. this condition may be seen in young dogs with congenital portosystemic shunting of blood flow. however, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. a well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy. pathogenesis. when the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (hardy, 1989; center, 1998) . these compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance 7-aminobutyric acid (gaba). clinical signs. the signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. diagnosis and differential diagnosis. a presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. serum glucose and protein levels may be low if hepatic function is severely impaired. a low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (hardy, 1989) . measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal. treatment. because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. during initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. the majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either 0.45% sodium chloride or 0.45% sodium chloride with 2.5% dextrose, supplemented with potassium chloride, is recommended (hardy, 1989) . the type of drug to be used for seizure control is controversial. the short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (maddison, 1995) . however, endogenous benzodiazepines mediate some of the cns signs seen with hepatic encephalopathy. therefore, the use of diazepam has been discouraged in favor of phenobarbital (johnson, 2000) . the drug selected for seizure control should be titrated carefully, given the altered liver metabolism. most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of h2 blockers such as cimetidine or ranitidine is suggested (swalec, 1993) . in addition, lactulose retention enemas should be performed (10-15 ml/lb of a 30% solution in water, retained for 20-30 min) (hardy, 1989) . lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. the decrease in colonic ph reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. lactulose will also cause an osmotic diarrhea. antibiotics such as neomycin (10 mg/lb, 3-4 times/ day) or metronidazole (9 mg/lb, 3 times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (hardy, 1989) . when the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. diets suitable for dogs with renal insufficiency are recommended initially. this type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. these requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. to maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (center, 1998) . in addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels. the prevalence of cancer in the general canine population has increased over the years (dorn, 1976) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, 1982) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. (1994) found death rates similar to the death rate of the at-large dog population (bronson, 1982) . approximately 22% of the male beagles died of cancer. the majority of the tumors were lymphomas (32%) and sarcomas (29%), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer (26% of the population studied), three-quarters had either mammary cancer (40%), lymphomas (18%), or sarcomas (15%). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, 1975; benjamin et al., 1996) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. an instrument such as a tru-cut needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a 1 mm x 1 to 1.5 cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least 1 cm around the tumor, and 3 cm if mast cell tumors are suspected (morrison et al., 1993) . incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology. lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide 2,4-dichlorophenoxyacetic acid (2,4-d) as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, 1991) . clinical signs. multicentric and alimentary lymphomas account for most cases of canine lymphoma. in multicentric lymphoma, animals usually present with enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. the tumors may occur as nodules, plaques, ulcers, or dermatitis. approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement. epizootiology. the incidence of lymphoma is highest in dogs 5-11 years old, accounting for 80% of cases. although the neoplasm generally affects dogs older than 1 year, cases in puppies as young as 4 months have been reported (dorn et al., 1967) . pathologic findings. enlarged neoplastic lymph nodes vary in diameter from 1 to 9 cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. pathogenesis. all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is 4-6 weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, 1991) . hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. diagnosis and differential diagnosis. differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed. treatment. therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. macewen and young (1991) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. research complications. given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. etiology. the fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (pulley and stannard, 1990) . frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas. clinical signs. although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. fibrosarcomas are extremely variable in size and can grow to be quite large. in general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. epizootiology. most fibrosarcomas develop in adult and aged animals but can affect dogs as young as 6 months or less. pathogenesis. fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. less frequently, spread to local lymph nodes is observed. diagnosis and differential diagnosis. differential diagnoses for fibrosarcomas vary with the location of the tumor. histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms. treatment. treatment of any soft-tissue sarcoma would begin with wide surgical excision. if the tissue margins indicate incomplete resection, radiotherapy could be used. for any highgrade tumors, adjuvant chemotherapy would be recommended (see macewen and withrow, 1991a , for a complete discussion). research complications. because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies. etiology. neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas. clinical signs. these growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. generally they are well circumscribed, encapsulated, and soft on palpation. further, the skin is freely movable over the tumor. epizootiology. lipomas occur principally in aged animals (average 8 years), and the incidence increases with age (pulley and stannard, 1990) . the tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed. pathologic findings. histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present. pathogenesis: lipomas are typically slow-growing and do not recur after complete surgical excision. diagnosis and differential diagnosis. lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. generally, the distinction can be made from the clinical history. treatment. treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. in such cases, surgical excision is usually curative. research complications. lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation. etiology. histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (pulley and stannard, 1990) . clinical signs. the most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. the masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure 1-2 cm in diameter. epizootiology. histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than 2 years of age. pathologic findings. histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. the cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. despite being benign lesions, histiocytomas characteristically have a high mitotic index. pathogenesis. this tumor typically exhibits rapid growth (1-4 weeks) but does not spread. most histiocytomas will spontaneously regress in less than 3 months. diagnosis and differential diagnosis. histiocytomas must be distinguished from potentially metastatic mast cell tumors. this is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple. treatment. although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. research complications. histiocytomas should not interfere with most studies. etiology. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (bostock, 1986) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. clinical signs. well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, 1 to 10 cm nodules in the skin. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-guinal and scrotal areas. mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. epizootiology. these tumors tend to affect middle-aged dogs but have been observed in dogs ranging from 4 months to 18 years (pulley and stannard, 1990 ). pathologic findings. because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. (1986) has become widely accepted. in this system, grade i has the best prognosis, and grade iii the worst prognosis. grade i tumors are well differentiated, with round to ovoid uniform cells. the nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. the cytoplasmic granules are few, but mitotic figures are much more frequent. in addition to skin lesions, mast cell tumors have been associated with gastric ulcers. these lesions are most likely secondary to tumor production of histamine. histamine stimulates the h2 receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (>75%) of dogs with mast cell tumors (howard et al., 1969) . the ulcers can be found in the fundus, pylorus, and/or proximal duodenum. although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis. mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple. treatment. initial treatment for mast cell tumors is generally wide surgical excision (1 to 3 cm margins). even with wide surgical margins, approximately 50% of mast cell tumors may recur. if the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. other alternatives include amputation (if on a limb) or radiation therapy alone. as an adjunct to surgery, grier et al. (1990 grier et al. ( , 1995 found that deionized water injected into surgical margins reduced tumor recurrence by hypo-osmotically lysing any mast cells left behind. this technique has recently been refuted by jaffe et al. (2000) . for systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (graham and o'keefe, 1994) . research complications. because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every 3-6 months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology. hemangiosarcomas are malignant tumors that originate from endothelial cells. clinical signs. these tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. clinical signs are associated with the site of involvement. vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. heart failure can be observed secondary to tumor burden or hemopericardium. when found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from 1 to 10 cm in diameter. the most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. epizootiology: hemangiosarcomas occur most frequently in 8-to 13-year-old dogs. the german shepherd dog is most commonly affected. pathologic findings. grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (fig. 7) . the masses are spherical and reddish black and can range in size up to 15-20 cm in diameter. on cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. when the masses are found in the heart, the endocardium may be covered by a thrombus, giving the 2 to 5 cm tumors a reddish gray or yellow appearance. histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. these spaces may be filled with blood or thrombi. the neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures. pathogenesis. hemangiosarcomas can be found in one or many sites. in cases where multiple sites are involved, it may be impossible to identify the primary tumor. this neoplasia is highly malignant and spreads easily. metastasis occurs most frequently to the lungs but can be found in any tissue. diagnosis and differential diagnosis. splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. when the heart is affected, other causes of heart failure must be ruled out. echocardiography is a valuable tool for identifying the primary lesion. histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors. treatment. surgery is generally the first choice of treatment for hemangiosarcoma. dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. research complications. dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. the other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting. etiology. also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (nielsen and kennedy, 1990) . the origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (macewen, 1991) . clinical signs. the tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to 10 cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (fig. 8) . less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission. transmissible venereal tumors are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, 1990) . extragenital lesions are believed to be a result of trauma prior to exposure to the tumor. pathogenesis. tumor growth is rapid after implantation but later slows. metastasis is rare (<5% of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. diagnosis and differential diagnosis. transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology. prevention. thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control. removing affected individuals from a breeding program should stop further spread through the colony. treatment. surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine (0.5-0.7 mg/m 2) iv once weekly for 4 -6 treatments will induce remission and cure in greater than 90% of the cases (macewen, 1991). research complications. experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific igg (cohen, 1972) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology. dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs. single nodules are found in approximately 75% of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. epizootiology. mammary tumors are uncommon in dogs under 5 years of age with the incidence rising sharply after that. median age at diagnosis is 10-11 years. mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, 1977) . extensive discussions of classification, staging, and histopathologic correlations can be found in macewen and withrow (199 lb) and moulton (1990) . pathogenesis. mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in 60-70% of tumors. futher, schneider et al. (1969) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. dogs spayed prior to the first estrus had a risk of 0.8%, whereas dogs spayed after the first and second estrus had risks of 8% and 26%, respectively. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs. diagnosis and differential diagnosis. both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis. prevention. mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. this is commonly done in the general pet population at 6 months of age. recently, the topic of spaying sexually immature dogs (8-16 weeks of age) has received much attention for the control of the pet population. kustritz (1999) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age. treatment. surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (macewen and withrow, 199 lb). research complications. because 50% of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-tinue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. etiology. beagles are among the breeds with the highest prevalence of thyroid carcinomas. benjamin et al. (1996) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle. clinical signs. thyroid carcinomas generally present as palpable cervical masses. affected animals may experience dysphagia, dyspnea, and vocalization changes. precaval syndrome resulting in facial edema is also observed in some cases. epizootiology and transmission. the mean age of dogs presented with thyroid carcinomas is 9 years, with equal distribution of cases between the sexes. pathologic findings. grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. they tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. the masses are unilateral twice as often as bilateral (capen, 1990) . histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see capen, 1990 , for complete discussion). pathogenesis. thyroid carcinomas tend to grow rapidly and invade local structures. early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein. diagnosis and differential diagnosis. nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. often a preliminary diagnosis can be made by fineneedle aspirate. treatment. surgery is the treatment of choice for thyroid carcinomas that have not metastasized. when the tumor is freely movable, surgery may be curative. surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. when bilateral tumors are observed, preservation of the parathyroid glands may not be possible. in such cases, treatment for hypoparathyroidism will be necessary. both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (ogilvie, 1991) . in the research setting, treatment of this tumor may not be rewarding. only freely movable tumors can be practically treated without seriously affecting research efforts. euthanasia is warranted in the more advanced cases when clinical illness is apparent. beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, 2000b) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm1 gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). in addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor viii deficiency (von willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. at a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia (1.82% of births) and open fontanelle (1.44% of births) (r. scipioni and j. ball, personal communication, 1999) . other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than 1.0% incidence. etiology. cataract is an opacification of the lens or the lens capsule. it is the pathologic response of the lens to illness or injury, because the lens has no blood supply. cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. the ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract. clinical signs. the first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (helper, 1989) . moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. epizootiology and transmission. certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. dogs in studies for diabetes mellitus should be observed regularly for cataract development. toxicological studies may also induce formation of cataracts. pathogenesis. lens fibers respond to all biological or chemical insults by necrosis and liquefaction (render and carlton, 1995) , because they have no blood supply with which to recruit an inflammatory and repair process. disruption of these fibers by any means, therefore, leads to opacification. the exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. in diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. as these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (capen, 1995) . diagnosis and differential diagnosis. the ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. prevention. most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. treatment. because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. many dogs adjust quite well to the visual impairment caused by persistent cataracts. lens removal can be performed for dogs seriously affected by cataracts, but this would not be anticipated for dogs in the research setting. information on surgical lens extraction procedures can be found in helper (1989) or other veterinary ophthalmology textbooks. research complications. research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. etiology. hip dysplasia is a degenerative disease of the coxofemoral joint. a specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (pedersen et al., 2000) , modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (smith et al., 1995) . clinical signs. the initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. this may present as a gait abnormality without any indication of lameness or stiffness. eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. epizootiology and transmission. hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. in the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. diagnosis and differential diagnosis. hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (pedersen et al., 2000) . hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. this may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. radiographic calculation of the distraction index (di) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (smith et al., 1995) . prevention. because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. dogs should be provided a good plane of nutrition but not be allowed to become overweight. dogs that were limit-fed at 75% of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (kealy et al, 1997) . treatment. in the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. more advanced cases may require continuous analgesia. sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (pedersen et al., 2000) . surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. neither surgical treatment is likely in a research setting. research complications. long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. in studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. etiology. benign prostatic hyperplasia (bph) is an agerelated condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs. bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for bph. epizootiology and transmission. bph typically affects older dogs (>4 years), although it has been seen as early as 2 years of age. pathologic findings. in its early stages, canine b ph is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis. bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to 5a-dihydrotestosterone (kustritz and klausner, 2000) . diagnosis and differential diagnosis. bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement. prevention. castration is the primary means for prevention of benign prostatic hyperplasia. treatment. the first and foremost treatment for b ph is castration. in pure cases of b ph, castration results in involution of the prostate gland detectable by rectal palpation within 7-10 days. for most dogs in research studies this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. newer drugs marketed for human males have also shown promise in treating canine bph. finasteride (proscar) is a 5a-reductase inhibitor that limits metabolism of testosterone to 5a-dihydrotestosterone. treatment at daily doses of 1-5 mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (kustritz and klausner, 2000) . dogs given 1.0 mg/kg were proven to still be fertile. there are also indications that lower doses may be effective in relieving b ph. androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of bph while maintaining libido and fertility (kustritz and klausner, 2000) . unfortunately, both the 5areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the united states. research complications. bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. it is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. etiology. juvenile polyarteritis syndrome (jps) is a painful disorder seen in young beagles (occasionally reported in other breeds). the lesion consistent with the syndrome is systemic necrotizing vasculitis. the cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs. clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to extend the neck ventrally. most dogs seem to be in pain when touched, especially in the neck region. the syndrome typically has a course of remissions and relapses characterized by 3-7 days of illness and 2-4 weeks of remission (scott-moncrieff et al., 1992) . there may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission. jps typically affects young beagles (6-40 months), with no sex predilection. pathologic findings. on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., 1995) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. these lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (fig. 9a) . the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., 1995) . fibrinous thrombosis of the affected arteries is also seen (fig. 9b) . a subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis. the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a2-globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in 1/7 affected pups (scott-moncrieff et al., 1992) . diagnosis and differential diagnosis. differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). are known at this time. no prevention and control measures brane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs. the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (fig. 10) . excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. treatment. clinical signs can be abated by administration of corticosteroids. prednisone administered orally at 1.1 mg/kg, q12 hr, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of 0.25-0.5 mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. pathologic findings. typically the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis. prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, 1989) . research complications. because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology. "cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-prevention. hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment. corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, 1989 ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications. in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. etiology. interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (fig. 11 ). the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). clinical signs. dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission. interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, 1993) . pathologic findings. histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis. initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis. bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment. if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at 1 mg/kg ql2h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require fig. 11 . interdigital cyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, 1993) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, 1993) . research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on 68 patients treated primarily with tetracycline and/or doxycycline dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog saunders manual of small animal practice neurologic manifestations associated with hypothyroidism in four dogs neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats platelet function, antithrombin-iii activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis ehrlichia platys infection in dogs the rickettsioses monoclonal gammopathy associated with naturally occurring canine ehrlichiosis variation in age at death of dogs of different sexes and breeds leptospira interrogans serovar grippotyphosa infection in dogs efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis infection, tetracycline therapy, and challenge inoculation comparison of campylobacter carriage rates in diarrheic and healthy pet animals. zentralbl advances in dietary management of obesity in dogs and cats effect of level and source of dietary fiber on food intake in the dog effect of amount and type of dietary fiber on food intake in energy-restricted dogs external parasites: identification and control tumors of the endocrine glands thomson's special veterinary pathology infectious diseases of the dog and cat nutritional support for dogs and cats with hepatobiliary disease specific amplification of ehrlichia platys dna from blood specimens by two step pcr detection of humoral antibody to the transmissible venereal tumor of the dog dirofilaria immitis: heartworm products contract rat trachea in vitro dogs: laboratory animal management management of septicemia in rhesus monkeys with chronic indwelling catheters client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis dirofilariasis in dogs and cats use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma study of obesity in dogs visiting veterinary practices in the united kingdom miller's anatomy of the dog update on diagnosis of canine hypothyroidism helicobacter-associated gastric disease in ferrets, dogs, and cats the role of helicobacter species in newly recognized gastrointestinal tract disease of animals serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test hemorrhagic streptococcal pneumonia in newly procured research dogs control of ticks platelet aggregation studies in dogs with acute ehrlichia platys infection health benefits of animal research: the dog as a research subject soft tissue sarcomas and mast cell tumors textbook of veterinary internal medicine infectious diseases of the dog and cat canine lyme borreliosis mast cell tumor destruction by deionized water mast cell tumour destruction in dogs by hypotonic solution transmission of ehrlichia canis to dogs by ticks (rhipicephalus sanguineus) textbook of veterinary internal medicine diseases of the liver and their treatment cyclic thrombocytopenia induced by a rickettsia-like agent in dogs shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of veterinary internal medicine canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats thiacetarsamide and its adverse effects infectious diseases of the dog and cat pediatrics: puppies and kittens canine viral diseases textbook of veterinary internal medicine antibodies to ehrlichia canis, ehrlichia platys, and spotted fever group rickettsia in louisiana dogs mastocytoma and gastroduodenal ulceration complications in the use of indwelling vascular catheters in laboratory animals deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours helicobacter infection textbook of veterinary internal medicine textbook of veterinary internal medicine hygroma of the elbow in dogs thermal injuries dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? depression of endotheliumdependent relaxation by filarial parasite products three cases of canine leptospirosis in quebec cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs role of bordetella bronchiseptica in infectious tracheobronchitis in dogs kirk's current veterinary therapy 12: small animal practice the fire of life kirk's current veterinary therapy 13: small animal practice coinfection with multiple tick-borne pathogens in a walker hound kennel in north carolina tarsal joint contracture in dogs with golden retriever muscular dystrophy clinical and hematological findings in canine ehrlichiosis early spay-neuter in the dog and cat textbook of veterinary internal medicine chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs the brown dog tick rhipicephalus sanguineus and the dog as experimental hosts of ehrlicha canis the clinical chemistry of laboratory animals double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease thomson's special veterinary pathology effects of four preparations of .05% chlorhexidine diacetate on wound healing in dogs transmissible venereal tumors kirk's current veterinary therapy 11: small animal practice soft tissue sarcomas tumors of the mammary gland canine lymphoma and lymphoid leukemias kirk's current veterinary therapy 12: small animal practice effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein tick paralysis in north america and australia saunders manual of small animal practice thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think urea protects helicobacter (campylobacter) pylori from the bactericidal effect of acid characterization of a new isolate of ehrlichia platys using electron microscopy and polymerase chain reaction decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension vaccination against canine bordetellosis: protection from contact challenge dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs clinical trial of dvm derm caps in the treatment of allergic diseases in dogs: a nonblinded study diagnosis of neoplasia tumors of the mammary gland dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior survey of conjunctival flora in dogs with clinical signs of external eye disease hyperoxia exacerbates microvascular injury following acid aspiration nutrient requirements of dogs surgical closure of elbow hygroma in the dog tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases walker's mammals of the world tumors of the endocrine system beta hemolytic streptococcus isolated from the canine vagina comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part 2: clinical trial in 100 dogs clinical behavioral medicine for small animals hypothyroidism in dogs: 66 cases (1987-1992) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs joint diseases of dogs and cats target imbalance: disparity of borrelia burgdorferi genetic material in synovial fluid from lyme arthritis patients textbook of veterinary internal medicine tumors of the skin and soft tissue thomson's special veterinary pathology canine leptospirosis: a retrospective study of 17 cases dogs and cats as laboratory animals effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs epidemiology of thyroid diseases of dogs and cats factors influencing canine mammary cancer development and postsurgical survival muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology textbook of veterinary internal medicine canine infectious tracheobronchitis (kennel cough complex) saunders manual of small animal practice serum protein alterations in canine erhlichiosis bacterial factors and immune pathogenesis in helicobacter pylori evaluation of rhipicephalus sanguineus as a potential biologic vector of ehrlichia platys role of the eastern chipmunk (tamias striatus) in the epizootiology of lyme borreliosis in northwestern illinois evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs pathologic features of naturally occurring juvenile polyarteritis in beagle dogs textbook of veterinary internal medicine clinical manifestations, pathogenesis, and effect of antibiotic treatment on lyme borreliosis in dogs streptococcus zooepidemicus as the cause of septicemia in racing greyhounds trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs portosystemic shunts textbook of veterinary internal medicine lumbosacral stenosis in dogs experimental respiratory disease in dogs due to bordetella bronchiseptica dirofilaria immitis: depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro thyroiditis in a group of laboratory dogs: a study of 167 beagles of agriculture, animal and plant health inspection service thomson's special veterinary pathology a retrospective study of 27 cases of naturally occurring canine ehrlichiosis role of canine parainfluenza virus and bortedella bronchiseptica in kennel cough management of superficial skin wounds serum concentrations of thyroxine and 3,5,3'-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs ehrlichia platys in a michigan dog ehrlichial diseases of dogs canine ehrlichiosis. miss albert, r. e., benjamin, s. a., and shukla, r. (1994). life span and cancer mortality in the beagle dog and human. key: cord-022756-kdgo4rqb authors: nan title: hematopoietic tumors date: 2012-11-28 journal: withrow and macewen's small animal clinical oncology doi: 10.1016/b978-1-4377-2362-5.00032-3 sha: doc_id: 22756 cord_uid: kdgo4rqb nan the lymphomas (malignant lymphoma or lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. they usually arise in lymphoid tissues such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. although the annual incidence of lymphoma is difficult to predict in the absence of a national canine tumor registry, it is clear that it represents one of the most common neoplasms seen in the dog. the annual incidence has been estimated to range between 13 to 24 per 100,000 dogs at risk. [1] [2] [3] the annual incidence rates at specific ages are estimated to be 1.5 per 100,000 for dogs less than 1.0 year of age and 84 per 100,000 in the 10-to 11-year-old group. lymphoma comprises approximately 7% to 24% of all canine neoplasia and 83% of all canine hematopoietic malignancies. 4, 5 in a review of the veterinary medical data base program (vmdp) at purdue university from 1987 to 1997, the frequency of canine lymphoma patients presented to 20 veterinary institutions increased from 0.75% of total case load to 2.0%, and it appears the frequency is continuing to increase. a similar trend is present in physician-based oncology; non-hodgkin's lymphoma (nhl) represents 5% of all new cancer cases, the fifth leading cause of cancer death, and the second fastest growing cancer in terms of mortality in humans. 6 middle-aged to older (median age of 6 to 9 years) dogs are primarily affected. a decreased risk for lymphoma is reported for intact females. 7 breeds reported to have a higher incidence include boxers, bull mastiffs, basset hounds, st. bernards, scottish terriers, airedales, and bulldogs; breeds at lower risk include dachshunds and pomeranians. 8, 9 etiology the etiology of canine lymphoma is likely multifactorial and largely unknown; however, investigations are currently shedding significant light on the subject. recent advances in molecular cytogenetics (see chapter 1, section a), including gene microarray techniques, have been and are currently being applied to investigations of chromosomal aberrations in dogs with lymphoma. [10] [11] [12] [13] [14] [15] [16] the publication of the canine genome and the commercial availability of canine gene microarrays (genechip canine genome 2.0 array, affymetrix, inc.) have led to advances in our understanding of genetic events occurring in lymphoma. 17 breen's group has documented gain of dog chromosomes 13 and 31 and loss of chromosome 14 as the most common aberrations in a group of 25 cases analyzed. 11 chromosomal aberrations have also been associated with prognosis in dogs with lymphoma. a study of 61 dogs with lymphoma demonstrated a prognostic advantage in dogs with trisomy of chromosome 13 (25% of the dogs studied) as evidenced by increase in duration of first remission and overall survival time. 18 germline and somatic genetic mutations and altered oncogene/tumor suppressor gene expression, epigenetic changes (e.g., dna hypomethylation), signal transduction, and death-pathway alterations (e.g., bcl-2 family) are common in human lymphomas and have been reported in the dog as well (see chapter 1, section a, and chapter 14, section b). these include n-ras, p53, rb, and p16 cyclin-dependent kinase aberrations. [19] [20] [21] [22] [23] [24] additionally, differences in the prevalence of immunophenotypic subtypes of lymphoma among different breeds indicate heritable risks. 25 additionally, telomerase activity (see chapter 2) has been documented in canine lymphoma tissues. [26] [27] [28] the hypothesis that a retrovirus may be involved in the pathogenesis of canine lymphoma has not been confirmed. however, serologic detection of epstein-barr virus infection, linked to some forms of lymphoma in humans, has been documented in dogs with lymphoma and is currently being investigated. 29 in humans, a direct association between helicobacter sp. infections and development of gastric lymphoma has been made. 30 although this has not been definitively shown in dogs, there is evidence of helicobacter sp. infection in laboratory beagle dogs resulting in gastric lymphoid follicle formation that is considered a precursor of mucosa-associated lymphoid tissue (malt) lymphoma in humans. 31 in humans, evidence has accumulated implicating phenoxyacetic acid herbicides, in particular 2, 4-dichlorophenoxyacetic acid (2, 4-d) , in the development of nhl. a published hospital-based casecontrol study of dogs indicated that owners in households with dogs that develop malignant lymphoma applied 2, 4-d herbicides to their lawn and/or employed commercial lawn care companies to treat their yard more frequently than owners of dogs without lymphoma. 32 the risk of canine lymphoma was reported to rise twofold (odds ratio [or] = 1.3) with four or more yearly owner applications of 2, 4-d. the results of this study have come under criticism, and three additional follow-up investigations have not validated assertions of increased risk. [33] [34] [35] in another study, dogs exposed to lawn treatment within 7 days of application were greater than 50 times histopathologically, distinguishing between gi lymphoma and lpe can be difficult. some have suggested that lpe may be a prelymphomatous change in the gi tract. a syndrome of immunoproliferative intestinal disease characterized by lpe has been described in basenjis, which subsequently develop gi lymphoma. 49 in addition, plasma cell-rich areas with heterogeneous lymphomatous infiltration may resemble lesions of lpe. only a few reports specifically identify the immunophenotype of the lymphocyte subpopulations in alimentary lymphoma in dogs. historically, it was presumed that they most likely originate from b cells; however, recent evidence suggests that most gi lymphomas in dogs arise from t cells and often exhibit epitheliotropism. 48, 50 the boxer and shar-pei breeds may be overrepresented in cases of alimentary lymphoma. 50, 51 the mediastinal form of the disease occurs in approximately 5% of cases. 46 this form is characterized by enlargement of the cranial mediastinal lymph nodes, thymus, or both ( figure 32 -2). hypercalcemia is reported to occur in 10% to 40% of dogs with lymphoma and is most common with the mediastinal form. in a study of 37 dogs with lymphoma and hypercalcemia, 16 (43%) had mediastinal lymphoma. 52 the mediastinal form in dogs is most commonly associated with a t-cell phenotype. 53, 54 cutaneous lymphoma can be solitary or more generalized and usually is classified as epitheliotropic (mycosis fungoides) or nonepitheliotropic. 55 canine epitheliotropic cutaneous lymphoma originates from t-cells, [55] [56] [57] [58] [59] [60] similar to its development in humans. in dogs, these more commonly represent cd8 + cells, whereas in humans they are typically cd4 + cells. a rare form of cutaneous t-cell lymphoma, characterized by skin involvement with evidence of peripherally circulating large (15 to 20 µm in diameter) malignant t-cells with folded, grooved nuclei, has been described. in humans, this is referred to as sézary syndrome and has been reported in both dogs and cats. [61] [62] [63] nonepitheliotropic cutaneous lymphomas form single or multiple dermal or subcutaneous nodules or plaques; histologically, they spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis and subcutis. 55 more likely to have urine levels of 2, 4-d at 50 µg/l or higher. 36 the highest concentration was noted 2 days after application. in an environmental case-control study performed in europe, two variables, residency in industrial areas and use of chemicals (defined as paints or solvents) by owners, modestly increased the risk of developing lymphoma; however, no link was found with pesticide use. 37 a weak association between lymphoma in dogs and exposure to strong magnetic fields was observed in a preliminary epidemiologic study. 38 in this hospital-based case-control study, the risk of developing lymphoma categorized into high or very high exposure was increased (odds ratio = 1.8). more thorough studies are necessary to evaluate this association further. proximity to environmental waste was implicated in two european studies; however, it was felt to be a risk indicator rather than a risk factor and would require further case-control investigations. 39, 40 impaired immune function has also been implicated in dogs with lymphoma. immune system alterations in the dog such as immunemediated thrombocytopenia, independent of age and sex, have been associated with a higher risk of subsequently developing lymphoma when compared to the normal population. 41, 42 additional evidence comes from observations in human and feline transplantation patients. in a case-control study of cats undergoing renal transplant, 24% of cases developed cancer (36% of those were lymphoma) while on cyclosporine immunosuppressive therapy compared to 5.1% of control cats, none of which developed lymphoma (or, 6.1; p = 0.001). 43 a case of lymphoma developing in a dog following treatment with cyclosporine also exists. 44 one report suggests an association between the immunodysregulation observed in dogs with atopic dermatitis and the risk of developing epitheliotropic t-cell lymphoma; whether this is associated with the disease or the immunomodulatory treatments commonly applied is unknown. 45 classification of malignant lymphoma in dogs is based on anatomic location, histologic criteria, and immunophenotypic characteristics. the most common anatomic forms of lymphoma, in order of decreasing prevalence, are multicentric, gastrointestinal (gi), mediastinal, and cutaneous forms. 46 primary extranodal forms, which can occur in any location outside the lymphatic system, include the eyes, central nervous system (cns), bone marrow, bladder, heart, and nasal cavity. the pathologic characteristics of the various anatomic classifications will be discussed in this section and clinical characteristics will be described in subsequent sections. eighty-four percent of dogs with lymphoma develop the multicentric form, which is usually characterized by the presence of superficial lymphadenopathy (figure 32-1) . 46 the alimentary form of lymphoma is much less common, accounting for 5% to 7% of all canine lymphomas. this form is reported to be more common in male dogs than female dogs. 6 primary gi lymphoma in dogs may occur focally but more often affects multiple segments, with thickening of the wall, narrowing of the lumen, and frequently mucosal ulceration. 47, 48 histologically, there is infiltration of neoplastic lymphocytes throughout the mucosa and submucosa, with occasional transmural infiltration. liver and local lymph nodes are often secondarily involved. lymphocytic-plasmacytic enteritis (lpe) can be seen adjacent to or distant from the primary tumor. pathologically, some of these neoplasms may resemble plasma cell tumors, and aberrant production of immunoglobulins may occur. species. the national cancer institute (nci) working formulation 76 and the updated kiel system 77 have been adapted to canine tumors with some success. the world health organization (who) also publishes a histologic classification scheme, which uses the revised european american lymphoma (real) system as a basis for defining histologic categories of hematopoietic and lymphoid tumors in domestic animals. 78 this system incorporates anatomic, histologic, and immunophenotypic criteria (b-and t-cell immunophenotype), with the goal of enabling accurate and reproducible diagnosis of specific neoplastic disease entities. this theoretically should assist in better tailoring of treatment protocols, better correlation of prognosis, and better comparative capabilities. 79, 80 ; some of the less common categories in the who system were not represented and are not listed. the who system provides accurate and consistent reproducible diagnostic results similar to the system used in human pathology; accuracy among a group of pathologists examining 300 cases was at 83% agreement, and accuracy in evaluating the six most common diagnoses (80% of the cases) was 87%. 81 clinical studies are needed to correlate the various categories of disease with biologic behavior, response to treatment, and prognosis. preliminary results indicate dogs with indolent lymphoma (e.g., marginal zone lymphoma, follicular lymphoma, b-or t-cell small cell lymphoma, t-cell-rich b-cell lymphoma, and t zone lymphoma) maintain normal activity and appetite levels even during advanced stages of disease and experience long-term survival even with limited or no therapy. [81] [82] [83] [84] the working formulation (wf) was developed to allow investigators to "translate" among the numerous classification systems so that clinical trials could be compared in humans. most of the larger compilations agree that most canine lymphomas are intermediate or high grade; however, diffuse immunoblastic forms appear to predominate in the united states, whereas the follicular hepatosplenic lymphoma is a relatively uncommon, distinct presentation in the dog marked by a lack of significant peripheral lymphadenopathy in the face of hepatic, splenic, and bone marrow infiltration with malignant lymphocytes, usually of t-cell origin. 64, 65 biologically, this form of lymphoma is extremely aggressive and poorly responsive to therapy. in humans the tumor usually is composed of γδt-cells (i.e., t-cells that express the γδt-cell receptor), and this immunophenotype has been confirmed in at least one dog in the veterinary literature. 65 intravascular (angiotropic, angioendotheliomatosis) lymphoma is a distinct form of lymphoma defined as proliferations of neoplastic lymphocytes within the lumen and wall of blood vessels in the absence of a primary extravascular mass or leukemia. it has been reported several times in the veterinary literature, and in most cases it involves the cns and peripheral nervous system (pns), including the eye. [66] [67] [68] [69] [70] [71] the b-cell immunophenotype is most common in humans; however, in most reported cases in dogs, the origin is either t-cell or null cell (neither b-nor t-cell), although one case of a b-cell phenotype has been reported. pulmonary lymphomatoid granulomatosis (plg) is a rare pulmonary infiltrative and/or nodular disorder characterized by a heterogenous accumulation of lymphocytes (both b and t, although some evidence suggests primarily a t-cell origin), neutrophils, plasma cells, and macrophages, often arranged angiocentrically. [72] [73] [74] [75] whether this syndrome is a true lymphoma or a prelymphoma state is debatable. clinical signs are related to respiratory compromise, and various chemotherapeutic protocols have been used with reported results varying from rapid progression to long-term clinical remissions. lymphomas arise from clonal expansion of lymphoid cells with distinctive morphologic and immunophenotypic features. many histologic systems have been used to classify nhl in humans, and some of these have been applied to lymphoma in the dog and other percentage of canine lymphomas (5.3% to 29%) are considered low-grade tumors. high-grade lymphomas occur frequently if diffuse large-cell lymphomas, classified as intermediate grade in the wf, are considered high-grade, as in the updated kiel classification (in which they are labeled as diffuse centroblastic lymphomas). a documented difference exists in the prevalence of the various immunophenotypes based on breed. 25 for example, cocker spaniels and doberman pinschers are more likely to develop b-cell lymphoma, boxers are more likely to have t-cell lymphoma, and golden retrievers appear to have an equal likelihood of b-and t-cell tumors. to be clinically useful, these classification systems in the end must yield information about response to therapy, maintenance of remission, and survival. some studies suggest that the subtypes in the wf can be correlated with survival, and the kiel system may be useful for predicting relapse. 86, 87 in most studies, high-grade lymphomas achieve a complete response (cr) to chemotherapy significantly more often than low-grade tumors. however, dogs with low-grade tumors may live a long time without aggressive chemotherapy. 83, 84 dogs with t-cell lymphomas have shown a lower rate of cr to chemotherapy and shorter remission and survival times than dogs with b-cell tumors (with the exception of lowgrade t-cell subtypes). 53, 54, 86, 88 furthermore, t-cell lymphomas tend to be associated with hypercalcemia. 89, 90 large cell variations predominate in europe. a comparison of european and american classifications is warranted based on this discrepancy. the wf categorizes tumors according to pattern (diffuse or follicular) and cell type (e.g., small cleaved cell, large cell, immunoblastic), but it does not include information about the immunophenotype of the tumor. 76 the wf subtypes are related to the biology of the tumor and patient survival. the updated kiel classification includes the architectural pattern, morphology (centroblastic, centrocytic, or immunoblastic), and immunophenotype (b-cell or t-cell) of the tumor cells. 77 in both systems, the tumors can then be categorized as low-grade, intermediate grade, or highgrade malignancies. low-grade lymphomas composed of small cells with a low mitotic rate typically progress slowly and are associated with long survival times but are ultimately incurable. highgrade lymphomas with a high mitotic rate progress rapidly but are more likely to respond initially to chemotherapy and, in humans, are potentially curable. several features of canine lymphomas become apparent when these classification systems are applied. the most striking difference between canine and human lymphomas is the scarcity of follicular lymphomas in the dog. 79, 80 some diffuse lymphomas in the dog may initially be follicular, but these may progress to the more aggressive, diffuse form by the time of diagnostic biopsy. the most common form of canine lymphoma is diffuse large-cell lymphoma, a highgrade tumor most commonly of b-cell origin. 80, 81, 85 only a small particularly evident in dogs with hypercalcemia of malignancy. dogs may also be presented with clinical signs related to blood dyscrasias secondary to marked tumor infiltration of bone marrow (myelophthisis) or paraneoplastic anemia, thrombocytopenia, or neutropenia. these could include fever, sepsis, anemia, and hemorrhage. diffuse pulmonary infiltration is seen in 27% to 34% of dogs with the multicentric form, as detected by radiographic changes (figure 32-3) . 97 , 98 based on bronchoalveolar lavage, the actual incidence of lung involvement may be higher. 99, 100 in the veterinary literature, 60% to 80% of canine lymphomas are of b-cell origin; t-cell lymphomas account for 10% to 38%; mixed b-and t-cell lymphomas account for as many as 22%; and null cell tumors (i.e., neither b-cell nor t-cell immunoreactive) represent fewer than 5%. 53, 54, [91] [92] [93] the development of monoclonal antibodies to detect specific markers on canine lymphocytes has made immunophenotyping of tumors in dogs routinely available in many commercial laboratories. such techniques can be performed on paraffin-embedded samples, from tissue microarrays, on cytologic specimens obtained by fine-needle aspiration (fna) of lesions, or by flow cytometric analysis of cellular fluid samples (e.g., peripheral blood, effusions) and lesion aspirates. the rappaport classification system, proposed in 1956 for human nhl, describes the architectural pattern (follicular or diffuse) and the cytologic features (well differentiated, poorly differentiated, or histiocytic) of lymphoma. 94, 95 this system has not proved useful in providing prognostic information or in guiding therapy in dogs with lymphoma because of the low number of follicular lymphomas in dogs, the problematic "histiocytic" subgroup, and the failure to account for different morphologic and immunologic cell types. one criticism of the rappaport, kiel, and wf classification systems is that they fail to include extranodal lymphomas as a separate category. the who system does include anatomic location as a factor in determining certain categories. although differences between nodal and extranodal tumors in biologic behavior and prognosis are well recognized, comparative information about the histogenesis of these tumors is lacking. for example, in humans small-cell lymphomas arising from malt are composed of cells with a different immunophenotype than that of other small-cell lymphomas (i.e., malt lymphomas typically are negative for both cd5 and cd10). except for cutaneous lymphoid neoplasms, detailed characterization of extranodal lymphomas in dogs has not been done. although cutaneous lymphoma is a heterogeneous group of neoplasms that includes an epitheliotropic form resembling mycosis fungoides and a nonepitheliotropic form, most cutaneous lymphomas have a t-cell phenotype. 64, 96 to summarize, it is important to determine the histologic grade of canine lymphomas as low (small lymphocytic or centrocytic lymphomas) or intermediate to high (diffuse large cell, centroblastic, and immunoblastic lymphomas) and the architecture as diffuse or follicular. furthermore, determining the immunophenotype of the tumor provides useful information. response rates to chemotherapy are, in general, better in animals with b-cell tumors and intermediate-to high-grade lymphomas. dogs with low-grade lymphomas can have long survival times without aggressive therapy. the clinical signs associated with canine lymphoma are variable and depend on the extent and location of the tumor. multicentric lymphoma, the most common form, is usually distinguished by the presence of generalized painless lymphadenopathy (see figure 32 -1). enlarged lymph nodes are usually painless, rubbery, and discrete and may initially include the mandibular and prescapular nodes. in addition, hepatosplenomegaly and bone marrow involvement occur commonly. most dogs with multicentric lymphoma present without dramatic signs of systemic illness (who substage a) (box 32-1); however, a large array of nonspecific signs such as anorexia, weight loss, vomiting, diarrhea, emaciation, ascites, dyspnea, polydipsia, polyuria, and fever can occur (who substage b). dogs presented with t-cell lymphoma are more likely to have constitutional (i.e., substage b) signs. polydipsia and polyuria are i involvement limited to a single node or lymphoid tissue in a single organ. † ii involvement of many lymph nodes in a regional area (±tonsils). iii generalized lymph node involvement. iv liver and/or spleen involvement (±stage iii). syndrome, characterized by pitting edema of the head, neck, and forelimbs secondary to tumor compression or invasion of the cranial vena cava (figure 32-4) . signs in dogs with extranodal lymphoma depend on the specific organ involved. cutaneous lymphoma is usually generalized or multifocal. [55] [56] [57] tumors occur as nodules, plaques, ulcers, and erythemic or exfoliative dermatitis with focal hypopigmentation and alopecia. epitheliotropic t-cell lymphoma (e.g., mycosis fungoides) typically has a clinical course with three apparent clinical stages. initially, there will be scaling, alopecia, and pruritus ( figure 32 -5, a), which can mimic a variety of other skin conditions. as the disease progresses, the skin becomes more erythematous, dogs with gi or alimentary lymphoma are usually presented with nonspecific gi signs, such as vomiting, diarrhea, weight loss, and malabsorption. 47, 101, 102 mesenteric lymph nodes, spleen, and liver may be involved. the mediastinal form of lymphoma is characterized by enlargement of the cranial mediastinal structures and/or thymus (see figure 32 -2), and clinical signs are associated with the extent of disease with resulting respiratory compromise or polydipsia/ polyuria from hypercalcemia. commonly, dogs are presented with respiratory distress caused by a space-occupying mass and pleural effusion, exercise intolerance, and possibly regurgitation. additionally, dogs with mediastinal lymphoma may present with precaval a c b intravascular lymphoma usually present with signs relative to cns, pns, or ocular involvement. [66] [67] [68] [69] [70] [71] these include paraparesis, ataxia, hyperesthesia, seizures, blindness, lethargy, anorexia, weight loss, diarrhea, polyuria, polydipsia, and intermittent fever. finally, dogs with pure hepatosplenic lymphoma usually are presented with nonspecific signs of lethargy, inappetence, and weakness and often are icteric. 64, 65 the differential diagnosis of lymphadenopathy depends on the dog's travel history (i.e., relative to infectious disease) and the size, consistency, and location of affected lymph nodes. other causes of lymphadenopathy include infections caused by bacteria, viruses, parasites (toxoplasma sp., leishmania sp.), rickettsial organisms (salmon-poisoning, ehrlichia sp.), and fungal agents (blastomyces and histoplasma sp.). the potential for hypercalcemia to accompany systemic fungal diseases may further complicate differentiation from lymphoma. discrete, hard, asymmetric lymph nodes, particularly if they are fixed to underlying tissues, may indicate metastatic tumors such as mast cell tumor or carcinoma. immunemediated diseases (e.g., pemphigus, systemic lupus erythematosus) also may result in mild-to-moderately enlarged lymph nodes. the thickened, ulcerated, and exudative. the final stage is characterized by proliferative plaques and nodules with progressive ulceration (figure 32-5, b) . oral involvement may also occur and this can appear as multicentric erythematous plaque-like lesions or nodules associated with the gum and lips ( figure 32 -5, c). extracutaneous involvement can also occur, most often in the lymph nodes, spleen, liver, and bone marrow. nonepitheliotropic cutaneous lymphomas form single or multiple dermal or subcutaneous nodules or plaques; histologically, they spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis and subcutis. 55 dogs with primary cns lymphoma may be presented with either multifocal or solitary involvement. [103] [104] [105] seizures, paralysis, and paresis may be noted. ocular lymphoma is characterized by infiltration and thickening of the iris, uveitis, hypopyon, hyphema, posterior synechia, and glaucoma. 106, 107 in one study of 94 cases of canine multicentric lymphoma, 37% had ocular changes consistent with lymphoma, and in a series of 102 cases of uveitis in dogs, 17% were secondary to lymphoma. 107 anterior uveitis was most commonly seen in advanced stage of disease (stage v). dogs with • figure 32-5 a, early epitheliotropic cutaneous lymphoma in the scaly, plaque stage in a dog. b, advanced epitheliotropic cutaneous lymphoma in the nodular stage in a dog. c, oral mucosal epitheliotropic cutaneous lymphoma in a dog. c count (cbc), with a differential cell count, including a platelet count; a serum biochemical profile; and urinalysis. optimally, ionized calcium should be measured. ultimately, obtaining tissue or cytologic specimens for a definitive diagnosis is essential. a thorough physical examination should include palpation of all assessable lymph nodes, including a rectal examination; in the authors' experience, a significant proportion of dogs will have rectal polyps consisting of aggregates of neoplastic lymphocytes. inspection of mucous membranes for pallor, icterus, petechiae, and ulceration should be undertaken as these signs may indicate anemia or thrombocytopenia secondary to myelophthisis or immunemediated disease or may be evidence of major organ failure or uremia. abdominal palpation may reveal organomegaly, intestinal wall thickening, or mesenteric lymphadenopathy. the presence of a mediastinal mass and/or pleural effusion can be suspected following thoracic auscultation. an ocular examination, including funduscopic assessment, may reveal abnormalities (e.g., uveitis, retinal hemorrhage, ocular infiltration) in approximately one-third to onehalf of dogs with lymphoma. 107, 111 anemia, the most common lymphoma-related hematologic abnormality, is usually normochromic and normocytic (nonregenerative), consistent with anemia of chronic disease. 108 however, hemorrhagic and hemolytic anemias may also occur, and regenerative anemias may reflect concomitant blood loss or hemolysis. additionally, if significant myelophthisis is present, anemia may be accompanied by thrombocytopenia and leukopenia. 112, 113 in animals with anemia or evidence of bleeding, in addition to a platelet count, a reticulocyte count and coagulation testing may be indicated. thrombocytopenia may be seen in 30% to 50% of cases, but bleeding is seldom a clinical problem. neutrophilia can be seen in 25% to 40% of dogs and lymphocytosis occurs in approximately 20% of affected dogs. 108 circulating atypical lymphocytes may be indicative of bone marrow involvement and leukemia. it is important to differentiate multicentric lymphoma with bone marrow involvement (i.e., stage v disease) from primary lymphoblastic leukemia (see later), as the prognosis for each is entirely different. hypoproteinemia is observed more frequently in animals with alimentary lymphoma. in dogs with a high total protein or evidence of an increased globulin fraction on a chemistry profile, serum proteins may be evaluated by serum electrophoresis. monoclonal gammopathies have been reported to occur in approximately 6% of dogs with lymphoma. 114 serum biochemical abnormalities often reflect the anatomic site involved, as well as paraneoplastic syndromes such as hypercalcemia. in cases of hypercalcemia of unknown origin, lymphoma should always be considered high on the differential disease list, and diagnostic testing directed at this possibility should be undertaken (see chapter 5) . in addition, the presence of hypercalcemia can serve as a biomarker for response to therapy and early recurrence. increased urea nitrogen and creatinine concentrations can occur secondary to renal infiltration with tumor, hypercalcemic nephrosis, or prerenal azotemia from dehydration. increases in liver-specific enzyme activities or bilirubin concentrations may result from hepatic parenchymal infiltration. increased serum globulin concentrations, usually monoclonal, occur infrequently with b-cell lymphoma. urinalysis is part of the minimum database used to assess renal function and the urinary tract. for example, isosthenuria and various differential diseases or conditions that can resemble canine lymphoma are listed in table 32-2. canine lymphoma also may be associated with paraneoplastic syndromes (see chapter 5) . anemia is the most common lymphoma-related paraneoplastic syndrome. 108 paraneoplastic hypercalcemia is also common and is characterized clinically by anorexia, weight loss, muscle weakness, lethargy, polyuria, polydipsia, and rarely cns depression and coma. lymphoma-induced hypercalcemia in most cases results from parathyroid hormonerelated peptide (pthrp), elaborated by neoplastic cells; however, it can also be related to the production of several other humoral factors, including interleukin-1 (il-1), tumor necrosis factor-α (tnf-α), transforming growth factor-β (tgf-β), and vitamin d analogs (e.g., 1,25-dihydroxyvitamin d). 89, 109, 110 as previously discussed, hypercalcemia is most commonly associated with the t-cell immunophenotype. other paraneoplastic syndromes that may be encountered include monoclonal gammopathies, neuropathies, and cancer cachexia. for dogs suspected of having lymphoma, the diagnostic evaluation should include a thorough physical examination; complete blood • variable, depending on organ/system involved *the existence of this disease is controversial; in most cases, the disease has been reclassified as a lymphoid neoplasm. prescapular or popliteal lymph nodes are preferable if also involved. also, lymphoid cells are fragile, and in preparing smears of aspirated material only gentle pressure should be applied in spreading material on the slides. in most cases, a diagnosis of lymphoma can be made on evaluation of fine-needle aspirates of affected lymph nodes or other tissues. typically, most of the cells are large lymphoid cells (>2 times the diameter of a red blood cell [rbc] or larger than neutrophils), and they may have visible nucleoli and basophilic cytoplasm (figure 32-6 , a) or fine chromatin with indistinct nucleoli. because tissue architecture is not maintained in cytologic specimens, effacement of the node or capsular disruption cannot be detected. therefore marked reactive hyperplasia characterized by increased numbers of large lymphoid cells may be difficult to distinguish from lymphoma, and small cell lymphomas may have few cytologic clues that point to malignancy. also, classification of lymphoma, which has been attempted using cytologic appearance and immunophenotypic analysis, 123 into subcategories that make up the low-, intermediate-, and high-grade forms is performed most accurately on histologic sections (discussed previously). proteinuria in the absence of an active sediment may indicate renal disease, and hematuria may result from a hemostatic abnormality. it is important to remember that isosthenuria in azotemic dogs with hypercalcemia is not necessarily indicative of renal disease as the high calcium levels interfere with tubular concentration capabilities through disruption of antidiuretic hormone (adh) control. several abnormalities in serum have been explored as biomarkers of lymphoma in the dog. examples include alpha-fetoprotein, alpha-1 glycoprotein levels, zinc, chromium, iron, endostatin, vascular endothelial growth factor (vegf), lactate dehydrogenase, c-reactive protein haptoglobin, and antioxidants/oxidative stress markers. [115] [116] [117] [118] [119] [120] [121] [122] the clinical, biologic, and prognostic significance of these alterations is yet to be definitively characterized. morphologic examination of the tissue and cells that constitute the tumor is essential to the diagnosis of lymphoma. care should be taken to avoid lymph nodes from reactive areas (e.g., mandibular lymph nodes), unless those nodes are the only ones enlarged; the immunophenotyping is used to determine the type of cells that comprise the tumor, but this technique also can be helpful for making the initial diagnosis. 133-140,155 when a heterogenous population of lymphocytes is expected in a tissue, documentation of a homogeneous population of the same immunophenotype is supportive of a neoplastic process. the immunophenotype of a lymphocyte is identified by determining the expression of molecules specific for b-cells (e.g., cd79a, cd20) and t-cells (e.g., cd3). although tumor cells sometimes have morphologic characteristics that typify a particular immunophenotype, exceptions occur, and morphologic appearance cannot be used as the sole determinant of immunophenotype. for example, in a series of nine high-grade t-cell lymphomas and leukemias in dogs, the cells had a plasmacytoid appearance, typically associated with b-cell lymphoma. 156 similarly, anatomic location does not always predict the immunophenotype. for accurate determination of immunophenotype, antibodies against lymphocyte markers are applied to tissue sections (immunohistochemistry), cytologic specimens (immunocytochemistry), or individual cells in a fluid medium (flow cytometry). flow cytometric evaluation of cells obtained by needle aspiration is also feasible. for t-cells, markers include cd3 (pan t), cd4 (helper t), and cd8 (cytotoxic t); for b-cells, the markers are cd79a ( figure 32-6, b) , cd20, and cd21. increasingly, aberrant expression of cd molecules has been reported in canine lymphoma. in a study of 59 dogs with lymphoma, tumor cells from six dogs were positive for both t-and b-cell markers; however, a clonality assay (see later) revealed clonality either of the t-cell or the immunoglobulin receptor but not both. this indicates that in some cases, the malignant cells may co-express b-and t-cell markers. 93 antibodies against these molecules are used to determine the immunophenotype; however, they also have potential utility as a therapeutic modality if tumor cells could be targeted using these antibodies. assessments of markers of multidrug resistance and apoptotic pathways (e.g., p-glycoprotein, p53, bcl-2 proteins) have been evaluated in dogs with lymphoma. 19,79,142,156a,156b however, their clinical significance and utility await further evaluation. occasionally, diagnosis of lymphoma and differentiation of malignant versus benign proliferation of lymphocytes are not possible based on standard histologic and cytologic criteria. in these cases, advanced molecular analyses may be helpful to confirm a diagnosis. clonality is the hallmark of malignancy; that is, the malignant cell population theoretically should be derived from expansion of a single malignant clone characterized by a particular dna region unique to that tumor. for example, in a dog with t-cell lymphoma, all the malignant cells theoretically should have the same dna sequence for the variable region of the t-cell receptor gene. for accurate histopathologic evaluation, an entire lymph node, including the capsule, should be removed, placed in buffered formalin, and submitted to a pathologist. although needle core biopsies may be satisfactory, it is important to avoid crush artifact or inadequate sample size. most pathologists prefer whole node biopsies because they provide the maximal amount of information. effacement of normal nodal architecture by neoplastic lymphocytes and capsular disruption are characteristic findings (figure 32 -6, c and d). diagnostic ultrasonography and ultrasound-guided fna or needle biopsy have been useful for evaluation of involvement of the liver, spleen, or abdominal lymph nodes. [124] [125] [126] aspiration of ultrasonographically normal splenic tissue is rarely contributory to a diagnosis. 124 if possible, the diagnosis should be made by sampling peripheral nodes, avoiding percutaneous biopsies of the liver and spleen. however, if there is no peripheral node involvement, it is appropriate to biopsy affected tissues in the abdominal cavity. when gi lymphoma is suspected, an open surgical wedge biopsy of the intestine is preferred in most cases to differentiate lymphoma from lymphocytic enteritis. if associated abdominal lymph nodes also appear involved, image-guided biopsies may be associated with less morbidity than intestinal biopsies. multiple samples may be necessary to accurately diagnose segmental disease. endoscopic biopsies may be inadequate as only a superficial specimen is obtained; however, more aggressive endoscopic biopsy techniques combined with more accurate histopathologic, immunophenotypic, and molecular assessments are improving the diagnostic yield of these less invasive techniques. [127] [128] [129] [130] in many dogs with primary gi lymphoma, an inflammatory nonneoplastic infiltrate (i.e., lpe) may be misdiagnosed on biopsy specimens that are too superficial. the application of assays for clonal expansion (e.g., parr-see next section on molecular diagnostic techniques) does not appear as yet to be as accurate for endoscopically derived intestinal biopsies as with other solid lymphoid tumors in dogs. cytologic examination of cerebrospinal fluid (csf), thoracic fluid, or mass aspirates is indicated in animals with cns disease, pleural effusion, or an intrathoracic mass, respectively. in one study of dogs with cns involvement, csf analysis was diagnostic in seven of eight dogs. 103 characteristics of the csf included an increased nucleated cell count in the seven dogs, and 95% to 100% of the cells were atypical lymphocytes. the csf protein concentration was increased in five of the dogs, ranging from 34 to 310 mg/ dl (reference interval: <25 mg/dl). for cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative, but not secondarily infected, skin lesions. application of immunophenotypic and clonality assessments of cutaneous biopsies can aid in differentiating lymphoma from benign lymphocytic lesions. 58, 131, 132 molecular techniques can be used to establish a diagnosis of lymphoma or to further characterize the tumor after the initial diagnosis is made. tissues and cells from peripheral blood, lymph nodes, nonlymphoid sites, and effusions can be analyzed by various molecular means to aid in cases that represent a more difficult diagnostic challenge, particularly in cases where reactive lymphocytosis and lymphoma are both possible based on standard histologic or cytologic assessment. these include histochemical and cytochemical, immunohistochemical and immunocytochemical, flow cytometric, and polymerase chain reaction (pcr) techniques. for example, a bone marrow aspirate or biopsy (from proximal humerus or iliac crest) is recommended for complete staging and prognostication and is indicated in dogs with anemia, lymphocytosis, peripheral lymphocyte atypia, or other peripheral cytopenias. in one study of 53 dogs with lymphoma, 28% had circulating malignant cells and were considered leukemic, whereas bone marrow examination indicated involvement in 57% of the dogs. 162 the presence of a few prolymphocytes and large lymphocytes with nucleoli in the circulation of dogs with lymphoma may indicate bone marrow involvement. it is important to remember these cells also can be seen with gi parasitism, immune-mediated hemolytic anemia, and other immune-mediated and infectious diseases. as discussed previously, tumor cells within the peripheral and bone marrow compartments can also be identified using clonality assays (parr) that are more sensitive than routine microscopic examination in detecting malignant cells; however, the prognostic significance of the knowledge gained with more sensitive staging methodologies is yet to be determined. although bone marrow evaluation may offer prognostically valuable information, it is not necessary to perform the procedure if the client is committed to treat regardless of stage. evaluation of thoracic and abdominal radiographs may be important in determining the extent of internal involvement ( figure 32-7) . approximately 60% to 75% of dogs with multicentric lymphoma have abnormalities on thoracic radiographs, with one-third having evidence of pulmonary infiltrates (see figure 32 -3) and twothirds having thoracic lymphadenopathy (sternal and tracheobronchial lymph nodes [see ) and widening of the cranial mediastinum (see figure 32 -2). 97, 98 pulmonary infiltrates usually are represented by an interstitial and/or alveolar pattern; however, nodules (rarely) and bronchial infiltrates can also occur. 163 pleural effusion may also be present. cranial mediastinal lymphadenopathy is detected in 20% of dogs with lymphoma. 97, 163 abdominal radiographs reveal evidence of involvement of medial iliac (sublumbar) and/or mesenteric lymph node, spleen, or liver in approximately 50% of cases. in the authors' practice, for the typical cases of canine multicentric lymphoma, imaging is limited to thoracic likewise, in a dog with b-cell lymphoma, the tumor cells should have identical dna sequences in the variable region of the immunoglobulin (ig) receptor gene. conversely, in reactive lymphocytosis, the cells are polyclonal for their antigen receptors. using this knowledge, investigators have used pcr technology to amplify the variable regions of the t-cell and immunoglobulin receptor genes to detect the presence of clonal lymphocyte populations in dogs (see figure 8 -4 of chapter 8). these techniques are reviewed in chapter 8 and elsewhere. 150 in physician-based medicine, such assays of clonality are approximately 70% to 90% sensitive and have a false-positive rate of approximately 5%, and recent studies report similar rates in dogs. false-negative and false-positive results can occur with clonality assays. for example, cells from a dog with lymphoma may be negative for clonality if the clonal segment of dna is not detected with the primers used, if the malignant cells are natural killer (nk) cells (rare), or if the malignant cells are present in too low a frequency to be detected. false positives occur rarely in some infectious diseases (e.g., ehrlichiosis and lyme disease). in these cases, a diagnosis should be made only after considering the results of all the diagnostic tests, including histologic/cytologic evaluation, immunophenotyping, and clonality studies in conjunction with signalment and physical examination findings. these molecular techniques, although helpful for diagnosis, could also have utility in detecting early recurrence and in determining more accurate clinical stage and so-called "molecular remission rates" because they are more sensitive than standard cytologic assessment of peripheral blood, bone marrow, or lymph nodes (covered subsequently in section on treatment response). proteomics comprises, simplistically, methodologies that analyze the entire protein component or protein signature of cells (the proteome). protein components of a cell (normal or malignant) change over time with upregulation and downregulation of gene expression in response to varied stimuli (e.g., growth factors, environmental cues). it may therefore be possible to use the field of proteomics to identify serum biomarkers of malignancy (i.e., cancer-specific protein markers) and to further analyze response to therapy or even to predict which therapies are appropriate for an individual patient's tumor. although in its infancy in veterinary oncology, preliminary investigations of the proteome of dogs with lymphoma have been reported 157-160 ; however, they have yet to reach the level of sophistication in which useful output would have an impact on clinical decision making. after a diagnosis has been established, the extent of disease should be determined and categorized by the clinical stage of disease. the who staging system routinely used to stage dogs with lymphoma is presented in box 32-1. most dogs (>80%) are presented in advanced stages (iii to iv). diagnostic imaging and assessment of bone marrow involvement may be indicated for staging. the degree to which thorough staging is implemented depends on whether the result will alter the treatment plan, whether relevant prognostic information is gleamed, and whether the clients need to know the stage prior to initiating (or declining) a treatment plan. additionally, when comparing different treatment protocols with respect to efficacy, consistent and similar staging diagnostics should be used to avoid so-called "stage migration, " which results when one staging methodology is more accurate than another. 161 the impact of stage migration on prognosis should be considered when comparing different published outcomes. radiographs as there is no prognostic difference between dogs with stage iii and iv disease (i.e., liver/spleen involvement), whereas the presence of cranial mediastinal lymphadenopathy is of prognostic significance (see prognosis section). however, if there are clinical signs attributable to abdominal disease or if complete staging is necessary (e.g., for clinical trial inclusion), further imaging of the abdomen is warranted. abdominal ultrasonography can be important for obtaining ultrasound-guided intraabdominal samples for diagnosis. it may also be useful for the diagnosis of gi, abdominal nodal, and hepatosplenic lymphoma. 125 ultrasonographic (including doppler ultrasound) assessment of peripheral lymph nodes has also been explored 126 ; however, its clinical applicability is questionable because cytologic assessment of peripheral nodes is easy, inexpensive, and of higher diagnostic utility. advanced imaging modalities, including computed tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), or pet/ct imaging, are becoming more commonplace in veterinary practice and their utility is only now being determined. 164-169 pet/ct imaging is the current standard of care for following and indeed predicting durability of treatment response in human patients with lymphoma, and both [18f]fluorothymidine (18flt) pet/ct and [18f]fluoro-d-glucose (18fdg) pet imaging have been reported in dogs with lymphoma. 164-166 18flt-pet/ct functional and anatomic imaging shows promise for the evaluation of response to cytotoxic chemotherapy in dogs with lymphoma and for predicting relapse before standard clinical and clinicopathologic confirmation (figure 32-8) . the therapeutic approach to a particular patient with lymphoma is determined by the stage and substage of disease, the presence or absence of paraneoplastic disease, the overall physiologic status of the patient, financial and time commitment of the clients, and their level of comfort with respect to likelihood of treatment-related success and/or side effects. without treatment, most dogs with lymphoma will die of their disease in 4 to 6 weeks after diagnosis, although significant variability exists. 114 with few exceptions, canine lymphoma is considered a systemic disease and therefore requires systemic therapy in order to achieve remission and prolong survival. the majority of canine multicentric lymphomas are intermediate to high grade, and, currently, histopathologic and immunophenotypic characterization has not played a significant role in determining the initial treatment protocol. it is hoped that in the near future, sufficient data will emerge to better tailor treatment protocols chosen for dogs with lymphoma based on these and other yet to be characterized parameters. that being said, systemic multiagent chemotherapy continues to be the therapy of choice for canine lymphoma. in general, combination chemotherapy protocols are superior in efficacy to single-agent protocols. single-agent protocols result in lower response rates that are not as durable as combination chemotherapy, which is summarized in table 11 -2 in chapter 11. in rare cases in which lymphoma is limited to one site (especially an extranodal site), the animal can be treated with a local modality such as surgery or radiation therapy (rt) as long as the client and clinician are committed to diligent reevaluation to document subsequent progression to systemic involvement, should it occur. many chemotherapeutic protocols for dogs with lymphoma have been developed over the past 15 to 20 years (table 32-3) . 91, significant limitations arise when comparing efficacy studies in the that is, clinical trials are inherently costly, and because most of the known effective drugs are unregistered off-label human generic (i.e., off patent) drugs, the incentive for pharmaceutical-funded, sufficiently powered, randomized field trials is low, resulting in a general lack of comparative data. despite the plethora of available combination protocols, most are modifications of chop protocols initially designed for human oncologic use, and currently randomized prospective evidence does not exist to clearly recommend one over the other as long as the basic "chop" components are present. chop represents combinations of cyclophosphamide (c), doxorubicin (h, hydroxydaunorubicin), vincristine (o, oncovin), and prednisone (p). conventional chop-based chemotherapy induces remission in approximately 80% to 95% of dogs, with overall median survival times (msts) of 10 to 12 months. approximately 20% to 25% of treated dogs will be alive 2 years after initiation of these protocols (figure 32-9 ). veterinary literature for the various published protocols. few of these studies include sufficient numbers for adequate statistical power and even fewer compare treatment protocols in a randomized prospective fashion. in addition, staging, inclusion, and response criteria vary considerably between reports. therefore evaluations of efficacy among various protocols are subject to substantial bias, making direct comparisons difficult and indeed precarious. a recurring theme in the concluding statement in most of these published protocols is some variation of "prospective randomized trials will be required to confirm these suggestive findings. " in an attempt to better standardize response criteria and outcome reporting of future trials, the veterinary cooperative oncology group (vcog) has recently published response evaluation criteria (v1.0) 169 (see subsequent response evaluation section). the greatest obstacle to the performance of prospective randomized comparative lymphoma trials in veterinary oncology is financial; • ("treatment holidays") are not uncommonly required in individual cases, only a minority of dogs develop significant adverse events requiring hospitalization. 191, 192 studies assessing client perceptions of medical treatment for cancer in general and lymphoma in particular report a positive experience; most owners feel treatment was worthwhile, that it resulted in improvement in the well-being of their pet, and that quality of life during treatment was good. 193, 194 very few clients express regret about treating lymphoma using a multidrug protocol. with lymphoma, the fundamental goals of chemotherapy are to induce a complete durable (>6 months) first remission (termed induction), to reinduce a remission when the tumor recrudesces (or the patient relapses) following achievement of a remission (termed reinduction), and, finally, to induce remissions when the cancer fails to respond to induction or reinduction using drugs not present in the initial protocols (termed rescue). an unanswered question in the treatment of lymphoma has been whether long-term maintenance chemotherapy is useful following an initial course of aggressive induction chemotherapy lasting 6 months or less. long-term maintenance chemotherapy has not been shown to be of significant value in humans with most forms of nhl; however, in humans, the initial induction course of chemotherapy is much more aggressive than that used in veterinary patients. although no randomized prospective studies have been performed to address the therapeutic benefit of long-term maintenance chemotherapy in dogs, most comparisons of dogs treated with chop-based protocols do not show any clear advantage for a maintenance or consolidation phase after induction therapy.* indeed, in most reports, dogs receiving shorter, less costly protocols that do not include a prolonged maintenance phase have comparable remission and progression-free survival (pfs) durations and appear to more readily achieve second remissions when they relapse following completion of chemotherapy than their counterparts receiving long-term maintenance. these data, taken together, suggest that maintenance therapy is not beneficial for most dogs with lymphoma. until well-designed randomized prospective trials indicate otherwise, the author (dmv) prefers protocols that utilize an aggressive induction without maintenance. the most effective, currently available chemotherapeutic agents for canine lymphoma include doxorubicin, l-asparaginase, vincristine, cyclophosphamide, and prednisone-most of which are represented to one degree or another in most first-line multiagent chemotherapy protocols. other drugs that have documented activity are often considered second-line agents and include lomustine, vinblastine, actinomycin-d, mitoxantrone, mustargen, chlorambucil, methotrexate, dacarbazine (dtic), 9-aminocamptothecin, ifosfamide, cytosine arabinoside, and gemcitabine. of these, cytosine arabinoside, 199 ifosfamide, 200 and gemcitabine 201 appear to have only minimal activity. with the exception of doxorubicin, induction therapy with single-agent chemotherapy does not typically result in durable remission durations when compared with standard combination protocols (see table 11 -2, chapter 11). incorporation of other standard cytotoxic drugs with single-agent activity into standard chop-based protocols has not resulted in significant gains, and most are reserved for subsequent rescue settings. response rates and duration of response vary according to the presence or absence of prognostic factors discussed subsequently in the section on prognosis in this chapter. the relative cost of the various protocols to the client depends on the drug(s) selected, the size of the animal, the frequency of administration, and the laboratory tests required to monitor adverse events and response. dogs responding to chemotherapy and undergoing complete remission are usually free of clinical signs associated with lymphoma and subsequently return to a very good quality of life. treating dogs with lymphoma is initially gratifying because a high percentage enjoy a complete response. most dogs tolerate chemotherapy well, and although dose reductions and treatment breaks initiation or at varying times throughout the protocol, several studies suggest this does not result in clinically relevant increases in remission rate, speed of attaining remission, or first-remission duration, and therefore the author reserves its use for rescue situations. 172, 195, 202, 203 if client or other considerations preclude a chop-based protocol, single-agent doxorubicin (30 mg/m 2 , intravenous [iv], every 3 weeks for 5 total treatments) is offered along with a 4-week tapering oral prednisone regimen (same prednisone regimen in box 32-2) as a less aggressive and less costly approach. the expected cr rate will range from 50% to 75%, with an anticipated median survival of 6 to 8 months. 171, 172, 204, 205 the addition of oral cyclophosphamide (50 mg/m 2 daily for 3 days starting on the same day as doxorubicin) to single-agent doxorubicin resulted in a numerically but not statistically superior outcome in a recent randomized trial 205 comparing doxorubicin/prednisone with doxorubicin/cyclophosphamide/ prednisone (pfs of 5.6 months versus 8.2 months, respectively). this trial was only powered to detect a threefold difference in pfs; therefore larger trials should be undertaken to confirm any benefit. if clients are reticent to include iv medications, the author often recommends a protocol of oral lomustine (ccnu; 70 mg/m 2 by mouth [po] every 3 weeks for 5 treatments) and prednisone. this protocol has been associated with short median remissions (40 days) in only one small case series 206 ; however, in the author's experience, a subset of dogs have remained in remission for several months on this protocol when clients decline iv medication. if financial or other client concerns preclude the use of systemic chemotherapy, prednisone alone (2 mg/kg po, daily) will often result in short-lived remissions of approximately 1 to 2 months. in these cases, it is important to educate clients that, should they decide to pursue more aggressive therapy at a later date, dogs receiving single-agent prednisone therapy are more likely to develop multidrug resistance (mdr) and experience shorter remission and survival durations with subsequent combination protocols. [207] [208] [209] this is especially true following long-term prednisone use or in dogs that have experienced a recurrence while receiving prednisone. therefore the earlier that clients opt for more aggressive therapy, the more likely a durable response will result. a cbc should be performed prior to each chemotherapy treatment. a minimum of 1500 neutrophils/µl (some oncologists use a cut-off of 2000 neutrophils/µl) and 50,000 platelets/µl should be present prior to the administration of myelosuppressive chemotherapy. if the neutrophil count is lower than 1500/µl, it is best to wait 5 to 7 days and repeat the cbc; if the neutrophil count has increased to more than 1500 cells/µl, the drug can be safely administered. a caveat to these restrictions is that for dogs presented prior to initiation of chemotherapy with low neutrophil and platelet counts due to bone marrow effacement, myelosuppressive chemotherapy is instituted in the face of cytopenias in order to clear the bone marrow of neoplastic cells and allow hematopoiesis to normalize. in those breeds likely to have mdr1 gene mutations (e.g., collies; see chapter 11) and therefore to be at risk for serious chemotherapeutic toxicity, 210 the author will initiate a chop protocol out of sequence, beginning with non-mdr1-associated drugs, such as cyclophosphamide. this ensures treatment of the lymphoma while allowing sufficient time for analysis of mdr1 gene mutations prior to initiating mdr1 substrate drugs. no specific protocols have been scrutinized for treating dogs that are doublemutant for mdr1; however, if using mdr1 substrate drugs, the author initiates at a 40% dose reduction. subsequent dose modifications (increased or decreased dosage) can be implemented, several factors should be considered and discussed with caregivers on a case-by-case basis when choosing the protocol to be used. these factors include cost, time commitment involved, efficacy, adverse event profiles, and experience of the clinician with the protocols under consideration. it is now clearly established that "standard of care" combination protocols used in dogs with lymphoma are essentially variations of "chop" protocols (see table 32 -3). specific details regarding dose and timing of the chop protocol currently preferred by the author (dmv) are outlined in box 32-2. this protocol does not have a maintenance therapy arm, and all treatments cease at 19 weeks, provided the animal is in complete remission. although several other chop-based protocols include l-asparaginase either at † in dogs <15 kg in body weight, a doxorubicin dose of 1 mg/kg is substituted for 30 mg/m 2 . molecular and biologic markers of minimal disease. advanced functional and anatomic imaging (i.e., pet/ct) are the current standard for assessing treatment response and early relapse of lymphoma in humans and have also been investigated in dogs (see . [164] [165] [166] 168 as this technology becomes available to a broader veterinary population, its clinical application will surely increase. molecular detection of mrd applies clonality and pcr techniques previously discussed in this chapter. beyond diagnostic applications, these techniques have been applied to determine cytoreductive efficacy of various chemotherapeutic drugs and to document and predict early relapse in patients prior to more conventional methods. 215-219 regarding biomarkers of mrd, preliminary investigations have suggested serum lactate dehydrogenase activity, 120 thymidine kinase 1 activity, 220 and serum c-reactive protein 221 may be candidates in the dog. as we become more proficient at defining mrd, the pressing clinical question becomes how we use this information. theoretically, such information could suggest when more aggressive therapy or alternative therapy should be instituted in patients who have not achieved a "molecular remission" or who are undergoing early relapse; however, until we determine what these interventions should be, their clinical utility remains theoretical. eventually, the majority of dogs that achieve a remission will relapse or experience recrudescence of lymphoma. this usually represents the emergence of tumor clones or tumor stem cells 222 (see chapter 2) that are inherently more resistant to chemotherapy than the original tumor, the so-called mdr clones that either were initially drug resistant or became so following exposure to selected chemotherapy agents. evidence suggests that in dogs with recurrent lymphoma, tumor cells are more likely to express the mdr1 gene that encodes the protein transmembrane drug pump often associated with mdr. 156a,156b,225 mdr1 represents only one of the plethora of mechanisms that lead to drug-resistant disease (see chapter 11) . other causes for relapse following chemotherapy include inadequate dosing and frequency of administration of chemotherapy, failure to achieve high concentrations of chemotherapeutic drugs in certain sites such as the cns, and initial treatment with prednisone alone. at the first recurrence of lymphoma, it is recommended that reinduction be attempted first by reintroducing the induction protocol that was initially successful, provided the recurrence occurred temporally far enough from the conclusion of the initial protocol (e.g., ≥2 months) to make reinduction likely. attention must be given to the cumulative dose of doxorubicin that will result from reinduction, and baseline cardiac assessment, the use of cardioprotectants, alternative drug choices, and client education should all be considered. in general, the length of the reinduction will be half that encountered in the initial therapy; however, a subset of animals will enjoy long-term reinductions, especially if the dog completed the initial induction treatment protocol and was currently not receiving chemotherapy for several months when relapse occurred. nearly 80% to 90% reinduction rates can be expected in dogs that have completed chop-based protocols and then relapse while not receiving therapy. 185, 226 the duration of a second chop-based remission in one report was predicted by the duration of the interval between protocols and the duration of the first remission. 226 if reinduction fails or the dog does not respond to the initial induction, the use of so-called "rescue" agents or "rescue" protocols may be attempted. these are single drugs or drug combinations that are typically not found in standard chop protocols and are depending on adverse event levels observed, particularly neutrophil counts at nadir. with some exceptions, multicentric t-cell lymphoma, when compared with multicentric b-cell lymphoma, is associated with similar initial response rates, but significantly lower response durability (e.g., pfs) following chemotherapy (including chop-based protocols).* additionally, the effectiveness of a single treatment of doxorubicin in the treatment of naïve dogs with lymphoma in one retrospective case series suggested a lower initial response rate for t-cell, compared with b-cell, immunophenotypes. 213 this has led many to question whether dogs diagnosed with t-cell lymphoma should be treated with standard chop-based protocols or with alternative protocols. this is a valid question; however, the answer remains elusive because adequately powered randomized controlled trials do not currently exist in the literature to show superiority for an alternate protocol in this scenario. a retrospective study of an l-asparaginase and mopp (m, mechlorethamine; o, oncovin; p, procarbazine; p, prednisone) protocol suggested improvement in pfs in dogs with either confirmed t-cell lymphoma or lymphoma with hypercalcemia and no immunophenotypic classification. 214 however, differences in determining pfs, response evaluation, and study population in this retrospective study did not definitively confirm superiority. 211 further, some have advocated early inclusion of lomustine (ccnu) into protocols for treating multicentric t-cell lymphoma based on moderate success of lomustine-based rescue protocols in dogs failing chop. as yet, no randomized trials have documented superiority with this approach. ultimately, superior protocol development for t-cell lymphoma awaits careful, randomized, prospective trial assessment. until such time, the author prefers to initiate chop-based induction and switch to lomustinebased rescue at the first sign of progression. vcog has recently published response evaluation criteria (v1.0) 169 to standardize reporting of outcome results and comparisons among protocols for peripheral nodal disease. the most important of these outcome measures and the preferred temporal outcome criterion for assessing protocol activity is now considered to be pfs, which is defined as being from the time of treatment initiation until tumor progression or death from any cause. this brings veterinary outcome reporting more in line with human standards. because the majority of dogs with lymphoma eventually experience recurrence following chemotherapy-induced remissions and because methodology for differentiating complete and partial responses is analysis dependent, pfs removes many sources of bias. further, overall survival in published reports invariably includes patients who go on to receive varied rescue protocols that bias the overall result, making it a less comparable outcome. widespread application of these standardized criteria should allow more suitable comparisons in the future. superior methods of detection of minimal residual disease (mrd) or early recurrence have been investigated in dogs with lymphoma and include advanced imaging and detection of despite the plethora of published chemotherapeutic protocols for dogs with lymphoma, it appears we have achieved as much as we can from currently available chemotherapeutics in standard settings. the 12-month median survival "wall" and the 20% to 25% 2-year survival rates have not improved dramatically. further advances in remission and survival durations await the development of new methods of delivering or targeting traditional chemotherapeutic drugs, new generations of chemotherapeutic drugs, or novel nonchemotherapeutic treatment modalities. mechanisms of avoiding or abrogating mdr, enhancing tumor apoptosis (programmed cell death), tumor ablation, and immune-system reconstitution, as well as novel immunomodulatory therapies for lymphoma, are all active areas of investigation in both human and veterinary medicine. drug resistance can be inherent in cancer cells or develop following exposure to selected chemotherapeutic agents and often is associated with increased expression of members of the adenosine triphosphate (atp)-binding cassette (abc) transporter superfamily (e.g., p-glycoprotein pump), many of which efflux various withheld for use in the drug-resistant setting. the most common rescue protocols used in dogs include single-agent use or a combination of actinomycin d, mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), dacarbazine (dtic), temozolomide, lomustine (ccnu), l-asparaginase, mechlorethamine, vincristine, vinblastine, procarbazine, prednisone, and etoposide. some rescue protocols are easy and convenient single-agent treatments, whereas others are more complicated (and expensive) multiagent protocols, such as mopp. overall rescue response rates of 40% to 90% are reported; however, responses are usually not durable, with median responses of 1.5 to 2.5 months being typical, regardless of the complexity of the protocol. a small (<20%) subset of animals will enjoy longer rescue durations. table 32 -4 provides a summary of canine rescue protocols and published results. 227-238 current published data from rescue protocols do not include sufficient numbers for adequate statistical power nor do they compare protocols in a randomized prospective fashion. therefore evaluations of efficacy among various protocols are subject to substantial bias, making direct comparisons difficult and indeed precarious. choice of a particular rescue protocol should depend on several factors, including cost, time commitment required, efficacy, toxicity, and experience of the clinician with the protocols in question. as the complexity of rescue protocols does not yet appear to be associated with significant gains in rescue durability, the author tends to choose simpler and less costly protocols (e.g., ccnu/l-asparaginase/prednisone) ( table 32-5) . however, the use of multiple varied rescue protocols, switching as needed based on response, continues as long as clients are • nr, not reported. *few of these protocols include sufficient numbers for adequate statistical power and fewer compare treatment protocols in a randomized prospective fashion. in addition, staging, inclusion, and response criteria vary considerably between protocols presented. therefore evaluations of efficacy between the various protocols are subject to bias, making direct comparisons difficult and indeed precarious. † various temporal response endpoints were used, including disease-free interval, time to progression, and progression-free survival. ‡ prednisone often used concurrently. performed. 243,244 although efficacy was established, enhancement of remission or survival durations over equivalent doses of native doxorubicin was not observed. in the past decade, enhanced durability of first remissions in humans with non-hodgkin's b-cell lymphoma has been achieved primarily through the institution of monoclonal antibody (mab)based therapies (so-called r-chop protocols); the "r" refers to rituximab, a recombinant chimeric murine/human antibody directed against the cd20 antigen, a hydrophobic transmembrane protein located on normal pre-b and mature b lymphocytes. following binding, rituximab triggers a host cytotoxic immune response against cd20-positive cells. unfortunately, rituximab does not have therapeutic activity in dogs due to a lack of external recognition of a similar antigen on canine lymphoma cells and the inherent antigenicity of human-derived antibodies in dogs. 245, 246 another immunotherapy approach involved mab-231, a murinederived anticanine mab (igg2a). it mediates antibody-dependent cellular cytotoxicity (addc) and complement-mediated cellular cytotoxicity (cmcc). 247-249 it also prevented outgrowth of canine lymphoma xenografts in nude mice. in a noncontrolled clinical study of 215 dogs treated with chop-based chemotherapy and mab-231, enhanced overall survival was observed; however, the antibody was removed from the commercial market in the mid-1990s without definitive randomized trials being performed. several laboratories throughout the world are currently working to characterize and develop effective mab therapies for use in dogs. several antitumor vaccine approaches have been applied in dogs with lymphoma. a tumor vaccine extract using killed lymphoma cells combined with freund's adjuvant was administered to a small number of dogs after remission induction with combination chemotherapy. 250 prolongation of median survival was noted in the treatment group; however, a subsequent study revealed that prolongation was likely due to the freund's adjuvant. 251 an autologous killed lymphoma tumor cell vaccine has been intralymphatically administered to dogs placed in remission using a combination chemotherapy protocol, and, although modest gains were reported in remission times, no survival advantage was found. 252 an exploratory vaccine study targeting telomerase 253 (see chapter 14, section d) and one using rna-loaded cd40-activated b cells 254 in dogs with lymphoma have also been conducted. these studies involved small numbers of nonrandomized patients and lacked controlled populations for comparison. in a randomized study of 60 dogs with lymphoma comparing chop-based chemotherapy with chopbased chemotherapy and a human granulocyte-macrophage colony-stimulating factor (gm-csf) dna cationic-lipid complexed autologous whole tumor cell vaccine, a small measure of immunomodulation was documented by delayed-type hypersensitivity; however, significant improvement in clinical outcome was not noted. 255 although little well-supported activity is reported to date with these immunomodulatory approaches, our basic understanding of methodologies is expanding. most dogs with lymphoma have multicentric disease and therefore require systemic chemotherapy to effectively treat their disease. chemotherapeutic compounds from cells (see chapter 11). 239 p-glycoprotein is under the control of the mdr1 gene. mdr has been reported in canine lymphoma following exposure to chemotherapy. 156a,225,240 expression levels of mrna encoding the canine mdr1 gene have been characterized in canine cell lines and lymphomas. although expression of mdr1 mrna correlated with in vitro drug sensitivity, it did not correlate with in vivo doxorubicin sensitivity in dogs with lymphoma in this study. additionally, quantitative analysis of mrna for 10 different drug-resistance factors was performed in 23 dogs with lymphoma. 241 these dogs were divided into drug "sensitive" and "resistant" categories based on response to a chop-based protocol; however, significant differences in expression were not observed in this small study. methods of increasing the time that tumor cells are exposed to chemotherapeutics should theoretically enhance tumor killing. these methods could include long-term continuous infusions (impractical in many veterinary situations), increasing the frequency of treatments, or enhancing the circulation time of drugs used. in one study, dogs with lymphoma received lower dose doxorubicin weekly rather than a higher dose every 3 weeks (thereby decreasing c max , which is associated with cardiotoxicity) in order to potentially increase the time of drug exposure. 242 no benefit was noted, and, in fact, remission rates were inferior. studies evaluating pegylated long-circulating doxorubicin-containing liposome drug delivery systems in dogs with lymphoma have also been • 3. increase in alt activity >2× upper limit of normal (or 2× baseline if higher than upper limit of normal at initiation)-institute drug discontinuation and reinstitution/dose reduction depending on normalization of alt. practice. 266,267 because of the high cost, limited accessibility to relatively sophisticated equipment, and management requirements, these types of procedures are limited to preliminary investigations at a few centers. currently, long-term results in significant numbers of treated cases have yet to be presented. in general, the veterinary literature suffers from a paucity of information on treating various extranodal forms of lymphoma in dogs, and our ability to predict outcome is thus limited. in general, it is recommended that, following extensive staging, in those cases where disease is shown to be localized to a solitary site, local therapies (e.g., surgery, local rt) can be used. in contrast, if multiple extranodal sites are involved or they are part of a more generalized process, systemic chemotherapy should be chosen. most dogs with alimentary lymphoma are presented with diffuse involvement of the intestinal tract, and involvement of local lymph nodes and liver is common. chemotherapy in dogs with diffuse disease has been reported to be unrewarding for the most part 47,268,269 ; however, more aggressive chop-based protocols used extensively for multicentric lymphoma in dogs have resulted in durable remissions in a small subset of cases. solitary alimentary lymphoma is rare in the dog; however, if the tumor is localized and can be surgically removed, results (with or without follow-up chemotherapy) can be encouraging. cns lymphoma in dogs usually results from extension of multicentric lymphoma. however, primary cns lymphoma (pcnsl) has been reported. [103] [104] [105] if tumors are localized, local rt should be considered. few studies have reported the use of chemotherapy. in one study, cytosine arabinoside (ara-c) at a dosage of 20 mg/m 2 was given intrathecally; this treatment was combined with systemic chemotherapy and cns radiation. 103 overall, the response rates are low and of short duration (several weeks to months). treatment of cutaneous lymphoma depends on the extent of disease. solitary lesions may be treated with surgical excision or rt. fractionated rt (to a total dose of 30 to 45 gy) has been associated with long-term control. 270 diffuse cutaneous lymphoma is best managed with combination chemotherapy, although the rate and durability of response is generally less than in multicentric lymphoma. the most widely used protocols for epitheliotropic cutaneous t-cell lymphoma include ccnu (60 to 70 mg/m 2 po, every 3 weeks) along with prednisone. 271,272 although response rates approach 80%, median remission is approximately 3 months; occasionally, durable remissions are encountered. the author has added l-asparaginase to this protocol (see table 32 -5), and although anecdotally it appears to improve response, comparative data are not available. sporadic reports of other therapies for cutaneous lymphoma in small numbers of cases include the use of coap (cyclophosphamide, vincristine [oncovin], ara-c, and prednisone), 273 retinoic acid analogs (e.g., accutane, etretinate), 274 l-asparaginase and pegylated l-asparaginase, 275 topical mechlorethamine (mustargen), 276 and recombinant human α-interferon. 277 all of these reports involved small numbers of cases and resulted in limited response rates with short durations. a form of cutaneous lymphocytic infiltration has recently been characterized as an indolent t-cell lymphoma based on clonality. 131 however, surgery has been used to treat solitary lymphoma (early stage i) or solitary extranodal disease. careful staging is necessary in such cases to rule out multicentric involvement prior to treating local disease. the benefit of surgical removal of the spleen in dogs with massive splenomegaly remains unclear. 82, 83, 256 in an older report, 16 dogs with lymphoma underwent splenectomy to remove a massively enlarged spleen and were subsequently treated with chemotherapy. 256 within 6 weeks of splenectomy, 5 of the 16 dogs died of disseminated intravascular coagulation (dic) and sepsis. the remaining 11 dogs (66%) had a cr, and 7 dogs had a mst of 14 months. no staging or histologic information was provided, so the information appears of limited usefulness, although those with follow-up lived approximately 1 year. in two reports of indolent nodular lymphoma of the spleen (marginal zone lymphoma [mzl] and mantle cell lymphoma [mcl]), outcome was available on seven mzl cases, including three cases that did not receive adjuvant chemotherapy after surgery, 82, 83 and only one died of lymphoma following splenectomy. in a recent report of indolent lymphomas, four splenic lymphomas (three mzl and one mcl) underwent splenectomy alone and all survived greater than 1 year with none dying of their primary disease. 84 splenectomy should be considered if the lymphoma is not documented in other sites following thorough staging, if lymphoma is an indolent form histologically, or if splenic rupture has occurred. of note, no control population consisting of dogs that did not undergo splenectomy exists, so the natural history of indolent splenic lymphoma remains uncertain. radiation therapy, although its use is limited in the treatment of lymphoma, may be indicated in selected cases. 257 indications are as follows: 1. curative intent therapy for stage i lymph node and solitary extranodal disease (i.e., nasal, cutaneous, spinal lymphoma). 2. palliation for local disease (e.g., mandibular lymphadenopathy, rectal lymphoma, mediastinal lymphoma where precaval syndrome is present, localized bone involvement). 3. total body radiation combined with bone marrow or stem cell transplantation. 4. whole or staged half-body rt following chemotherapyinduced remissions. in the latter case, staged half-body irradiation sandwiched between chemotherapy cycles or following the attainment of remission by induction chemotherapy has been preliminarily investigated as a form of consolidation or maintenance. 198,258-262 radiation therapy was delivered to either the cranial or the caudal half of the dog's body in 4 to 8 gy fractions, and following a 2-or 4-week rest the other half of the body was irradiated in a similar fashion. although these preliminary investigations were not randomized, they suggest that rt applied when dogs are in either complete or partial remission is safe and warrants further investigation to determine if a significant therapeutic gain can be realized. a pilot study of low-dose (1 gy) single-fraction total body irradiation in seven dogs with relapsed drug-resistant lymphoma, although safely applied, resulted in only partial nondurable (1 to 4 week) remissions. 263 total body irradiation (and/or ablative chemotherapy) for complete or partial bone marrow ablation followed by reconstitution with bone marrow or stem-cell transplant in dogs, although a recognized model in comparative research settings, 264,265 is still in its early phases of development and application in clinical veterinary it is associated with slow progression and long-term survival following corticosteroid management; however, it does have the potential to progress to high-grade lymphoma. the prognosis for dogs with lymphoma is highly variable and depends on a wide variety of factors documented or presumed to affect response to therapy. although rarely curable (<10% of cases), crs and a good quality of life during extended remissions and survival are typical. factors that have been shown to influence treatment response and survival are summarized in tables 32-6* and 32-7. † the two prognostic factors most consistently identified are immunophenotype and who substage (see figure 32 -9). many reports have confirmed that dogs with cd3-immunoreactive tumors (i.e., t-cell derivation) are associated with significantly shorter remission and survival durations. ‡ this holds true primarily for dogs with multicentric lymphoma because the immunophenotype of solitary or extranodal forms of lymphoma has not been thoroughly investigated with respect to prognosis. additionally, it has been shown that dogs with b-cell lymphomas that express lower than normal levels of b5 antigen (expressed in 95% of nonneoplastic lymphocytes) also experience shorter remission and survival durations. 54 recently, low levels of class ii mhc expression on b-cell lymphoma predicted poor outcomes. 279 dogs presented with who substage b disease (i.e., clinically ill) also do poorly when compared with dogs with substage a disease. 53, 86, 91, 181, 278 dogs with stage i and ii disease have a better prognosis than those dogs in more advanced stages (stage iii, iv, and v). histologic grade (subtype) has been found to influence prognosis in some studies; however, our ability to predict outcome based on subtype is still quite limited. dogs with lymphoma classified as intermediate or high grade (large cell, centroblastic, and immunoblastic) tend to respond to chemotherapy but can relapse early. dogs with low-grade lymphomas (small lymphocytic or centrocytic) have a poorer response rate to chemotherapy, yet have a survival advantage over dogs with intermediate-and high-grade lymphomas ( figure 32 -10) in that the disease may be more indolent. several case compilations have documented that dogs with indolent lymphoma (e.g., mzl, mcl, t-zone) experience prolonged survivals, often in the absence of any or aggressive chemotherapy. [82] [83] [84] proliferative assays such as analysis of bromodeoxyuridine (brdu) uptake, ki67 antibody reactivity, and argyrophilic nucleolar organizer region (agnor) indices to measure proliferative activity of tumor cells have been shown to provide prognostic information in dogs treated with combination chemotherapy. results of different studies are contradictory, however. in two trials, dogs having tumors with short doubling times, high agnor frequencies, or high ki67 immunoreactivity had a better prognosis than those with tumors with long doubling times or low agnor frequencies. 53, 142 in other trials, the low-proliferating tumor groups were associated with a better prognosis. 283,284 additionally, in one trial, the proportion of tumor cells undergoing apoptosis was modestly predictive of remission duration. 142 the anatomic site of disease is also of considerable prognostic importance. primary diffuse cutaneous, diffuse gi, hepatosplenic, and primary cns lymphomas tend to be associated with a poor ‡ rights were not granted to include this figure in electronic media. please refer to the printed publication. prognosis. dogs with indolent cutaneous t-cell lymphocytic infiltration experience long-term survivals. 131 sex has been shown to influence prognosis in some studies. 175, 181 neutered females tend to have a better prognosis; male dogs may have a higher incidence of the t-cell phenotype, which may account for the poorer prognosis. reported biomarkers of prognosis, summarized in table 32 -7, include circulating levels of glutathione-s-transferase, thymidine kinase, lactate dehydrogenase, serum c-reactive proteins, and vegf. finally, one report suggests that a history of chronic inflammatory disease of several types predicts likelihood of early relapse. 291 these putative prognostic indicators require further confirmation in larger trials. lymphocytic leukemia is typically defined as proliferation of neoplastic lymphocytes in bone marrow. neoplastic cells usually originate in the bone marrow, but occasionally in the spleen, and may or may not be circulating in the peripheral blood. although our ability to diagnose lymphocytic leukemias using flow cytometric and molecular diagnostic techniques has increased significantly in the past decade, little information on treatment and prognosis is available except for chronic lymphocytic leukemia (cll). differentiating between true leukemia and stage v lymphoma can be difficult and arbitrary and is often based on lack of significant lymphadenopathy, degree of blood and bone marrow involvement, and immunophenotypic characteristics. lymphocytic leukemia is more common than acute myeloid leukemia and myeloproliferative disorders (mpd), but the true incidence is unknown. german shepherd dogs and golden retrievers may be overrepresented. 137,292 lymphocytic leukemia can occur in dogs of any age but typically occurs in middle-aged to older dogs (mean of 7 to 10 years); cll usually occurs in older dogs (mean of 10 years). 137,280,292,293 a significant sex predilection is not reported. as with lymphoma, the etiology of lymphocytic leukemia is for the • acute (large cells with an immature cytologic phenotype). immunophenotypic assessment using flow cytometric and molecular assays can further characterize these two major subtypes; however, some discordance exists in the veterinary literature. three primary subtypes of cll are reported in dogs, based primarily on immunophenotyping 137,280,293 : (1) t-cll, which is the most common form, with cells in the majority of cases being cd8 + granular lymphocytes; (2) b-cll, which is the next most common subtype; and (3) atypical cll, which represents a combination of immunophenotypes (cd3 − , cd8 + ; cd3 + , cd4 − , cd8 − ; cd3 + , cd4 + , cd8 + ; and cd3 + + cd21 + *). this is in contrast to cll in humans, which is primarily a disease of b-cells. in cll, lymphocytes often are indistinguishable morphologically from normal small lymphocytes ( figure 32 most part unknown. genetic factors likely play a role and have been compared between dogs and humans. 16 retroviruses have been implicated in diverse animal species such as cats, cattle, fish, snakes, birds, rodents, nonhuman primates, and humans; however, there is no proven evidence implicating a retroviral cause in dogs. in humans, acute lymphocytic leukemia (all) has been associated with genetic factors and exposure to radiation, benzene, phenylbutazone, and antineoplastic agents. extrapolation of predisposing factors across species is not warranted; in fact, etiologic factors in dogs may be quite different from those for humans given the difference in the predominant immunophenotype of the neoplastic cells (see later). lymphocytic leukemias can be subdivided based on cell size, maturity, genetic aberrations, microrna expression, and immunophenotype.* the simplest classification divides leukemia into two groups: chronic (small cells with a mature cytologic phenotype) and *note that either cd21 or cd79 can be used for assessing b-cell lineage in this context. *references 16, 137, 150, 280, 292-294. chromatin from disintegrated cells also is visible. (wright's stain, ×60 objective.) b, peripheral blood from a dog with chronic lymphocytic leukemia (cll). note the small lymphocytes of normal morphology (smaller than the neutrophil). (wright's stain, ×60 objective.) constitute the remaining fraction. in the t-cell fraction, helper t-cells (cd4 + ) outnumber cytotoxic t-cells (cd8 + ). 296 lymphocytic leukemia should be a consideration if atypical lymphocytes are in circulation, the immunophenotype of the lymphocytes in circulation is homogenous as determined by flow cytometric analysis, a phenotype typically present in low frequency has increased, or if clonality is documented (e.g., by parr analysis). other differential diagnoses for lymphocytosis include infectious diseases, such as chronic ehrlichiosis, postvaccinal responses in young dogs, il-2 administration, and transient physiologic or epinephrine-induced lymphocytosis. in some cases, reactive and neoplastic lymphocytosis are difficult to distinguish. expansion of neoplastic lymphocytes in bone marrow is the hallmark of all and, in most cases, cll. careful examination of peripheral blood and bone marrow by an experienced cytopathologist is important in establishing a diagnosis of lymphoid leukemia; in cases of marked lymphocytosis with atypia, peripheral blood can be used for analysis of immunophenotype and clonality, and examination of bone marrow is not essential. if diagnostic bone marrow cannot be adequately obtained by aspiration, bone marrow core biopsy should be performed. in all, lymphoblasts predominate in the bone marrow and are also present in peripheral blood, and other lineages are decreased. in b-and t-cell cll, the lymphocytes are small mature cells that occur in excessive numbers in bone marrow (≥30% of all nucleated cells) early in the disease. 295 in t-cll, lymphocytes may contain pink granules. infiltration becomes more extensive as the disease slowly progresses, and eventually the neoplastic cells replace normal marrow. a separate clinical staging system has not been developed for lymphoid leukemia. currently, all dogs with leukemia are classified as stage v based on the who staging system for lymphoma as presented in table 32 -2. because of the indolent and often asymptomatic nature of cll, the decision to treat is often based on the clinical and laboratory findings in the individual dog. most oncologists recommend active surveillance (monthly or bimonthly physical examination and cbc) over active therapy for patients when cll is identified incidentally, there are no accompanying clinical signs, and other significant hematologic abnormalities are not identified. if the animal is significantly anemic or thrombocytopenic, is showing evidence of significant lymphadenopathy or hepatosplenomegaly, or has an excessively high lymphocyte count (e.g., >60,000/µl), therapy should be instituted. the definition of "excessively high" varies among oncologists, and a standard has not been established in veterinary medicine. the author (dmv) prefers to base treatment decisions on the presence of significant constitutional signs and peripheral cytopenias. currently, the most effective drug available for treatment of cll is chlorambucil. 295 chlorambucil is given orally at a dose of 0.2 mg/kg or 6 mg/m 2 po once daily for 7 to 14 days; the dose can then be reduced to 0.1 mg/kg or 3 mg/m 2 po daily. for long-term maintenance, a dose of 2.0 mg/m 2 every other day can be used. the dose is adjusted based on clinical response and bone marrow tolerance. oral prednisone is used concurrently with chlorambucil at doses of 1 mg/kg daily for 1 to 2 weeks, then 0.5 mg/kg every other day thereafter. the addition of vincristine or the substitution of cyclophosphamide for chlorambucil has been advocated in animals that do not respond to chlorambucil. origin (cd3 + ,cd4 − , cd8 − , cd21 − ). 137 in general, these cells tend to be intermediate-sized or large cells with moderate amounts of basophilic cytoplasm. perhaps the most distinguishing feature of lymphoblasts is the nuclear chromatin pattern, which typically is more condensed than the chromatin in myeloblasts. lymphoblasts are larger than neutrophils, have a high nuclear : cytoplasmic ratio, and contain blue cytoplasm that in some cases is intensely basophilic (see figure 32 -11). nucleoli, although present, are less prominent in lymphoblasts than in myeloblasts. nevertheless, these cells cannot be distinguished easily from blast cells of other hematopoietic lineages, and identification of lineage-specific markers by immunocytochemical or flow cytometric analysis is required to ascertain the lineage. if the cells express cd34, a stem cell marker, an acute phenotype is implied 137,280 ; however, both myeloid and lymphoid lineages express cd34, and our ability to differentiate all from acute myeloid leukemia (aml) relies on detection of other markers, including t-and b-cell markers and myeloperoxidase, a myeloid marker. dogs with cll are often asymptomatic, but some owners report lethargy and decreased appetite. mild lymphadenopathy and splenomegaly may be present, although late in the disease splenomegaly may be marked. 295 the white blood cell (wbc) count is usually greater than 30,000 cells/µl but can vary from normal to greater than 100,000 cells/µl because of an increase in circulating mature lymphocytes. lymphocytosis is persistent and granulocytes are usually present in normal numbers. other than lymphocytosis, hemograms of dogs with cll tend to have few abnormalities when lymphocytes are less than 30,000/µl. 137, 280, 293 in some dogs, the disease is identified incidentally when the animal is undergoing evaluation for an unrelated problem. mild anemia, neutropenia, and thrombocytopenia are common but may become marked as the disease progresses and lymphocyte counts increase above 30,000/µl. despite the well-differentiated appearance of the lymphocytes in cll, these cells may function abnormally. paraneoplastic syndromes include monoclonal gammopathies, immunemediated hemolytic anemia, pure red cell aplasia, and, rarely, hypercalcemia. 296,297 in one report of 22 dogs with cll, 68% had monoclonal gammopathies (usually igm or iga). 296 the immunophenotypes were not reported, but a monoclonal gammopathy would be more likely to occur in b-cll. dogs with all usually are presented with clinical signs of anorexia, weight loss, and lethargy. splenomegaly is typical and other physical abnormalities may include hemorrhage, lymphadenopathy, and hepatomegaly. 298 infiltration of bone marrow by neoplastic lymphoblasts may be extensive, resulting in significant depression of normal hematopoietic elements or myelophthisis. 137,280,292,298,299 anemia, neutropenia, and thrombocytopenia are typically much more severe than with cll and may become life threatening. infiltration of extramedullary sites such as the cns, bone, and gi tract may also occur and can result in neuropathies, bone pain, and gi signs, respectively. consideration of signalment, history, physical findings, and morphologic appearance and immunophenotype of cells is essential in making an accurate diagnosis. it is helpful to know the profile of lymphocyte subsets in the peripheral blood of normal dogs to determine if a particular subset has expanded. approximately 80% of circulating lymphocytes in normal dogs are t-cells, and about 15% are b-cells. nk cells and double-negative (cd4 − , cd8 − ) t-cells animal, nat cancer inst treatment of cll is primarily palliative with rare complete remissions. owing to the indolent nature of this disease, however, survival times have been in the range of 1 to 3 years with a good quality of life. 295,300 the phenotypic expression of cll is usually stable over months to years. however, the disease may evolve into an acute phase, and some dogs will develop a form of lymphoma that is rapidly progressive and characterized by the presence of pleomorphic immunoblasts; in humans, this is termed richter's syndrome. 301 the prognosis for response to treatment is poor for this form of lymphoma. much of the morbidity in dogs with all results from effacement of bone marrow (myelophthisis) and subsequent life-threatening peripheral cytopenias. neutropenia, thrombocytopenia, and anemia may be severe. patients often require supportive therapy, such as fresh whole-blood transfusions, broad-spectrum antibiotics, fluid therapy, and nutritional support. careful monitoring for sepsis, hemorrhage, and dic is important. specific treatment of all requires aggressive chemotherapy. consistently efficacious protocols for all have not been developed in veterinary medicine, and there are few published reports. chop-based protocols, similar to those used for lymphoma (see table 32 -4), have been used by the author (dmv) for dogs with all; however, responses and durability of response are generally disappointing. the standard of care in humans with acute leukemia generally involves bone marrow ablative treatments with stem cell or marrow replacement, a protocol not generally available in veterinary oncology. in general, cll is a slowly progressive disease, and some animals will not require therapy for some time after diagnosis; one dog was reported to survive almost 2 years without treatment. 302 for those dogs that are treated, normalization of lymphocyte counts can be expected in 70% of cases. in one report of 17 dogs treated with vincristine, chlorambucil, and prednisone, mst was approximately 12 months with an expected 30% survival at 2 years. 295 in larger compilations of cases that include immunophenotypic analysis, treatment protocols were poorly documented, although most received chlorambucil and prednisone; in 43 dogs with follow-up, for dogs with t-cll, b-cll, and atypical cll, median survival was 930, 480, and only 22 days, respectively. 293 in this group of dogs, young age and anemia were also associated with a poor prognosis. in another series with limited treatment information, dogs with cll of a cd8 + immunophenotype that presented with less than 30,000 lymphocytes/µl or greater than 30,000 lymphocytes/µl had median survivals of 1098 and 131 days, respectively. prognosis for dogs with all is generally very poor. in a study of 21 dogs treated with vincristine and prednisone, the dogs achieving complete or partial remission (29%) had a mst of 120 days, and few dogs survived longer than 8 months with that protocol. 298 in one report of 46 cases of all with a cd34 + phenotype, dogs had a median survival of 16 days (ranged from 3 to 128 days), even though the majority received a chop-based treatment protocol. 280 additionally, dogs with b-cell all (cd21 + ) in which the lymphocytes were large cells (forward scatter lymphocyte/forward scatter neutrophil ratio of >0. 58 the lymphomas (malignant lymphoma or lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. they usually arise in lymphoid tissues such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. lymphoma is one of the most common neoplasms seen in the cat. epidemiologic reports prior to 1990 suggested that lymphoma accounted for 50% to 90% of all hematopoietic tumors in the cat, 1,2 and since hematopoietic tumors (lymphoid and myeloid) represent approximately one-third of all feline tumors, it was estimated lymphoid neoplasia accounted for an incidence of 200 per 100,000 cats at risk. 3 in one series of 400 cats with hematopoietic tumors, 61% had lymphoma and 39% had leukemias and mpds, of which 21% were categorized as undifferentiated leukemias, most likely myeloid in origin. 4 mediastinal form that is not felv associated and represents a younger population (median of 2 years). felv was the most common cause of hematopoietic tumors in the cat in the so-called "felv era" of the 1960s through the 1980s when approximately two-thirds of lymphoma cases were associated with felv antigenemia. several studies have documented the potential molecular means by which felv can result in lymphoid neoplasia (see chapter 1, section c). as one would predict, along with a shift away from felv-associated tumors came a shift away from traditional signalment and relative frequency of anatomic sites. this is also supported outside of north america by similar signalment and anatomic frequency data observed in australia where felv infection is quite rare. 10, 11 the median age of approximately 11 years now reported in north america is considerably higher than the median ages of 4 to 6 years reported in the felv era. 1,2,5-7 the median age of cats within various anatomic tumor groupings has not changed, and anatomic forms traditionally associated with felv such as the mediastinal form still occur in younger, felv antigenemic cats. similarly, the alimentary form occurs most often in older, felvnegative cats. table 32 -8 presents an overview of the characteristics, including felv antigenemic status, of the various anatomic sites of lymphoma in cats. as our ability to interrogate felv associations on a molecular basis has improved (e.g., pcr amplification and fluorescent in-situ hybridization), several reports exist defining the role or potential role of felv in cats with and without felv antigenemia. [12] [13] [14] [15] [16] [17] collectively, these studies indicate felv proviral insertion exists in a significant proportion of feline lymphoma tissues and is more common in those of t-cell origin, particularly the thymic and peripheral lymph node anatomic forms. they also suggest that several common felv integration sites exist. there is also evidence that feline immunodeficiency virus (fiv) infection can increase the incidence of lymphoma in cats. [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] in contrast to the direct role of felv in tumorigenesis, most evidence available felv vaccines appearing in the late 1980s (see the later section on viral etiology). the decline in felv-associated lymphoma was mirrored by a decline in the overall prevalence per year of felv positivity in cats tested as characterized by reports, including the tufts veterinary diagnostic laboratory from 1989 to 1997, 5, 6 and by the louwerens group, who reported a decline in felv association in over 500 cases of lymphoma in cats presenting to the university of california at davis veterinary teaching hospital. 7 in these reports, felv antigenicity declined to represent only 14% to 25% of cats presenting with lymphoma. importantly, louwerens' study revealed that despite a sharp drop in felv-associated lymphoma, the overall prevalence of lymphoma in cats is increasing. the increased prevalence appears due to an increase in the number and relative frequency of the alimentary (and in particular the intestinal) anatomic form of lymphoma in the species. this is supported by an epidemiologic survey of 619 cases of feline intestinal lymphoma; 534 (86%) were from the 20 years following 1985 and only 14% were from cases diagnosed in the 20 years prior to 1985. 8 the true annual incidence rate for lymphoma in cats is currently unknown. with respect to feline pediatric tumors, a study in the united kingdom (n = 233 pathology specimens) found that 73 (31%) represented hematopoietic tumors, of which 51 (70%) were lymphoma-note that felv status was unavailable for this compilation of cases. 9 the typical signalment for cats with lymphoma cannot be uniformly stated as it varies widely based on anatomic site and felv status and therefore will be discussed individually under sitespecific discussions. in general, based on two large compilations (n = 700) of cases in north america, 5, 7 siamese cats appear overrepresented and although a 1.5 : 1 male to female ratio was observed in one, no association with sex or neutering status was observed in the other. in a large compilation of australian cases, male cats and the siamese/oriental breeds were overrepresented, 10 and similar breed findings have been observed in north america, although similar sex predilections have not been found. within the siamese/ oriental breeds, there appears to be a predisposition for a • felv, feline leukemia virus; id, insufficient data; cns, central nervous system. common = >50% of clinical presentations; moderate = 20%-50% of clinical presentations; uncommon = 5%-20% of clinical presentation; rare = <5% of clinical presentations. *data may include overlap or mixing of sites and represents the post-felv era. † as the primary site of presentation, rather than extension or progression. ‡ includes those reported as "intraabdominal" in which intestinal is a documented component. support for this concept. 47 additionally, an association between gastric helicobacter infection and gastric malt lymphoma in cats is suggested in one study, and because this is a recognized syndrome in humans, it warrants further investigation. 47a although no direct evidence exists, a link between diet and the development of intestinal lymphoma in cats has been suggested. 7 support is offered by the relative and absolute increase in the alimentary form of lymphoma in the past 20 years and the fact that several dietary modifications in cat food have occurred in a similar timeframe in response to diseases, such as urinary tract disease. further investigation is warranted to prove or disprove such assertions. lymphoma can be classified based on anatomic location and histologic and immunophenotypic criteria; often, the two are intimately associated because certain histologic and immunophenotypic types are commonly associated with specific anatomic locations necessitating discussions within the individual anatomic categories that follow. the largest compilation of feline cases subjected to rigorous histologic classification was reported by valli and others 48 using the nci wf. who has also published a histologic classification system that uses the real system as a basis for defining histologic categories of hematopoietic tumors of domestic animals. 49 this system incorporates both histologic criteria and immunohistologic criteria (e.g., b-and t-cell immunophenotype). regarding anatomic location, as discussed previously, a profound change in presentation, signalment, felv antigenemia, immunophenotype, and frequency of anatomic sites has occurred in cats with lymphoma in the "post-felv" era (see table 32 -8). because of this shift, characteristics of feline lymphoma discussed in this chapter will be primarily limited to reports collected from cases presenting after 1995. several anatomic classifications exist for lymphoma in the cat, and some categorize the disease as mediastinal, alimentary, multicentric, nodal, leukemic, and individual extranodal forms. others have combined various nodal and extranodal forms into categories of atypical, unclassified, and mixed, and others have combined intestinal, splenic, hepatic, and mesenteric nodal forms into one category termed intraabdominal. some discrepancies in the discussion of frequency will inevitably result from the variations in classification used in the literature. the relative frequency of anatomic forms and their associated immunophenotype may also vary with geographic distribution and may be related to genetic and felv strain differences, as well as prevalence of felv vaccine use. alimentary/gi lymphoma can present as a purely intestinal infiltration or a combination of intestinal, mesenteric lymph nodes and liver involvement. the tumors can be solitary but more commonly diffuse throughout the intestines. some reports limit the alimentary form to gi involvement with or without extension to the liver. lymphoma is the most common tumor type found in the intestines of cats, representing 55% of cases in an epidemiologic survey of 1129 intestinal tumors in the species. 8 the siamese breed is reported at increased risk. 7, 8 while lymphoma may occur in cats of any age, it is primarily a disease of aged cats with a mean age of approximately 13 years for t-cell alimentary lymphoma and 12 years for b-cell lymphoma. 7, 8, [50] [51] [52] no consistent sex bias is noted. anatomically, alimentary lymphoma is nearly 4 times more likely to occur in the small intestine than the large intestine. 52 in a series of colonic points toward an indirect role for fiv secondary to the immunosuppressive effects of the virus. shelton 18 determined that fiv infection alone in cats was associated with a fivefold increased risk for development of lymphoma. coinfection with felv will further potentiate the development of lymphoproliferative disorders. experimentally, cats infected with fiv have developed lymphoma in the kidney, alimentary tract, liver, and multicentric sites. fivassociated lymphoma is more likely that of the b-cell immunophenotype rather than the t-cell predominance associated with felv. it has been suggested that fiv infection may be associated more commonly with alimentary lymphoma of b-cell origin, 28, 29 and this may be related to chronic dysregulation of the immune system or the activation of oncogenic pathways; however, fiv antigenemia was only rarely associated with alimentary lymphoma in other large compilations of cases. 5,30-33 as discussed earlier in section a, recent advances in molecular cytogenetics (see chapter 1, section a, and chapter 8), including gene microarray techniques, have and are currently being applied to investigations of chromosomal aberrations in veterinary species with lymphoma. indeed a predisposition of the oriental cat breeds to develop lymphoma suggests a genetic predisposition and indicates heritable risk. 7,10 altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and cell deathpathway alterations are common in lymphomas of humans and are likely also involved in the cat. several genetic factors have already been discussed as they relate to felv associations. additionally, n-ras aberrations have been implicated, although they are rare in cats. 34 furthermore, telomerase activity (see chapter 2) has been documented in feline lymphoma tissues. 35, 36 alterations in cellular proliferation and in cell-cycle and death (apoptosis) pathways, in particular the cyclin-dependent kinase cell-cycle regulators and the bcl-2 family of proapoptotic and antiapoptotic governing molecules, have also been implicated in feline lymphoma. [37] [38] [39] evidence for exposure to environmental tobacco smoke (ets) as a risk factor for lymphoma in humans has prompted investigations in cats. in one report, the relative risk of developing lymphoma in cats with any exposure to ets and with 5 or more years of exposure to ets was 2.4 and 3.2, respectively. 40 a large european study documenting an association between proximity of waste management and cancer in dogs failed to show increased risk in cats. 41 immune system alterations in the cat such as those accompanying fiv infection has been implicated in the development of lymphoma. [18] [19] [20] 25 as is the case in immunosuppressed human organ transplantation patients, two reports of immunosuppressed feline renal transplant recipients document increased risk of lymphoma following transplant and associated immunosuppressive therapy. 42, 43 in both studies, nearly 10% of transplanted cats developed de novo malignant lymphoma. although definitive proof is lacking, there is a growing body of indirect evidence to suggest that lymphoma can be associated with the presence of chronic inflammation, which theoretically could be the case with intestinal and nasal lymphoma. in particular, an association has been suggested between intestinal lymphoma and inflammatory bowel disease 7,44-46 ; however, others have not found with malignant pleural effusion and a mediastinal mass present. peripheral blood involvement was present in 10% of cases in one report 59 and 86% in another. 60 affected cats are generally felv/fiv negative. the mediastinal form can involve the thymus, mediastinal, and sternal lymph nodes. pleural effusion is common. in two large compilations, 63% of cats with thymic disease and 17% of cats with pleural effusion were documented as having lymphoma. 62, 63 hypercalcemia occurs frequently with mediastinal lymphoma in dogs but is rare in cats. the majority of cats with mediastinal lymphoma are young (median age 2 to 4 years), felv positive, and the t-cell immunophenotype. 5, 7, [9] [10] [11] the disease is confined to the mediastinum in most cases. 7 there also exists a form of mediastinal lymphoma occurring primarily in young, felv-negative siamese cats that appears to be less biologically aggressive and more responsive to chemotherapy than felv-associated forms. 64 involvement limited to peripheral lymph nodes is unusual in cats with lymphoma, representing approximately 4% to 10% of cases. 5, 7 in contrast, approximately one-quarter of all other anatomic forms of lymphoma have some component of lymph node involvement. one-third of cats with nodal lymphoma are t-cell immunophenotype and felv antigenemic; however, complete categorizations have not occurred in the post-felv era and this may no longer be true. 5, 7, 11, 55 peripheral nodal lymphoma was the most common anatomic form of lymphoma reported in a recent compilation of cases in cats under the age of 1 year, representing a full third of cases in this age group. 9 as lymphoma progresses, bone marrow and hepatic infiltration may develop. an uncommon and distinct form of nodal lymphoma in cats referred to as "hodgkin's-like" lymphoma has been reported. 65, 66 this form typically involves solitary or regional nodes of the head and neck (figure 32-12) and histologically resembles hodgkin's lymphoma in humans. affected cats generally present with enlargement of one or two mandibular or cervical nodes initially, and tumors are immunophenotypically classified as t-cell-rich, b-cell lymphoma. one case each of inguinal node, multicentric nodal, and neoplasia in cats, lymphoma was the second most common malignancy (41%), second only to adenocarcinoma. 33 there is some discordance in the literature regarding the histologic type (primarily cell size: small versus large), immunophenotype, and architecture involved with gi lymphoma. while studies (often older or smaller reports) suggest a majority of b-cell immunophenotypes, 5,53 larger, more recent reports 51, 54, 55 indicate the majority represent mucosal low-grade t-cell immunophenotypes. conversely, the vast majority of b-cell gi lymphomas in cats are large cells and intermediate or high grade. 51, 53 the largest compilation to date (n = 120), by moore and others, 51 classified gi lymphomas based on immunophenotype, then as either mucosal (infiltrate confined to mucosa and lamina propria with minimal submucosal extension) or transmural (significant extension into submucosa and muscularis propria). they then compared infiltration patterns with the who classification scheme, 56 as well as documenting anatomic location, cell size, presence of epitheliotropism, clonality, and outcome data. this information is summarized in table 32-9. of the 120 cases, none tested serologically positive for felv and only 3 for fiv. four cats had concurrent large b-cell lymphoma (stomach, cecum, or colon) and small t-cell lymphoma of the small intestine. topographically, t-cell variants are much more likely to occur in the small intestine (94%) and rarely in the stomach or large intestine. conversely, b-cell variants were often multiple and often occurred simultaneously within the stomach, small intestine, and ileocecocolic junction. the vast majority of t-cell variants were mucosal (equivalent to who enteropathy-associated t-cell lymphoma [who eatcl] type ii), and the vast majority of b-cell tumors were transmural (equivalent to who eatcl type i classification). regarding cell size, nearly all mucosal t-cell tumors were composed of small lymphocytes, and slightly more than half of transmural t-cell and all b-cell variants were composed of larger cells. epitheliotropism is present in approximately 40% of t-cell tumors but is rare in b-cell tumors. other abdominal organ involvement is common, and in one report of 29 cases of low-grade t-cell intestinal lymphoma, liver and mesenteric involvement was documented in 53% and 33% of cases, respectively. 57 hepatic lymphoma can occur concurrently with gi lymphoma or be confined solely to the liver. 52, 58 most are t-cell and clonal or oligoclonal based on pcr analysis. a less common, distinct form of alimentary lymphoma, large granular lymphoma (lgl), also occurs in older (median age 9 to 10 years) cats. 51,53,59-61 these granulated round cell tumors have been termed globule leukocyte tumors, although they are likely variations of the same disease. lgl is characterized by lymphoblasts described as 12 to 20 µm in diameter with a round, clefted, or cerebriform nucleus; variably distinct nucleoli; finely granular to lacey chromatin; and a moderate amount of basophilic granular cytoplasm that was occasionally vacuolated. 59 prominent magenta or azurophilic granules are characteristic (see figure 7 -34, chapter 7). they are granzyme b positive by immunohistochemistry. 51 this population of cells includes cytotoxic t-cells and occasionally nk cell immunophenotypes-most are cd3 + , cd8 + , and cd20 − and have t-cell receptor gene rearrangement. 51, 60 in one report, nearly 60% expressed cd103 (integrin). 60 approximately 10% express neither b-or t-cell markers and are thus classified as nk cells. these nk tumors commonly originate in the small intestine, especially the jejunum, are transmural, often exhibit epitheliotropism, and at least two-thirds present with other organs involved-most with mesenteric lymph node involvement and many with liver, spleen, kidney, peritoneal malignant effusions, and bone marrow infiltration. also, thoracic involvement may occur • figure 32 -12 a cat presented with mandibular lymphadenopathy that was confirmed to be hodgkin's-like lymphoma following histologic assessment. • frequent sequela to renal lymphoma and occurs in 40% to 50% of treated cats. 74 cns lymphoma can be intracranial, spinal, or both. cns lymphoma made up 14% of 110 reported cases of extranodal lymphoma, 68 15% to 31% of intracranial tumors, 75, 76 and 39% of spinal cord tumors, 77 making it one of the most common malignancies encountered in the cns in cats. although some discordance exists in the literature, cats with cns lymphoma are younger (median ages of 4 to 10.5 years reported), and 17% to 50% of cases are felv antigenemic. [76] [77] [78] approximately two-thirds of intracranial cases are part of a multicentric, extracranial process, and approximately 40% of spinal lymphoma cases occur in multiple spinal cord sites with one-third also involving intracranial locations. [76] [77] [78] in a compilation of 160 cases of intracranial tumors in cats, diffuse cerebral and diffuse brainstem involvement was most common for lymphoid malignancies. 76 spinal lesions are usually both extradural and intradural, although they can be limited to one or the other compartment. 77 feline cns lymphoma may be primary but more commonly (approximately 80%) represents a multicentric process (especially renal or bone marrow). 76 ,78 a paucity of information exists on the immunophenotype of cns lymphoma. laryngeal lymphoma made up 10% of 110 cases of extranodal forms in one report and represented 11% of all laryngeal disease in the species. 68, 79 it occurs in older cats (median age 9 years), is not associated with felv, and may be a solitary lesion or occur in the presence of other multicentric sites. no information on immunophenotype is available. cutaneous lymphoma is a rarely encountered anatomic form in the cat. it is usually seen in older cats (median age 10 to 13.5 years), with no sex or breed predominance, and is not associated with felv/fiv. 80, 81 it can be solitary or generalized, often affecting the head and face and is generally a slow chronic disease. two forms of cutaneous lymphoma have been distinguished histologically and immunohistochemically. most reports in the cat are epitheliotropic and consist of t-cells, although unlike the disease in dogs, adnexal structures are often spared. a report of nonepitheliotropic cutaneous lymphoma in cats also found five of six cases to be of t-cell derivation. 82 cutaneous "lymphocytosis, " an uncommon disease histologically resembling well-differentiated lymphoma, was characterized in 23 cats. 83 solitary lesions were most common, and all were composed primarily of t-cells, with two-thirds having some b-cell aggregates. cutaneous lymphocytosis was characterized as a slowly progressive disorder; however, a few cases went on to develop internal organ infiltration. two case reports exist of cats with cutaneous t-cell lymphoma and circulating atypical lymphocytes. 84, 85 the circulating cells were lymphocytes with large, hyperchromatic, grooved nuclei, and one case was immunophenotyped as a cd3/ cd8 population. in humans, cutaneous t-cell lymphoma with circulating malignant cells is termed sézary syndrome. ocular lymphoma was identified in 5 of 110 cases of extranodal lymphoma in one report. 68 in a compilation of 75 cases of intraocular tumors, 15 (20%) were lymphoma (7 b-cell and 4 t-cell). 86 it was presumed but not proved that the majority were part of a systemic multicentric process. the clinical signs associated with feline lymphoma are variable and depend on anatomic location and extent of disease. the alimentary form is most commonly associated with nonspecific signs associated with the intestinal tract. in the more conjunctival involvement have been reported. 66, 67 histologically, lymph nodes can be effaced by either nodular or diffuse small to blastic lymphocytes with characteristic bizarre or multinucleated cells (reed-sternberg-like cells) (figure 32-13 ). no association with felv or fiv has been documented. the most common extranodal sites for lymphoma in cats include nasal (including nasopharyngeal and sinonasal), kidney, cns, laryngeal and tracheal, ocular, retrobulbar, and skin. nasal lymphoma is the most common extranodal lymphoma in cats. 68 it is usually a localized disease; however, 20% have local extension or distant metastasis at necropsy. 69 the majority of nonviral nasal/paranasal disease in cats are neoplasias, and lymphoma represents nearly one-third to half of these cases. [70] [71] [72] it occurs primarily in older (median age 9 to 10 years; range 3 to 17 years) felv/ fiv-negative cats and at least three-quarters are b-cell in origin, although t-cell and mixed b-cell/t-cell immunophenotypes can be seen in approximately 10% to 15% of cases. 5, 68, 69, 73 siamese cats appear overrepresented, and one report 73 observed a 2 : 1 male-tofemale ratio. most are of intermediate-or high-grade histology. 69, 73 epitheliotropism is common if the epithelium is present in the biopsy. renal lymphoma is the second most common form of extranodal lymphoma after the nasal form, occurring in approximately one-third of cases. 68 it can present as primary to kidney lymphoma or occur concurrent with alimentary lymphoma. in more contemporary reports, the median age at presentation is 9 years, although 6% occurred in cats under 1 year of age. 68, 69 the vast majority of cases are not associated with either felv or fiv. the greater median age and lack of felv/fiv association are in contrast to reports compiled prior to the post-felv era; in earlier studies, the median age was approximately 7.5 years, 25% of cases were felv antigenemic, and the majority constituted a b-cell immunophenotype. little contemporary information exists on the immunohistologic classification of renal lymphoma; however, in australia where felv is not a significant problem, most renal lymphoma is b-cell and intermediate to high grade. 11 extension to the cns is a hodgkin's-like nodal lymphoma usually present without overt clinical signs. 65, 66 cats with nasal lymphoma are typically presented with nasal discharge (60% to 85%), sneezing (20% to 70%), upper respiratory noise (stridor, stertor, wheezing; 20% to 60%), facial deformity (0% to 20%), anorexia (10% to 60%), epiphora (10% to 30%), and occasionally increased respiratory effort and coughing. 68, 69, 73 the nasal discharge is usually mucopurulent, although epistaxis is present in up to one-third of cases. regional lymphadenopathy can also occur. the median duration of clinical signs prior to diagnosis is 2 months (range of 1 to 1800 days). cats with renal lymphoma present with signs consistent with renal insufficiency: inappetence, weight loss, and polyuria/ polydipsia. 68, 74 on physical examination, renomegaly (usually bilateral, lumpy, and irregular) is palpated in the majority of cases (figure 32-14) . cats with cns lymphoma can present with constitutional signs (anorexia, lethargy) and signs referring to intracranial lesions, spinal lesions, or both. 68, [75] [76] [77] 91, 92 intracranial signs may include ataxia, altered consciousness, aggression, central blindness, and vestibular abnormalities. in a study of cats with seizures, of those diagnosed with intracranial lesions, 8% were due to lymphoma. 75 clinical signs referring to spinal cord involvement may include paresis or paraplegia (>80%; tetraparesis in 20%), ataxia, pain, and constipation, and nonspecific constitutional signs (e.g., anorexia, lethargy, weight loss) are also common. 77, 92 in cats with spinal cord involvement, neurologic examination may further reveal tetraparesis, lower or upper motor neuron bladder, tail flaccidity, and absent deep pain; approximately one-third of signs will be asymmetric and most refer to thoracolumbar involvement. the neurologic dysfunction may be insidious or progress rapidly. common low-grade small cell forms, weight loss (83% to 100%), vomiting and/or diarrhea (73% to 88%), and anorexia (66%) are the most common findings, and icterus is uncommon (7%). 50, 52, 87 abdominal palpation is abnormal in approximately 70% of cases, with half consisting of intestinal wall thickening and one-third having a palpable mass. clinical signs are usually present for several months (median: 6 months). 87 in contrast, although the lymphoblastic high-grade forms tend to cause similar clinical signs, they progress more rapidly with signs present for days or weeks and are more likely to present with a palpable abdominal mass originating from the gi tract, enlarged mesenteric lymph nodes, or liver. 31, 50, 88 icterus is also more common in large cell forms. hematochezia and tenesmus may also be present if the colon is involved. 33 rarely, cats may present with signs consistent with an acute abdomen due to intestinal obstruction or perforation and concurrent peritonitis. cats with intestinal lgl are presented with anorexia, weight loss, lethargy, and vomiting. 59, 60 a palpable abdominal mass is present in approximately half of lgl cases, and hepatomegaly, splenomegaly, and renomegaly are common. abdominal effusions, pleural effusions, and icterus are observed in less than 10% of cases. the clinical signs associated with the mediastinal form of lymphoma include dyspnea, tachypnea, and a noncompressible anterior mediastinum with dull heart and lung sounds. 89 rarely, a horner's syndrome and precaval syndrome may be observed. pleural effusion is common and characterized by serohemorrhagic to chylous effusion, and in most cases, neoplastic cells (lymphoblasts) are identified. 63, 90 cats with the nodal form of lymphoma present with variable clinical signs depending on the extent of disease; however, they are often depressed and lethargic. peripheral lymphadenopathy, as the only physical finding, is an uncommon presentation. cats with • figure 32-14 a , ventrodorsal projection of a cat with renal lymphoma. massive, bilateral renomegaly is observed. b, necropsy specimens of a cat with bilateral renal lymphoma illustrating the diffuse cortical nature of the disease that is most common. the reader is referred to chapter 8 for a general discussion of flow cytometric analysis and molecular diagnostic techniques, as well as the molecular diagnostic techniques section in section a of this chapter for specific applications to lymphoma. parr applications in cats have been described as being approximately 80% sensitive for the diagnosis of feline lymphoma 96 ; however, assessment of specificity has not been clearly established. clonality assessment tools (e.g., primers) for both ig and t-cell receptor variable region genes have been developed in cats. [97] [98] [99] [100] assessments of tumor proliferation rates (e.g., ki67, pcna, agnor), telomerase activity, and serum protein electrophoresis can also be performed on involved tissues in cats; however, consistent prognostic value across the anatomic, histopathologic, and immunophenotypic variants of lymphoma in cats is not well characterized. if these ancillary assays are helpful with respect to prognosis or diagnosis, they will be discussed in site-specific discussions to follow. thorough staging, including a bone marrow aspiration or biopsy, peripheral lymph node assessment (clinically normal or abnormal nodes), and thoracic and/or abdominal imaging, is indicated when (1) solitary site disease is suspected (in particular, extranodal sites) and a decision between locoregional therapy (i.e., surgery and/or rt) versus systemic therapy (i.e., chemotherapy) is being considered; (2) it provides prognostic information that will help a caregiver make treatment decisions; and (3) complete staging of the extent of disease is required as part of a clinical trial. bone marrow evaluation may be of particular interest if anemia, cellular atypia, and leukopenia are present. a who staging system exists for the cat that is similar to that used in the dog (see box 32-1); however, because of the high incidence of visceral/extranodal involvement in the feline species, a separate staging system has been evaluated and is often used (box 32-3). 101 because lymphoma in cats is more varied with respect to anatomic locations, staging systems are generally less helpful for predicting response. alimentary/gastrointestinal lymphoma the diagnosis of large cell, high-grade alimentary/gi lymphoma is generally less complicated than for the more common low-grade gi type. the former (including lgl) is often diagnosed with physical examination, abdominal imaging (e.g., ultrasound), and cytologic or histologic assessment of needle aspirate or needle biopsy samples from intestinal masses, enlarged mesenteric lymph nodes, or liver because mass lesions and gross lymphadenopathy are more commonly present. if obvious abdominal masses are present on physical examination, transabdominal needle aspiration may be possible without the aid of abdominal imaging. less commonly, abdominal exploration is necessary if lesions are more subtle or not amenable to transabdominal sampling. further staging via thoracic imaging, peripheral lymph node aspiration, and bone marrow assessment may be performed, but rarely contributes prognostic information or alters treatment decisions because the disease is already widespread and systemic therapy is required. in contrast, low-grade, small cell gi lymphoma is more commonly associated with modest (or palpably absent) intestinal thickening without mass effect and is clinically similar if not identical in presentation to benign inflammatory bowel disease (ibd). cytologic assessment alone is often not sufficient for diagnosis; in one study, eight of nine cases in which mesenteric lymph nodes were confirmed histologically as lymphoma, cytologic assessment incorrectly indicated benign lymphoid hyperplasia. 52 the key elements necessary for the diagnosis of low-grade, small cell gi lymphoma signs associated with laryngeal lymphoma in cats most commonly include dyspnea, dysphonia, stridor, gagging or retching, and rarely, coughing. 68, 79 cutaneous lymphoma may be solitary or diffuse with a varied presentation. 80, 83 in decreasing order of likelihood, lesions may include erythematous patches, alopecia, scaling, dermal nodules, or ulcerative plaques. nasal hypopigmentation, miliary dermatitis, and mucosal lesions are rarely observed. peripheral lymphadenopathy may also be present. in most cats, the duration of signs will be prolonged, lasting several months. cats with ocular lymphoma are presented with uveitis or iridial masses, as well as signs related to systemic involvement of disease. 68 all cats with lymphoma, regardless of site, may be presented with nonspecific constitutional signs that may include anorexia, weight loss, lethargy, or depression. secondary bone marrow infiltration may lead to anemia-at least 50% of affected cats have moderateto-severe nonregenerative anemia. signs related to paraneoplastic hypercalcemia (pu/pd) can occur in cats, however, much less commonly than in the dog. in one survey of hypercalcemia in cats, approximately 10% were diagnosed with lymphoma of various anatomic types. 93 for most cats with suspect lymphoma, the diagnostic evaluation should include a baseline assessment consisting of a cbc with differential cell count, platelet count, serum chemistry profile, urinalysis, and retroviral (felv/fiv) screen. serum chemistry profiles can help establish the overall health of the animal, as well as, in some cases, suggest site-specific tumor involvement; for example, increased activities of liver enzymes may indicate hepatic infiltration and increased blood urea nitrogen (bun) and creatinine may indicate renal lymphoma. for cats with alimentary lymphoma, hypoproteinemia and anemia are reported to occur in up to 23% and 76% of cases, respectively. 31, 52, 94 hypercalcemia is rarely seen in cats but has been reported in cats with lymphoma at various anatomic sites. hypoglycemia was reported in approximately one-third of cats with lymphoma in one australian study. 94 in a series of cats with various anatomic forms of lymphoma, serum albumin concentrations were significantly lower and β-globulin concentrations (as measured by protein electrophoresis) were significantly higher than a healthy control population. 95 the use of various imaging modalities in cats with lymphoma depends on the anatomic site and will be discussed in site-specific discussions to follow. cytopathologic or histopathologic evaluation of lymph node or involved organ tissue, procured via needle aspirate cytology (see chapter 7), surgical, endoscopic, or needle-core biopsy (see chapter 9) is required for a definitive diagnosis. fna cytology alone may not be sufficient in some cases, owing to difficulties encountered in distinguishing lymphoma from benign hyperplastic or reactive lymphoid conditions. in such cases, whole lymph node excision and/or involved organ biopsy is preferred because orientation and information regarding invasiveness and architectural abnormalities may be necessary for diagnosis. additionally, involved tissue, needle aspirate, and fluid samples can be further interrogated by various histochemical, immunohistochemical, flow cytometric analysis (e.g., size and immunophenotypic assessment), and molecular techniques (e.g., parr to assess clonality) to further characterize the disease process and refine the diagnosis in equivocal cases. the stomach, liver, spleen, colon, and pancreas, and occasionally, mild effusions are observed. as mentioned previously, although aspirate cytology may be sufficient for diagnosis of large cell intermediate-or high-grade alimentary lymphoma in cats, it is rarely diagnostic for low-grade, small cell intestinal lymphoma, and tissue procurement for histologic and ancillary assessment is required for diagnosis (and differentiation from ibd). the debate still rages as to whether endoscopically obtained tissue is sufficient for diagnosis or if fullthickness tissue procured during laparotomy or laparoscopy is necessary in light of similarities with ibd. 51, 54, 103, 104 as previously discussed, histologic features that help differentiate intestinal lymphoma from ibd include lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. 51, 54 although the presence of transmural involvement is highly suggestive of lymphoma, the lack of transmural infiltration is not pathognomonic for ibd; transmural infiltration is common with b-cell and large t-cell (including lgl) intestinal lymphoma but is observed in the minority of low-grade t-cell intestinal lymphomas that represent the largest group in cats (see table 32 -9). 51 for these reasons, if the differentiation of lymphoma and ibd is equivocal after standard histopathologic assessment, the addition of immunophenotypic and parr analysis in a stepwise fashion, as proposed by kiupel and others, 54 may be ultimately necessary for a definitive diagnosis. their study of 63 cats with either lymphoma or ibd found that, although standard histopathology was highly specific for diagnosis of lymphoma (99% specific, 72% sensitive), sensitivity was enhanced by the addition of immunophenotypic analysis (99% specific, 78% sensitive) and further enhanced by parr analysis (99% specific, 83% sensitive). for cats with mediastinal lymphoma, diagnostic suspicion may begin with a noncompressible cranial thorax on physical examination and confirmation of a mediastinal mass/pleural effusion on thoracic radiograph. fna of the mass or cytologic evaluation of pleural fluid may be sufficient to establish a diagnosis. in most cats, the finding of a monotonous population of intermediate-or high-grade cells will establish a diagnosis. however, definitive diagnosis of lymphoma in cats with a mediastinal mass and concurrent chylothorax can be challenging. ct appearance may be helpful but generally does not contribute to a definitive diagnosis. if lymphoblasts are not identified in the pleural chylous effusion, then cholesterol and triglyceride concentrations can be measured. 105 in chylous effusions, the pleural fluid triglyceride concentration will be greater than in the serum; however, anorectic cats will have lower triglyceride levels in the pleural fluid. a major differential for mediastinal lymphoma is thymoma. the cytologic features of thymoma can be distinct from lymphoma in many cases, but the diagnosis can be challenging because of a preponderance of small lymphocytes in thymoma. mast cells can also be seen in up to 50% of aspirations from thymomas. the addition of immunophenotypic and clonality assessment may be helpful in equivocal cases. if nasal lymphoma is suspected, advanced imaging (ct, mri), rhinoscopy, and biopsy are usually necessary for diagnosis (see chapter 23, section b). ct or mri is useful to determine the extent of involvement and to help plan biopsy procurement and rt if that treatment option is pursued. ct characteristics associated with sinonasal tumors in cats include the presence of a unilateral or bilateral nasal/sinus mass or fluid, bulla (and differentiation from ibd) include abdominal imaging (usually ultrasound), procurement of tissue for histopathology, and if necessary, assessment of immunophenotype and clonality. abdominal ultrasound will be abnormal in approximately 60% to 90% of cats with low-grade, small cell gi lymphoma. 31, 52, 102, 103 diffuse small intestinal wall thickening is the most common finding; 50% to 70% of cats with lymphoma will have ultrasonic evidence of wall thickening, which predominantly involves the muscularis propria, and submucosa, although mucosal thickening can also occur. focal mural masses are uncommon. mesenteric lymphadenopathy is also common and reported in 45% to 80% of affected cats. these ultrasonographic findings are by no means pathognomonic for lymphoma, however, because 10% to 50% and 15% to 20% of cats with ibd also have ultrasonographic evidence of intestinal wall thickening and lymphadenopathy, respectively. 102, 103 mucosal thickening is more common, and muscularis propria thickening is less common in ibd than lymphoma. less commonly, cats with low-grade intestinal lymphoma will have ultrasonographic abnormalities in other abdominal organs such as stage 1 • a single tumor (extranodal) with regional lymph node involvement • two or more nodal areas on the same side of the diaphragm • two single (extranodal) tumors with or without regional lymph node involvement on the same side of the diaphragm • a resectable primary gastrointestinal tract tumor, usually in the ileocecal area, with or without involvement of associated mesenteric nodes only • two single tumors (extranodal) on opposite sides of the diaphragm • two or more nodal areas above and below the diaphragm • all extensive primary unresectable intraabdominal disease • all paraspinal or epidural tumors, regardless of other tumor site or sites • stages 1-3 with liver and/or spleen involvement yield enough tissue for a cytologic diagnosis. ct or mr also reveals multifocal disease in the majority of cats with intracranial lymphoma. 75 ,76 csf analysis may be helpful but is rarely definitive for lymphoma. one of 11 cats with confirmed spinal lymphoma in one study 77 and 6 of 17 with confirmed intracranial lymphoma in another 76 had evidence of lymphoblasts in the cns, and an increased protein content was commonly found. in cats suspected of cns lymphoma, bone marrow and renal involvement are often present, and cytologic assessment of these or other more accessible organs is generally more easily attainable than from spinal sites. for cats suspected of cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative sites, while avoiding overtly infected skin lesions. immunophenotypic and parr analysis often are helpful in definitive diagnosis. complete staging to rule out systemic disease is also recommended for cats with cutaneous lymphoma because local therapies can be applied in cases of solitary disease. our knowledge base for treating cats with lymphoma is less well established, and outcomes are less predictable than that in dogs, primarily due to the greater variation in histologic type and anatomic location observed in the species. this is further complicated by the plethora of papers that "lump" very small numbers of cases representing multiple anatomic/immunophenotypic and histologic subtypes (e.g., small cell versus large cell variants) together when reporting survival analysis following chemotherapy. this provides only general observations rather than important specific outcome information (i.e., response rate and durability of response) that can vary significantly with respect to anatomic and histologic subtype. in general, canine lymphoma is most commonly intermediate-high grade and nodal, whereas cats more commonly present with gi or extranodal (±nodal extension), small cell, low-grade, and/or indolent forms. as will be discussed subsequently, the author bases most treatment decisions on assessment of whether the individual case represents a low-grade (e.g., indolent, small cell variants) versus an intermediate-or high-grade (e.g., large cell) lymphoma. finally, much of the early work on chemotherapy protocol development for cats with lymphoma occurred during the felv era, and care should be exercised when applying this information in the post-felv era. in general, cats tolerate chemotherapy for lymphoma quite well, most clients are happy with their choice to initiate treatment, and quality of life generally improves following commencement of therapy. 109, 110 the chemotherapeutic agents used most commonly to treat intermediate-or high-grade lymphoma in cats are similar to those used for dogs and humans with lymphoma (see section a in this chapter) and include doxorubicin, vincristine, cyclophosphamide, methotrexate, l-asparaginase, ccnu (lomustine), and prednisone. most combination induction protocols currently employed in cats are modifications of chop protocols initially designed for human oncologic use. 5,110-116 chop represents combinations of cyclophosphamide (c), doxorubicin (h, hydroxydaunorubicin), vincristine (o, oncovin) and prednisone (p). in general, chopbased protocols are appropriate for cats with large cell, intermediate-and high-grade lymphoma involving any anatomic site (e.g., peripheral nodal, mediastinal, and renal forms) but should not be first-line therapy for small cell, low-grade variants. as in the dog (see section a in this chapter), a plethora of modifications are used with chop-based protocols, although virtually no quality comparative data exist to compare outcomes, and as such, the protocol used should be based on cost, ease, client/veterinarian preference, and level of comfort. the current chop-based protocol in use by effusion, and lysis of associated bony structures. 106, 107 a biopsy can be procured either by intranasal procurement (with or without rhinoscopy) or by flushing one hemicavity with a bulb syringe and saline while occluding the contralateral cavity and collecting samples flushed out of the nasopharynx (figure 32-15 ). thorough staging (i.e., regional node assessment, thoracic and abdominal staging, and bone marrow assessment) to ensure the disease is confined to the nasal passages is recommended, if local rt without systemic chemotherapy is being considered. in the case of renal lymphoma, physical examination findings of massive and often bilateral renomegaly will raise the index of suspicion. radiographic appearance is smoothto-irregular renomegaly (see figure 32-14, a) . ultrasonographic imaging usually reveals bilateral (>80%), irregular renomegaly with hypoechoic subcapsular thickening. 108 approximately one-third of cases will have ultrasonographic evidence of other abdominal organ involvement. the disease is usually diffuse throughout the renal cortex (see figure 32-14, b) and transabdominal needle aspirate or core biopsy is diagnostic in most cases. in cats with suspected spinal lymphoma, survey radiographs of the spine will rarely reveal osseous lesions. myelograms, ct, or mri are indicated, and in approximately 75% of the cases, an extradural or intradural mass will be detected. 76, 77, 91, 92 most lesions occur at a thoracolumbar or lumbosacral location, and they are often found in more than one location. image-guided needle aspiration of epidural lesions may used in cats in europe, and one compilation reported similar results to chop. 64 a cop protocol commonly employed in cats is presented in table 32 -11. some studies with relatively few case entries have reported limited activity for doxorubicin as a single agent in cats with lymphoma 118, 119 ; however, larger studies using combination protocols have more consistently reported the addition of doxorubicin as necessary for the attainment of more durable responses. 5, 114 interestingly, in a report of 23 cats having relapsed following cop-based protocols (without doxorubicin), only 22% responded subsequently to doxorubicin-containing rescue therapy. 120 a small number of cats with lymphoma have been treated with single-agent oral ccnu (lomustine) at a dosage range of 30 to 60 mg/m 2 every 3 to 6 weeks. 121, 122 whereas activity was noted, only partial responses were reported. l-asparaginase, which is often included in protocols for lymphoma in cats, has a much shorter asparagine-depleting effect in cats (lost by 7 days) than in dogs and in one study in 13 cats with lymphoma resulted in only a 30% response rate. 123 in general, cats with intermediate-and high-grade lymphoma treated with chop-based or cop protocols do not enjoy the same level of success as dogs. bearing in mind that these reports group together a wide variety of subtypes having dissimilar prognoses (see subsequent site-specific treatment sections), the overall response rates tend to be in the 50% to 80% range with median remission and survival durations of 4 and 6 months, respectively. 5, 64, [110] [111] [112] [113] [114] [115] [116] 124 alimentary/gastrointestinal lymphoma representing the most common presentation for cats with lymphoma, the large majority have the small cell, mucosal, t-cell variant that carries a good prognosis, often with less aggressive chemotherapy protocols (e.g., oral chlorambucil and prednisone). 51, 52, 87, 125 chlorambucil (20 mg/m 2 po, every 2 weeks [preferred by the author] or 2 mg po every other day) and prednisone or prednisolone (initially 1 to 2 mg/kg po daily, reduced to 0.5 to 1.0 mg/kg every other day over several weeks) results in response rates (i.e., resolution of clinical signs) of greater than 90% and median survivals of approximately 2 years or longer. 52, 87, 125 cats who relapse with this protocol often will subsequently respond to alternative alkylators, such as cyclophosphamide or lomustine. 125 anecdotally, many will also respond the author is presented in table 32 -10. this protocol has been used in many cats with various forms of intermediate-and high-grade lymphoma and is generally well tolerated. at present, most canine lymphoma protocols (see section a in this chapter) discontinue chemotherapy by the 25th week, and we have sufficient data to show shorter, maintenance-free protocols are as good if not superior to longer maintenance protocols; however, similar comparative data do not exist in the cat. until such time as evidence to the contrary exists, the author presently recommends discontinuation of chemotherapy at week 25 in cats who have attained a complete remission. doxorubicin alone (25 mg/m 2 , every 3 weeks for 5 total treatments) or palliative prednisone therapy is offered if clients decline more aggressive chop-based therapy. cats are generally less tolerant of doxorubicin than are dogs; therefore a lower dosage (25 mg/m 2 iv or 1 mg/kg iv) is used (see chapter 11) . cardiac toxicity does not appear to be a clinically significant problem in cats, although renal toxicity is more commonly encountered in the species 117 and renal function should be monitored (i.e., serial bun, creatinine, and urine specific gravity) closely prior to and during therapy. the use of cop (i.e., chop without the addition of doxorubicin) is often • nasal cavity following thorough staging (node cytology, thoracic and abdominal imaging, bone marrow aspiration), then rt is the treatment of choice. crs in the order of 75% to 95% are reported, with reports of median survivals following rt of 1.5 to 3 years. 73, 129 cats that do not achieve a cr with rt have a median survival of approximately 4.5 months. total radiation dosage does affect survival durations, and a total dose greater than 32 gy is recommended. 73 the addition of chemotherapy has not been shown to enhance survival for cats with locally confined disease; combinations of rt and chemotherapy result in similar response rates and survival times. 73, 128, 129 chemotherapy (cop-or chop-based protocols) used in the absence of rt is a reasonable alternative, with complete response rates of approximately 75% and median survivals of approximately 2 years reported for cats achieving cr. 68 the author's preference is to initiate systemic chemotherapy only for (1) cases that have confirmed disease beyond the nasal passage, (2) cases that relapsed following rt, or (3) cases in which rt is unavailable or declined. ing treatment for cns lymphoma exist, and although an occasional case experienced durable response to systemic chemotherapy, generally less than 50% will respond and median survivals of 1 to 2 months can be expected. 68, 76, 77 laryngeal/tracheal lymphoma the vast majority of cats with laryngeal or tracheal lymphoma respond to either rt (if localized) or systemic chemotherapy (90% cr to cop-or chop-based protocols) (figure 32-16) . 68 whereas the authors experience is that most have durable responses and survival durations typically approach or exceed 1 year, the only case series (n = 8) reported a median survival of 5.5 months following achievement of a cr. ing the treatment of cutaneous lymphoma or mycosis fungoides in cats 80 ; however, a report of a cr to lomustine exists. 130 cats with a solitary disease could theoretically be treated with surgical excision to vinblastine chemotherapy if they no longer are responsive to alkylators. in contrast, cats with b-cell or large t-cell (including lgl) or small t-cell lymphoma that is transmural typically do not enjoy a durable response to therapy and survivals are much shorter. 31, 51, 59, 60 median survivals range from 45 to 100 days, even in cats treated with more aggressive cop-based protocols. in the author's experience, these variants are more likely to respond to chop-based protocols than chlorambucil/prednisone; however, durable responses occur only in a minority of cases. in particular, lgl appears to carry a grave prognosis 59, 60 ; in 2 compilations of 66 cats with lgl, median survivals of approximately 2 months were reported, including 23 cats receiving either cop or chop-based protocols, which resulted in only a 30% response rate. nutritional support is especially important for cats with gi lymphomas. it may be necessary to place a feeding tube in cats undergoing chemotherapy, particularly if prolonged anorexia is present (see chapter 15, section b). recently, two preliminary studies evaluated rt, either as rescue following recurrence or in addition to chemotherapy for the treatment of intestinal lymphoma in cats. 126, 127 eleven cats (6 small cell, 4 large cell, and 1 lgl) that progressed following chemotherapy received abdominal radiation (8 gy in 2 fractions over 2 days) and resulted in a median survival of 7 months, although numbers were small and 40% were lost to follow-up. 127 a second report of eight cats (seven with large cell lymphoma) underwent 6 weeks of chopbased combination chemotherapy, followed 2 weeks later by whole abdomen radiation consisting of 10 daily 1.5 gy fractions. 126 although three cats died within 3 weeks of rt, five enjoyed durable remissions. these preliminary promising outcomes warrant further investigation. felv-positive cats is generally associated with a poor prognosis, and survival times of approximately 2 to 3 months are expected following chop-or cop-based protocols . 5, 116 in contrast, young felv-negative siamese cats with mediastinal lymphoma experience remission rates approaching 90%, and responses tend to be more durable (median ≈9 months). 64 the treatment choice for peripheral nodal lymphoma in cats depends on whether the individual case represents a low-grade (e.g., indolent, small cell variants) versus an intermediate-or high-grade (e.g., large cell) lymphoma; the latter are best treated with chop-or cop-based protocols and carry a less favorable prognosis, whereas the former generally respond to less aggressive chlorambucil/corticosteroid protocols and enjoy durable responses. less is known regarding the treatment of hodgkin's-like lymphoma involving solitary or regional nodes of the head and neck. 65, 66 clinical outcome following surgical extirpation of the affected node (or nodes if a reasonable number) is often associated with long-term, disease-free intervals and survivals of approximately 1 year, suggesting it is a more indolent form of lymphoma. eventual recurrence in distal nodes following surgical excision is common, and the author currently offers clients the option of adjuvant chlorambucil/corticosteroids following surgery-this theoretically may have benefit; however, insufficient data exist to document a survival advantage with this approach. the use of chemotherapy to treat all has been disappointing. using cop-based protocols, cotter 124 reported a 27% cr rate. cll can be treated with chlorambucil (0.2 mg/kg po or 2 mg/cat qod) and prednisone (1 mg/kg po daily); however, little information exists regarding outcome. as in humans and dogs, if significant clinical signs or profound cytopenias are not present, treatment can be withheld-one cat with cll remained stable without chemotherapy for over a year. 140 the prognoses for acute nonlymphoblastic leukemias are generally very poor, although some exceptions exist in case report form in the historic literature. or rt, although clinical staging is necessary to rule out possible further systemic involvement. for multiple sites, combination chemotherapy may be considered. as previously discussed, the prediction of outcomes in cats with lymphoma is not generalizable due to the wide spectrum of histologic and anatomic subtypes encountered. much has been mentioned in the previous treatment sections, and tables 32-8 and table 32 -9 summarize prognostic parameters for lymphoma in cats. for a complete discussion of leukemias and mpds, including a general discussion of hematopoiesis, etiologies, lineage classification and descriptions, see section c of this chapter. the classification of leukemias in cats is difficult because of the similarity of clinical and pathologic features and the transition, overlap, or mixture of cell types involved. [131] [132] [133] [134] [135] most case-series reports are from the felv era and generally only single case reports exist from the more contemporary post-felv era, which further confuses our understanding of the biology and outcome. for this reason, only a simplistic discussion, primarily relating to the lymphoid leukemias will be presented here and the interested reader is again referred to section c for a general discussion of nonlymphoid leukemia. for cats with suspected leukemia, peripheral blood assessment (e.g., cbc with differential, flow cytometric analysis for size and immunophenotype, and parr [for lymphoid leukemias]), and bone marrow aspiration or biopsy may contribute to a diagnosis. the preferred sites for bone marrow aspiration are the proximal humerus or iliac crest. cats with acute leukemia are likely to have malignant cellular infiltrates in organs other than bone marrow. 134 a bone marrow aspirate with greater than 30% abnormal blast cells is sufficient to make a diagnosis of an acute leukemia. in cats with suspected cll, infiltration of the bone marrow with more than 20% mature lymphocytes helps confirm the diagnosis. all cats with leukemia should be tested for felv/fiv. determining the lineage of some leukemias can be challenging; most can be distinguished from one another by histologic appearance, histochemical stains, or immunohistochemical or flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage (see chapter 8 and section c in this chapter). 131, 133, 136 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. the french-american-british (fab) classification system is considered useful in cats with myelodysplastic syndromes and almost all will be felv antigenemic. 136, 137 lymphoid leukemia all was the most commonly encountered type of leukemia in cats in the felv era; however, it is much less common today. all is characterized by poorly differentiated lymphoblasts and prolymphocytes in blood and bone marrow. approximately 60% to 80% of cats with all were felv positive, and most malignant cells have t-cell immunophenotypes 138 ; however, little information is available in the contemporary literature. cll is rarely reported in cats and is characterized by welldifferentiated, small, mature lymphocytes in peripheral blood and 1 aberrant proliferation of cells with defective maturation and function leads to reduction of normal hematopoiesis and invasion of other tissues. these disorders have been classified based on biologic behavior, degree of cellular differentiation, and lineage of the neoplastic cells (granulocytic, monocytic, erythroid, megakaryocytic, or mixed). newer classification systems in humans have incorporated genetics and molecular genetic analysis; these are currently areas of active investigation in the study of animal leukemias. 2 in 1991 the animal leukemia study group made recommendations for classifying nonlymphoid leukemias in dogs and cats. 3 more recently, the oncology committee of the american college of veterinary pathologists (acvp) has been reexamining criteria for a classification system and spearheading large multiinstitutional studies to validate the criteria. long-term produced experimentally following irradiation. [22] [23] [24] in contrast to mpds in cats, no causative viral agent has been demonstrated in dogs, although retrovirus-like budding particles were observed in the neoplastic cells of a dog with granulocytic leukemia. 25 pathology and natural behavior a review of normal hematopoiesis will aid in understanding the various manifestations of mpds. hematopoiesis is the process of proliferation, differentiation, and maturation of stem cells into terminally differentiated blood cells. a simplified scheme is presented in figure 32 -17. pluripotent stem cells differentiate into either lymphopoietic or hematopoietic multipotent stem cells. 26 under the influence of specific regulatory and microenvironmental factors, multipotent stem cells in bone marrow differentiate into progenitor cells committed to a specific hematopoietic cell line, for example, erythroid, granulocytic-monocytic, or megakaryocytic. maturation results in the production of terminally differentiated blood cellserythrocytes, granulocytes, monocytes, and platelets-that are delivered to the circulation. in some cases, as in the maturation of reticulocytes to erythrocytes, final development may occur in the spleen. proliferation and differentiation of hematopoietic cells are controlled by a group of regulatory growth factors. 26, 27 of these, objectives of these studies are to define molecular lesions, establish prognostic markers, and target effective therapeutic approaches. 4 myeloid neoplasms are uncommon or rare in the dog and occur 10 times less frequently than lymphoproliferative disorders. 5 accurate information about incidence and other epidemiologic information await consistent use of a uniform classification system (see later discussion). there is no known age, breed, or sex predisposition, although in some retrospective studies, large-breed dogs have been overrepresented. [6] [7] [8] [9] [10] [11] [12] [13] [14] in dogs, the etiology of spontaneously occurring leukemia is unknown. it is likely that genetic and environmental factors (including exposure to radiation, drugs, or toxic chemicals) play a role. in humans, acquired chromosomal derangements lead to clonal overgrowth with arrested development. 15 at the end of the last century, chromosomal abnormalities were reported in dogs with aml, chronic myelogenous leukemia (cml), and lymphoid leukemia. 16, 17 however, because karyotyping is difficult to perform in dogs because of the large number and morphologic similarity of their chromosomes and their resistance to banding, defining genetic factors in canine myeloid neoplasms has awaited application of molecular technologies and use of the canine genome map. erythropoietin is the best characterized; it regulates erythroid proliferation and differentiation and is produced in the kidney, where changes in oxygen tension are detected. the myeloid compartment depends on a group of factors, collectively referred to as colonystimulating factors (csfs). these factors act at the level of the committed progenitor cells but also influence the functional capabilities of mature cells. some of these factors have a broad spectrum of activity; others are more restricted in their target cells and actions. csfs are produced in vitro by a multitude of cell types, including monocytes, macrophages, lymphocytes, and endothelial cells, and these cells likely play a role in the production and regulation of these factors in vivo. the gene for thrombopoietin also has been cloned, and it appears that this hormone alone can induce differentiation of megakaryocytes and platelet production. 28 recombinant forms of many of these hormones are increasingly available. clonal disorders of bone marrow include myeloaplasia (usually referred to as aplastic anemia), myelodysplasia, and myeloproliferation. a preleukemic syndrome, characterized by peripheral pancytopenia and bone marrow hyperplasia with maturation arrest, is more correctly termed myelodysplasia because the syndrome does not always progress to overt leukemia. this syndrome has been described in cats, usually in association with felv infection but has only rarely been recognized in dogs. [29] [30] [31] [32] these clonal disorders may be manifested by abnormalities in any or all lineages because hematopoietic cells share a common stem cell. in addition, transformation from one form to another may occur. 33 myeloid neoplasms are classified in several ways. the terms acute and chronic refer to the degree of cellular differentiation of the leukemic cells, but these terms also correlate with the biologic behavior of the neoplasm. 34 disorders resulting from uncontrolled proliferation or decreased apoptosis of cells incapable of maturation lead to the accumulation of poorly differentiated or "blast" cells. these disorders are included under the umbrella term, acute myeloid leukemia (aml). disorders resulting from unregulated proliferation of cells that exhibit progressive, albeit incomplete and defective, maturation lead to the accumulation of differentiated cells. these disorders are termed myeloproliferative neoplasms (mpn) and include polycythemia vera, cml and its variants, essential thrombocythemia, and possibly primary myelofibrosis. myeloid neoplasms are further classified by the lineage of the dominant cell type(s), defined by romanowsky stains, special cytochemical stains, ultrastructural features, flow cytometric analysis, and immunologic cell markers, and they have been classified into subtypes (see later discussion). aml has a more sudden onset and is more aggressive. in both acute and chronic disorders, however, abnormalities in proliferation, maturation, and functional characteristics can occur in any hematopoietic cell line. 1 in addition, normal hematopoiesis is adversely affected. animals with leukemia usually have decreased numbers of circulating normal cells. the pathogenesis of the cytopenias is complex and may result in part from production of inhibitory factors. eventually, neoplastic cells displace normal hematopoietic cells, and this is termed myelophthisis. anemia and thrombocytopenia are particularly common. neutropenia and thrombocytopenia result in infection and hemorrhage, which may be more deleterious to the animal than the primary disease process. aml is rare and is characterized by aberrant proliferation and/or decreased apoptosis of a clone of cells without maturation. this results in accumulation of immature blast cells in bone marrow and peripheral blood (figure 32-18, a to e) . the wbc count is variable and ranges from leukopenia to counts up to 150,000/µl. spleen, liver, and lymph nodes are frequently involved, and other tissues, including tonsils, kidney, heart, and the cns, may be infiltrated as well. there is no characteristic age, and even very young dogs may be affected. 35 the clinical course of these disorders tends to be rapid. production of normal peripheral blood cells is usually diminished or absent, and anemia, neutropenia, and thrombocytopenia are common with infection and hemorrhage occurring as frequent sequelae. occasionally, neoplastic blasts are present in bone marrow but not in peripheral blood. this is termed aleukemic leukemia, whereas subleukemic suggests a normal or decreased wbc count with some neoplastic cells in circulation. in 1985 the animal leukemia study group was formed under the auspices of the american society for veterinary clinical pathology to develop specific morphologic and cytochemical criteria for classifying acute nonlymphocytic leukemias. recognition of specific subtypes of leukemia is required to compile accurate and useful information about prognosis and response to treatment, as well as to compare studies from different sites. in 1991, this group proposed a classification system following adaptation of the french-american-british (fab) system and criteria established by the nci workshop. 3 group members examined blood and bone marrow from 49 dogs and cats with myeloid neoplasms. romanowsky-stained specimens were examined first to identify blast cells and their percentages. lineage specificity was then determined using cytochemical markers. the percentage of blasts and the information about lineage specificity were used in combination to classify disorders as acute undifferentiated leukemia (aul), acute myeloid leukemia (aml, subtypes m1 to m5 and m7), and erythroleukemia with or without erythroid predominance (m6 and m6er). a description of these subtypes is presented in table 32-12. canine karyotyping is difficult, but with advancements in molecular cytogenetic analysis, chromosome painting, and genomic hybridization, aml in dogs can now be analyzed at the base-pair level, 18, 19 and missense mutations in flt3, c-kit, and ras sequences have been identified in dogs with aml, similar to what has been found for human aml. 36 in addition to serving as diagnostic and prognostic markers, cytogenetic lesions may be therapeutic targets. as cytogenetic abnormalities continue to be identified, this information will need to be incorporated into classification schemes. with the exception of acute promyelocytic leukemia or m3, all of these subtypes have been described in dogs. however, because this modified fab system has been adopted only recently, the names given to these disorders in the literature vary considerably. in addition, in the absence of cytochemical staining, immunophenotyping, or electron microscopic evaluation, the specific subtype of leukemia has often been uncertain, making retrospective analysis of epidemiologic information, prognosis, and response to therapy confusing at best. although defining specific subtypes may seem to be an academic exercise owing to the uniformly poor prognosis of acute leukemias, this information is critical to improving the management of these diseases. because of the low incidence of aml, national and international cooperative efforts will be required to accumulate information on the pathogenesis and response to different treatment modalities of specific subtypes. utilization of a uniform classification system is an essential first step. different forms of aml are demonstrated in figure 32 polycythemia vera (pv) is a clonal disorder of stem cells, although whether the defect is in the pluripotent stem cell or the hematopoietic multipotent stem cell is still not clear. in humans, progenitor cells have an increased sensitivity to insulin-like growth factor 1, which stimulates hematopoiesis. 64 it is not known whether this hypersensitivity is the primary defect or is secondary to another gene mutation. in any case, the result is overproduction of red blood cells (rbcs). the disease is rare and must be distinguished from more common causes of polycythemia, including relative and secondary absolute polycythemia (see later discussion). in pv, there is neoplastic proliferation of the erythroid series with terminal differentiation to rbcs. the disease has been reported in dogs that tend to be middle-aged with no breed or sex predilection [65] [66] [67] [68] [69] [70] [71] [72] [73] and is characterized by an increased rbc mass evidenced by an increased packed cell volume (pcv), rbc count, and hemoglobin concentration. the pcv is typically in the range of 65% to 85%. the bone marrow is hyperplastic, although the myeloid : erythroid (m : e) ratio tends to be normal. in contrast to the disease in humans, other cell lines do not appear to be involved, and transformation to other mpns has not been reported. the disease in dogs may be more appropriately termed primary erythrocytosis. in humans, acquired jak2 gene mutations are identified in 90% of patients with primary polycythemia, and recently an identical mutation in the jak2 gene of one of five dogs with primary polycythemia was reported. 74 in dogs, cml is more similar to chronic neutrophilic leukemia, a rare form of mpn in humans, than to cml in humans because it is a neoplastic proliferation of the neutrophil series, although concurrent eosinophilic and basophilic differentiation can occur. cml can occur in dogs of any age. 35, [75] [76] [77] [78] [79] neutrophils and neutrophilic precursors accumulate in bone marrow and peripheral blood as well as in other organs. the peripheral wbc count is usually, but not always, greater than 100,000/µl. both immature and mature neutrophils are present, as demonstrated in figure 32 -18, f. mature forms are usually more numerous, but sometimes an "uneven" left shift is present. signs of dysplasia may be evident, including hypersegmentation, ringed nuclei, and giant forms. eosinophils and basophils may also be increased. the bone marrow is characterized by granulocytic hyperplasia, and morphologic abnormalities may not be present. erythroid and megakaryocytic lines may be affected, resulting in anemia, thrombocytopenia, or less commonly, thrombocytosis. this disorder must be distinguished from severe neutrophilic leukocytosis and "leukemoid reactions" caused by inflammation or immune-mediated diseases. leukemoid reactions can also occur as a paraneoplastic syndrome. in humans with cml, characteristic cytogenetic abnormalities are present in all bone marrow cells, signifying a lesion at the level of an early multipotent stem cell. typically, these individuals have a chromosomal translocation, resulting in the philadelphia chromosome or bcr-abl translocation between chromosomes 9 and 22. 80 the analogous chromosomes in dogs are chromosomes 9 and 26, and bcr-abl mutations have now been reported in three cases of cml in dogs. 2 variants of cml are chronic myelomonocytic leukemia (cmml) and chronic monocytic leukemia (cmol). [81] [82] [83] these diagnoses are made based on the percentage of monocytes in the leukemic cell leukemia (m4).* megakaryoblastic leukemia (m7) also is well recognized in dogs 10,47-58 and may be associated with platelet dysfunction. 51 monocytic leukemias have likely included those with and without monocytic differentiation (m5a and m5b), 11, 59 but in some cases the diagnosis may have been chronic myelomonocytic or chronic monocytic leukemia (see later discussion). there are few reports in dogs of spontaneously occurring erythroleukemia (m6) in which the leukemic cells include myeloblasts, monoblasts, and erythroid elements. [60] [61] [62] auls have uncertain lineages because they are negative for all cytochemical markers. these leukemias should be distinguished from lymphoid leukemias by flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 63 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. mpns, previously termed chronic myeloproliferative disorders, are characterized by excessive production of differentiated bone marrow cells, resulting in the accumulation of erythrocytes (polycythemia vera), granulocytes and/or monocytes (cml and its variants), or platelets (essential thrombocythemia). primary myelofibrosis as a clonal disorder of marrow stromal cells, characterized by proliferation of megakaryocytes and granulocytic precursors with accumulation of collagen in bone marrow, has been *references 5-9, 23-34, 37-46. • myelodysplastic syndrome with erythroid predominance out this disorder. however, spurious microcytosis may be reported if a dog has many giant platelets that are counted by an analyzer as small rbcs. 93 microscopic review of the blood film may be helpful in these cases. myelofibrosis primary myelofibrosis has been reported only rarely in dogs and is usually a secondary, or reactive, process. 94, 95 in humans, myelofibrosis is characterized by collagen deposition in bone marrow and increased numbers of megakaryocytes and granulocytic precursors, many of which exhibit morphologic abnormalities. in fact, breakdown of intramedullary megakaryocytes and subsequent release of factors that promote fibroblast proliferation or inhibit collagen breakdown may be the underlying pathogenesis of the fibrosis. 96 focal osteosclerosis is sometimes present. anemia, thrombocytopenia, splenomegaly, and myeloid metaplasia (production of hematopoietic cells outside the bone marrow) are consistent features. in dogs, myelofibrosis occurs secondary to mpds, radiation damage, and congenital hemolytic anemias. [97] [98] [99] [100] in some cases, the inciting cause is unknown (idiopathic myelofibrosis). there may be concurrent marrow necrosis in cases of ehrlichiosis, septicemia, or drug toxicity (estrogens, cephalosporins), and there is speculation that fibroblasts proliferate in response to release of inflammatory mediators associated with the necrosis. 94 myeloid metaplasia has been reported to occur in the liver, spleen, and lung. 100 extramedullary hematopoiesis is ineffective in preventing or correcting the pancytopenia that eventually develops. dysfunction of the hematopoietic system can be manifested by a variety of abnormalities that constitute myelodysplastic syndrome (mds). in dogs, in which the syndrome is rare, there usually are cytopenias in two or three lines in the peripheral blood (anemia, neutropenia, and/or thrombocytopenia). other blood abnormalities can include macrocytic erythrocytes and metarubricytosis. the bone marrow is typically normocellular or hypercellular, and dysplastic changes are evident in several cell lines. if blast cells are present, they make up less than 30% of all nucleated cells, 2 although this threshold is being changed to less than 20%. 4, 20 myelodysplasia is sometimes referred to as preleukemia because, in some cases, it may progress to acute leukemia. [29] [30] [31] based on reported cases, poor prognostic indices include increased percentage of blast cells, cytopenias involving more than one lineage, and cellular atypia. primary mdss are clonal disorders and are considered neoplastic. complex classification schemes for human mds, based on percentages of blasts in bone marrow, cytogenetic analysis, cytopenias, need for transfusions, and other variables, comprise at least nine subtypes; their applicability to veterinary medicine is unknown. 5 three subtypes are proposed for dogs and cats and include mds with excessive blasts (mds-eb), in which blast percentages are greater than 5% and less than 20%, and progression to aml may occur; mds with refractory cytopenia (mds-rc) with blast percentages less than 5% and cytopenias in one or more lineages; and mds with erythroid predominance (mds-er) in which the m : e ratio is less than 1 and prognosis is poor. 4 larger studies are needed to determine the utility of this classification scheme and other potential prognostic indices, such as sex, age, and felv positivity. in addition to accumulating enough cases, another confounding factor to studying and classifying mds is the presence of reversible mdss population. bcr-abl translocation has also been reported in a dog with cmol. 45 in addition to accumulating in bone marrow and peripheral blood, leukemic cells also are found in the red pulp of the spleen, the periportal and sinusoidal areas of the liver, and sometimes lymph nodes. other organs such as the kidney, heart, and lung are less commonly affected. in addition, extramedullary hematopoiesis may be present in the liver and spleen. death is usually due to complications of infection or hemorrhage secondary to neutrophil dysfunction and thrombocytopenia. in some cases, cml may terminate in "blast crisis, " in which there is a transformation from a predominance of well-differentiated granulocytes to excessive numbers of poorly differentiated blast cells in peripheral blood and bone marrow. this phenomenon is well documented in the dog. 75, 76, 78 basophilic and eosinophilic leukemia basophilic leukemia, although rare, has been reported in dogs and is characterized by an increased wbc count with a high proportion of basophils in peripheral blood and bone marrow. [84] [85] [86] hepatosplenomegaly, lymphadenopathy, and thrombocytosis may be present. all the dogs have been anemic. basophilic leukemia should be distinguished from mast cell leukemia (mastocytosis). whether dogs develop eosinophilic leukemia remains in question. reported cases have had high blood eosinophil counts and eosinophilic infiltrates in organs. 87, 88 one dog responded well to treatment with corticosteroids. the distinction between neoplastic proliferation of eosinophils and idiopathic hypereosinophilic syndrome remains elusive. disorders associated with eosinophilia such as parasitism, skin diseases, or diseases of the respiratory and gi tracts should be considered first in an animal with eosinophilia. one distinguishing feature should be clonality, with reactive eosinophilia comprising polyclonal cells and the neoplastic condition arising from a single clone. as clonality assays become more available, this discrepancy may be resolved. in humans, essential thrombocythemia, or primary thrombocytosis, is characterized by platelet counts that are persistently greater than 600,000/µl. there are no blast cells in circulation, and marked megakaryocytic hyperplasia of the bone marrow without myelofibrosis is present. thrombosis and bleeding are the most common sequelae, and most patients have splenomegaly. other mpds, especially pv, should be ruled out, and importantly, there should be no primary disorders associated with reactive thrombocytosis. 89 these include inflammation, hemolytic anemia, iron deficiency anemia, malignancies, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and splenectomy. in addition, certain drugs such as vincristine can induce thrombocytosis. essential thrombocythemia has been recognized in dogs. 33, [90] [91] [92] [93] in one dog, the platelet count exceeded 4 million/µl and bizarre giant forms with abnormal granulation were present. the bone marrow contained increased numbers of megakaryocytes and megakaryoblasts, but circulating blast cells were not seen. other findings included splenomegaly, gi bleeding, and increased numbers of circulating basophils. causes of secondary or reactive thrombocytosis were ruled out. 90 basophilia was also reported in a more recent case. 92 in another dog, primary thrombocytosis was diagnosed and then progressed to cml. 33 in some cases reported in the literature as essential thrombocythemia, the dogs had microcytic hypochromic anemias. because iron deficiency anemia is associated with reactive or secondary thrombocytosis, care must be taken to rule or thrombocytopenia are usually present. occasionally, neoplastic cells can be found in cerebrospinal fluid in animals with invasion of the cns. smears of aspirates from tissues such as the lymph nodes, spleen, or liver may contain blasts but usually contribute little to the diagnostic work-up. examination of blasts stained with standard romanowsky stains may give clues as to the lineage of the cells (figure 32-18 , a to c and e). in myelomonocytic leukemia, the nuclei of the blasts are usually pleomorphic, with round to lobulated forms. in some cells, the cytoplasm may contain large azurophilic granules or vacuoles. blasts in megakaryocytic leukemia may contain vacuoles and have cytoplasmic blebs. in addition, bizarre macroplatelets may be present. although these distinguishing morphologic features may suggest a definitive diagnosis, cytochemical staining or immunophenotyping are usually required to define the lineage of the blasts. several investigators have reported modification of diagnoses following cytochemical staining. 102, 103 it is especially important to distinguish aml from lymphocytic leukemia in order to provide accurate prognostic information to the owner and institute appropriate therapy. the animal leukemia group has recommended the following diagnostic criteria, summarized in figure 32 -19. 3 using wellprepared romanowsky-stained blood and bone marrow films, a minimum of 200 cells are counted to determine the leukocyte differential in blood and the percentage of blast cells in bone marrow and/or blood. in bone marrow, blast cells are calculated both as a percentage of all nucleated cells (anc) and nonerythroid cells (nec) and are further characterized using cytochemical markers. [102] [103] [104] neutrophil differentiation is identified by positive staining of blasts for peroxidase, sudan black b, and chloracetate esterase. nonspecific esterases (alpha-naphthyl acetate esterase or alpha-naphthyl butyrate esterase), especially if they are inhibited by sodium fluoride, mark monocytes. canine monocytes may also contain a few peroxidase-positive granules. acetylcholinesterase is a marker for megakaryocytes in dogs and cats. in addition, positive immunostaining for von willebrand's factor (factor viii-related antigen) and platelet glycoproteins on the surface of blasts identifies them as megakaryocyte precursors. 10, [49] [50] [51] [52] [53] alkaline phosphatase (ap) only rarely marks normal cells in dogs and cats but is present in blasts cells in acute myeloblastic and myelomonocytic leukemias. however, owing to reports of ap activity in lymphoid leukemias in dogs, its specificity as a marker for myeloid cells is not certain. omega exonuclease is a specific marker for basophils, which are also positive for chloracetate esterase activity. 86 blood and bone marrow differential counts and cytochemical staining should be performed and interpreted by experienced veterinary cytopathologists. if erythroid cells are less than 50% of anc and the blast cells are greater than 30%, a diagnosis of aml or aul is made. if erythroid cells are greater than 50% of anc and the blast cells are greater than 30%, a diagnosis of erythroleukemia (m6) is made. if rubriblasts are a significant proportion of the blast cells, a diagnosis of m6er, or erythroleukemia with erythroid predominance, can be made. it should be noted that in the human aml classification system, the blast threshold has been lowered to 20% and similar recommendations are being made for aml in dogs and cats. in some cases, electron microscopy is required to identify the lineage of the blast cells. for example, megakaryocyte precursors are positive for platelet peroxidase activity and contain demarcation membranes and alpha granules. 49, 53 both of these features are detected at the ultrastructural level. immunophenotyping, used to identify cell lineages in human patients, awaits development of that occur secondary to immune-mediated, infectious, and other diseases in both dogs and cats. dogs with myeloid neoplasms have similar presentations regardless of the specific disease entity, although animals with aml have a more acute onset of illness and a more rapid clinical course. a history of lethargy, inappetence, and weight loss is common. clinical signs include emaciation, persistent fever, pallor, petechiation, hepatosplenomegaly, and, less commonly, lymphadenopathy and enlarged tonsils. shifting leg lameness, ocular lesions, and recurrent infections are also seen. vomiting, diarrhea, dyspnea, and neurologic signs are variable features. serum biochemical analytes may be within the reference intervals but can change if significant organ infiltration occurs. animals with mds may be lethargic and anorectic and have pallor, fever, and hepatosplenomegaly. in pv, dogs often have erythema of mucous membranes owing to the increase in rbc mass. some dogs are polydipsic. in addition, neurologic signs such as disorientation, ataxia, or seizures may be present and are thought to be the result of hyperviscosity or hypervolemia. 69 hepatosplenomegaly is usually absent. peripheral blood abnormalities are consistently found. in addition to the presence of neoplastic cells, other abnormalities, including cytopenias of any lineage, may be present. low numbers of nucleated rbcs are present in the blood of about half the dogs with acute nonlymphocytic leukemia. 3 nonregenerative anemia and thrombocytopenia are present in most cases. anemia is usually normocytic and normochromic, although macrocytic anemia is sometimes present. pathogenic mechanisms include effects of inhibitory factors leading to ineffective hematopoiesis, myelophthisis, immune-mediated anemia secondary to neoplasia, and hemorrhage secondary to thrombocytopenia, platelet dysfunction, or dic. anemia is most severe in aml, although both anemia and thrombocytopenia may be milder in animals with the m5 subtype (acute monocytic leukemia). in myelofibrosis, the anemia is characterized by anisocytosis and poikilocytosis. in addition, pancytopenia and leukoerythroblastosis, in which immature erythroid and myeloid cells are in circulation, may be present. these phenomena probably result from replacement of marrow by fibrous tissue with resultant shearing of red cells and escape of immature cells normally confined to bone marrow. in pv, the pcv is increased, usually in the range of 65% to 85%. the bone marrow is hyperplastic, and the m : e ratio is usually in the normal range. neoplastic cells are often defective functionally. platelet dysfunction has been reported in a dog with acute megakaryoblastic leukemia (m7), 51 and in cml, neutrophils have decreased phagocytic capacity and other abnormalities. one exception to this was a report of cml in a dog in which the neutrophils had enhanced phagocytic capacity and superoxide production. 101 the authors hypothesized that increased synthesis of gm-csf resulted from a lactoferrin deficiency in the neoplastic neutrophils and mediated the enhanced function of these cells. in all cases of myeloid neoplasms, diagnosis depends on examination of peripheral blood and bone marrow. aml is diagnosed on the basis of finding blast cells with clearly visible nucleoli in blood and bone marrow. most dogs with acute leukemia have circulating blasts. these cells may be present in low numbers in peripheral blood, and a careful search of the smear, especially at the feathered edge, should be made. even if blasts are not detected in circulation, indications of bone marrow disease such as nonregenerative anemia increases in these cell types. in order to make a diagnosis of pv, it must first be established that the polycythemia is absolute rather than relative. in relative polycythemias, plasma volume is decreased from hemoconcentration, dehydration, or hypovolemia, and the absolute rbc mass is not increased. splenic contraction can also result in relative polycythemia. absolute polycythemia, in which rbc mass is increased, is usually secondary to tissue hypoxia, causing appropriate increased production of erythropoietin. rarely, erythropoietin may be produced inappropriately by a tumor (e.g., renal cell carcinoma) or in renal disease (pyelonephritis) or localized renal hypoxia. [109] [110] [111] these causes of polycythemia should be eliminated by appropriate laboratory work, thoracic radiographs, arterial blood gas analysis, and renal ultrasonography. in humans with pv, plasma erythropoietin (epo) levels are low. epo levels in dogs with pv tend to be low or low-normal, whereas in animals with secondary absolute polycythemia, the levels are high. 112, 113 samples for determination of epo concentrations should be taken prior to therapeutic phlebotomy used to treat hyperviscosity and, owing to fluctuations in epo levels, should be repeated if results are incongruous with other information. there are no pathognomonic features of cml in dogs, and other common causes for marked leukocytosis with a left shift ("leukemoid reaction") and granulocytic hyperplasia of bone marrow must be eliminated. these include infections, especially pyogenic ones; immune-mediated diseases; and other malignant neoplasms. in cml, maturation sometimes appears disorderly, and there may be variation in the size and shape of neutrophils at the same level of maturation. in addition, neoplastic leukocytes may disintegrate more rapidly and appear vacuolated. 35 because of the invasive nature of cml, biopsy of liver or spleen may also help to distinguish true leukemia from a leukemoid reaction, assuming the animal can tolerate the procedure. if characteristic cytogenetic abnormalities can be found in dogs with cml, this analysis may be helpful. basophilic leukemia is diagnosed by finding excessive numbers of basophils in circulation and in bone marrow. basophilic leukemia must be differentiated from mastocytosis based on the morphology of the cell type present. basophils have a segmented nucleus and variably sized granules, whereas mast cells have a round-to-oval nucleus that may be partially or totally obscured by small, round, metachromatic-staining granules. this distinction is usually easy to make; however, in basophilic leukemia, changes in the morphology of the nucleus and granules make the distinction less clear. 85 essential thrombocythemia has been diagnosed based on finding persistent and excessive thrombocytosis (>600,000/µl) without circulating blast cells and in the absence of another mpd (e.g., pv), myelofibrosis, or disorders known to cause secondary thrombocytosis. 89 these include iron deficiency anemia, chronic inflammatory diseases, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and absence of a spleen. thrombocytosis is transient in these disorders or abates with resolution of the primary disease. in essential thrombocythemia, platelet morphology may be abnormal, with bizarre giant forms and abnormal granulation. 90 in the bone marrow, megakaryocytic hyperplasia is a consistent feature, and dysplastic changes may be evident in megakaryocytes. 93 spurious hyperkalemia may be present in serum samples from dogs with thrombocytosis from any cause due to the release of potassium from platelets during clot formation. 114 measuring potassium in plasma is recommended in these cases and usually demonstrates a potassium concentration within reference interval. platelet aggregability has been appropriate markers for animal species (see later). increasingly, cytogenetic abnormalities are being identified in animal leukemias; cytogenetic analysis may yield important diagnostic and prognostic information and become a valuable tool for identifying targeted therapeutic approaches. although morphologic and cytochemical analyses have formed the mainstay of cell identification, newer technologies now are routinely used to classify leukemias by using monoclonal antibodies to detect antigens associated with certain cell types. cells can be immunophenotyped using flow cytometric analysis or immunocytochemistry. 20, 63, [105] [106] [107] [108] cells from both acute lymphoid leukemia and aml are positive for cd34. many lymphocyte markers, including cd3, cd4, cd8, cd18, cd21, cd45, cd79, and igg, are available for dogs and can be used to rule out lymphoblastic leukemia in dogs with acute leukemias. 63, 105 other markers include myeloperoxidase (mpo) and cd11b for myeloid cells and cd41 for megakaryoblasts. there is some overlap in expression of these cellular antigens. for example, canine (but not human) granulocytes express cd4. it is best to use a panel of antibodies (similar to using a battery of cytochemical stains) because antigens are often expressed on multiple lineages, and lineage infidelity can occur. these tests have become more valuable with the availability of canine reagents. currently, the acvp oncology committee recommends that the following immunophenotyping panel be done on bone marrow and/ or blood smears to characterize animal leukemias: for b lymphocytes, cd79a; for t lymphocytes, cd3; for myeloid cells, mpo and cd11b; for megakaryoblasts, cd41; for dendritic cells, cd1c; and for acute leukemias, cd34. 20 because of the degree of differentiation of cells in mpn, these disorders must be distinguished from nonneoplastic causes of lymphoma, could be used as maintenance therapy. 9, 118 another protocol that has been used in treating acute myeloblastic leukemia is presented in table 32 -13. regardless of the chemotherapy protocol used, significant bone marrow suppression will develop, and intensive supportive care will be necessary. transfusions of whole blood or platelet-rich plasma may be required to treat anemia and thrombocytopenia, and infection should be managed with aggressive antibiotic therapy. because of the generally poor response, the major thrust of therapy may be to provide palliative supportive care. in treating pv, therapy is directed at reducing rbc mass. the pcv should be reduced to 50% to 60% or by one-sixth of its starting value; phlebotomies should be performed as needed, administering appropriate colloid and crystalloid solutions to replace lost electrolytes; 20 ml of whole blood/kg of body weight can be removed at regular intervals. 67 in humans, phlebotomy continues to be the therapeutic approach used most frequently. radiophosphorus ( 32 p) has been shown to provide long-term control but can only be used in specialized centers. 121 the chemotherapeutic drug of choice is hydroxyurea, an inhibitor of dna synthesis. this drug should be administered at an initial dose of 30 mg/kg for 10 days and then reduced to 15 mg/kg po daily. 69 the major goal of treatment is to maintain the pcv as close to normal as possible. cml is best managed with chemotherapy to control the proliferation of the abnormal cell line and improve the quality of life. hydroxyurea is the most effective agent for treating cml during the chronic phase. 75, 122 the initial dosage is 20 to 25 mg/kg twice daily. treatment with hydroxyurea should continue until the leukocyte count falls to 15,000 to 20,000 cells/µl. 75, 79, 84 then the dosage of hydroxyurea can be reduced by 50% on a daily basis or to 50 mg/ kg given biweekly or triweekly. in humans, the alkylating agent busulfan can be used as an alternative. 123 an effective dosage has not been established in the dog, but following human protocols, 0.1 mg/kg/day po is given until the leukocyte count is reduced to 15,000 to 20,000 cells/µl. variably reported as impaired 90 or enhanced. 93 in the one dog in which it was measured, the plasma thrombopoietin (tpo) concentration was normal. 92 it is unclear whether tpo plays a role in essential thrombocythemia or is suppressed by the high platelet mass. elucidation of the pathogenesis of this disorder should be aided by the recent cloning of the genes for thrombopoietin and its receptor, the proto-oncogene mpl. 115 in mds, abnormalities in two or three cell lines are usually manifested in peripheral blood as neutropenia with or without a left shift, nonregenerative anemia, or thrombocytopenia. other changes include macrocytosis and metarubricytosis. the bone marrow is typically normocellular or hypercellular with an increased m : e ratio, and blasts cells, although increased, constitute less than 20% of nucleated cells; in a report of 13 dogs with primary or secondary mds, in all but one dog the blast cell percentage was less than 20%. 116 dysplastic changes can be detected in any cell line. dyserythropoiesis is characterized by asynchronous maturation of erythroid cells typified by large hemoglobinized cells with immature nuclei (megaloblastic change). if the erythroid component is dominant, the mds is called mds-er (see table 32 -12) . 3, 32 in dysgranulopoiesis, giant neutrophil precursors and abnormalities in nuclear segmentation and cytoplasmic granulation can be seen. finally, dysthrombopoiesis is characterized by giant platelets and micromegakaryocytes. myelofibrosis should be suspected in animals with nonregenerative anemia or pancytopenia, abnormalities in erythrocyte morphology (especially shape), and leukoerythroblastosis. bone marrow aspiration is usually unsuccessful, resulting in a "dry tap. " this necessitates a bone marrow biopsy taken with a jamshidi needle. 117 the specimen is processed for routine histopathologic examination, and if necessary, special stains for fibrous tissue can be used. because myelofibrosis occurs secondary to other diseases of bone marrow such as chronic hemolytic anemia or bone marrow necrosis, the clinician should look for a primary disease process. treatment of acute nonlymphocytic leukemias has been unrewarding to date. however, we have little information on the response of specific subtypes of leukemia to uniform chemotherapeutic protocols, in part due to the rarity of these disease processes and the paucity of cases in the literature. the veterinarian is advised to contact a veterinary oncologist for advice on new protocols and appropriate management of these cases. the therapeutic goal is to eradicate leukemic cells and reestablish normal hematopoiesis. currently, this is best accomplished by cytoreductive chemotherapy, and the agents most commonly utilized include a combination of ara-c plus an anthracycline, such as doxorubicin or cyclophosphamide, vincristine, and prednisone.* in humans, the introduction of cytosine arabinoside has been the single most important development in the therapy of acute nonlymphocytic leukemia. 120 in dogs, ara-c, 100 to 200 mg/m 2 , given by slow infusion (12 to 24 hrs) daily for 3 days and repeated weekly, has been used, as well as several other variations using subcutaneous injections of cytosine (see chapter 11) . doxorubicin, 30 mg/m 2 iv every 2 to 3 weeks, can be administered at intervals alternating with ara-c. if remission is achieved, as evidenced by normalization of the hemogram, the coap protocol (cyclophosphamide, vincristine (oncovin), ara-c, and prednisone), as described for canine • benzene (chronic exposure), and alkylating agents. 130 new classification systems have incorporated genetic mutations, more accurately reflect prognoses, and facilitate use of consistent categorization among institutions. 131 therapeutic modalities under investigation or development include combination chemotherapy, immunotherapy, cytokine therapy, drug-resistance modulators, proapoptotic agents, antiangiogenic factors, signal transduction-active agents, and bone marrow transplantation. the prognosis for mpn is better than for aml. for acute nonlymphocytic leukemias, the prognosis is better for children than adults, with only 10% of adults receiving chemotherapy maintaining remissions for more than 5 years. 130 the spontaneous canine diseases probably occur too infrequently to serve as useful models. myeloid neoplasms have been induced experimentally in the dog by irradiation and transplantation in an attempt to create models for study. many similarities between human and canine myeloid neoplasms exist, and veterinary medicine may benefit from any therapeutic advances made in the human field. despite response to chemotherapy and control for many months, most dogs with cml will eventually enter a terminal phase of their disease. in one study of seven dogs with cml, four underwent terminal phase blast crisis. 75 in humans, blast crisis may be lymphoid or myeloid. 124 in dogs, it is usually difficult to determine the cell of origin. these dogs have a poor prognosis, and the best treatment to consider, if any, would be that listed in table 32-13. it has now been documented that a subset of cml in dogs may be associated with a bcr-abl chromosomal abnormality (the so-called "raleigh chromosome") similar to the "philadelphia chromosome" translocation responsible for a large majority of cml in humans. 2 while imatinib mesylate (gleevec) is known to be an effective therapy for cml in humans, bcr-abl kinase inhibitors have, as yet, not been investigated for this subset of cml in dogs. few cases have been reported, but one dog was treated successfully with a combination chemotherapy protocol that included vincristine, ara-c, cyclophosphamide, and prednisone. 91 treatment is controversial in humans because of the lack of evidence that asymptomatic patients benefit from chemotherapy. patients with thrombosis or bleeding are given cytoreductive therapy. hydroxyurea is the drug of choice for initially controlling the thrombocytosis. 89 there is no standard therapeutic regime for mds. often, humans receive no treatment if the cytopenias do not cause clinical signs. transfusions are given when necessary, and patients with fever are evaluated aggressively to detect infections. growth factors, such as epo, gm-csf, g-csf, and il-3, are sometimes used in patients who require frequent transfusions to increase their blood cell counts and enhance neutrophil function. 125, 126 in one case report, human epo was administered (100 u/kg sq, every 48 hours) to a dog with mds because of profound anemia. the rationale for use of epo was to promote terminal differentiation of dysplastic erythrocytes. the pcv increased from 12% to 34% by day 19 of epo treatment. this dog remained in remission for more than 30 months. 32 other factors that induce differentiation of hematopoietic cells include retinoic acid analogs, 127 1,25 dihydroxyvitamin d3, 128 interferon-α, and conventional chemotherapeutic agents, such as 6-thioguanine and ara-c. 129 the propensity of these factors to enhance progression to leukemia is not known in many cases, but the potential risk exists. in general, the prognosis for animals with mpn is better than for dogs with aml, in which it is grave. the prognosis for pv and cml is guarded, but significant remissions have been achieved with certain therapeutic regimes and careful monitoring. animals commonly survive a year or more. 75, 84 development of blast crisis portends a grave prognosis. the pathophysiology and therapy of nonlymphocytic leukemia in humans are being studied intensively. myeloid neoplasms have been demonstrated to be clonal, with abnormalities evident in all hematopoietic cell lines. leukemogenesis is likely caused by mutation or amplification of proto-oncogenes in a two-step process that initially involves a single cell and is followed by additional chromosomal alterations that may involve oncogenes. 1, 15 these alterations are manifested as cytogenetic abnormalities. environmental factors known to cause leukemia are exposure to high-dose radiation, blurring of the distinction between mm and multicentric noncutaneous emp in cats and these two mrds will be discussed together in this species. although mm represents less than 1% of all malignant tumors in animals, it is responsible for approximately 8% of all hematopoietic tumors and 3.6% of all primary and secondary tumors affecting bone in dogs. 1, 2 in a compilation of bone marrow disorders in dogs (n = 717), mm represented 4.4% and 19.8% of all abnormal samples and neoplastic processes, respectively. 3 further, in a compilation of serum protein electrophoretic samples (n = 147 dogs), mm accounted for 4.3% of abnormal and 28.5% of neoplastic processes encountered, respectively. 4 early studies suggested a male predisposition, 5 although subsequent reports have not supported this. 1, 6 older dogs are affected with an average age of between 8 and 9 years. 1, 5, 6 in one large case series, german shepherd dogs were overrepresented based on the hospital population. 1 the true incidence of mm in the cat is unknown; however, it is a more rare diagnosis than in the dog, representing only 1 of 395 and 4 of 3248 tumors in two large compilations of feline malignancies and 0.9% of all malignancies and 1.9% of hematologic malignancies in another report. 7-9 mm represented 1.4% and 14% of abnormal and malignant serum protein electrophoretic samples, respectively, in a compilation of 155 feline samples. 10 mm occurs in aged cats (median age 12 to 14 years), most commonly in domestic short hairs and no sex predilection has been consistently reported, although a male preponderance may exist. 6, 9, 11, 12 mm has not been associated with corona virus or felv or fiv infections. the etiology of mm is for the most part unknown. genetic predispositions, molecular aberrations (e.g., c-kit), viral infections, chronic immune stimulation, and exposure to carcinogen stimulation have all been suggested as contributing factors. 6,13-18 suggestion of a familial association in cats follows cases reported among siblings. 12 evidence exists that molecular mechanisms of cellular control, including overexpression of cell cycle control components like cyclin d1 (see chapter 2) and receptor tyrosine kinase dysregulation may be involved in canine myeloma and plasma cell tumors. 17, 18 in rodent models, chronic immune stimulation and exposure to implanted silicone gel have been associated with development of mm, 13, 14 as have chronic infections and prolonged hyposensitization therapy in humans. 15 viral aleutian disease of mink results in monoclonal gammopathies in a small percentage of cases. 16 exposure to the agricultural industry, petroleum products, and irradiation are known risk factors for development in humans. [19] [20] [21] additionally, progression of solitary plasma cell tumors to mm has been reported in both dogs and cats, and a single case of a b-cell lymphoma progressing to mm exists in the dog. 22, 23 pathology and natural behavior multiple myeloma is a systemic proliferation of malignant plasma cells or their precursors arising as a clone of a single cell that usually involves multiple bone marrow sites in dogs. in cats, as previously stated, a blurring of the distinction of mm and multicentric noncutaneous emp within the mrd occurs because widespread abdominal organ involvement without significant bone marrow infiltration has been described in a significant proportion of cases in european compilations. 11, 24 because both mm and multicentric noncutaneous emp have a similar clinical course and widespread systemic involvement with hyperglobulinemia in cats, they will be  section d ig-secreting lymphomas and leukemias (including plasma cell leukemia). mm is the most important mrd based on clinical incidence and severity. there appears to be some discordance and below 37° c and require blood collection and clotting to be performed at 37° c prior to serum separation. if whole blood is allowed to clot at temperatures below this, the protein precipitates in the clot and is lost. pure light-chain m component is rare but has been reported in both dogs and cats. 38, 39 the pathology associated with mm is a result of either high levels of circulating m component, organ or bone infiltration with neoplastic cells, or both. associated pathologic conditions include bone disease, bleeding diathesis, hyperviscosity syndrome, renal disease, hypercalcemia, immunodeficiency (and subsequent susceptibility to infections), cytopenias secondary to myelophthisis, and cardiac failure. bone lesions can be isolated, discrete osteolytic lesions (including pathologic fractures) (figure 32-22, a) or diffuse osteopenias, or both (figure 32-23) . approximately one-quarter to two-thirds of dogs with mm have radiographic evidence of bony lysis or diffuse osteoporosis. 1, 5, 6 the incidence of radiographic skeletal lesions in cats varies tremendously within reports, from as few as 8% in european case series to as high as 65% in north american case series. 8, 9, 11, 12, 26 those bones engaged in active hematopoiesis are more commonly affected and include the vertebrae, ribs, pelvis, skull, and proximal or distal long bones. skeletal lesions are rare with igm (waldenström's) macrogammaglobulinemia, in which malignant cells often infiltrate the spleen, liver, and lymph tissue rather than bone. 6, [40] [41] [42] bleeding diathesis can result from one or a combination of events. m components may interfere with coagulation by (1) inhibiting platelet aggregation and the release of platelet factor-3; (2) causing adsorption of minor clotting proteins; (3) generating abnormal fibrin polymerization; and (4) producing a functional decrease in calcium. 6 to very large anaplastic round cells (often referred to as plasmablasts), with a high mitotic index representing early stages of differentiation. 5, 6, 9, 24, 25 binucleate and multinucleate cells are often present (see figure 7 -32, chapter 7). in 16 cats with mm in a north american case series, the majority (83%) of plasma cells were immature and had marked atypia, including increased size, multiple nuclei, clefted nuclei, anisocytosis, anisokaryosis, variable n : c ratios, decreased chromatin density, and variable nucleoli; nearly one-quarter had "flame cell" morphology characterized by peripheral eosinophilic cytoplasmic processes. 9 however, in a european compilation of feline multicentric noncutaneous mrd cases (n = 17), 78% had well-differentiated morphologies. 24 the authors of this latter case series developed a grading system dependent on the percentage of plasmablasts within the neoplastic cells in which well-differentiated, intermediate-grade, and poorly differentiated have less than 15%, 15% to 49%, and 50% or more plasmablasts, respectively. malignant plasma cells typically produce an overabundance of a single type of or component of immunoglobulin, which is referred to as the m component (figure 32-21) . the m component can be represented by any class of the entire immunoglobulin or only a portion of the molecule, such as the light chain (bence jones protein) or heavy chain (heavy chain disease) of the molecule. in the dog, the m component is usually represented by either iga or igg immunoglobulin types in nearly equal incidence, whereas the ratio of igg : iga in cats is approximately 5 : 1 in some reports and approximately 1 : 1 in others. 1, [5] [6] [7] [8] [9] 24, 26 that being said, in the author's (dmv) experience, the vast majority of canine cases are of the iga type. if the m component is the igm type, the term macroglobulinemia (waldenström's) is often applied. several cases of biclonal gammopathy in dogs and cats have been reported. 9, 11, 12, [27] [28] [29] [30] [31] [32] several cases of nonsecretory mm have been reported in dogs. 33, 34 rarely, cryoglobulinemia occurs in dogs with mm and igm macroglobulinemia and has been reported in a cat with igg myeloma. 6, [35] [36] [37] cryoglobulins are paraproteins that are insoluble at temperatures a b nonneoplastic immunoglobulin production. in the case of mm, an unbalanced excess of light-chain products may be produced. light chains are of low molecular weight and are normally filtered by the renal glomerulus, and their presence in urine can result in protein precipitates and subsequent renal tubular injury. the presence of light chains in urine without a concomitant monoclonal spike in serum, although rare, is indicative of pure light-chain disease. 38 tubules become obstructed by large laminated casts containing albumin, immunoglobulin, and light chains. 6, 38, 43, 44 bence jones proteinuria occurs in approximately 25% to 40% of dogs with mm. 1, 5, 6 bence jones proteinuria is reported to occur in approximately 40% of cats with mm/mrd. 9, 11 hypercalcemia is reported in 15% to 20% of dogs with mm and is thought to result primarily from the production of osteoclast-activating factor by neoplastic cells. 1, 6, 54 other factors, including increased levels of various cytokines, tnf, il-1, and il-6 have been implicated in human mm. in two dogs with mm and hypercalcemia, serum elevations in circulating n-terminal pthrp were noted. 55 hypercalcemia may also be exacerbated by associated renal disease. hypercalcemia, initially thought to be a rare event in cats with mm, occurred in 10% to 25% of recently reported cases. 9, 11, 12, 56 susceptibility to infection and immunodeficiency have long been associated with mm and are often the ultimate cause of death in affected animals. 1, 6, 26 infection rates in humans with mm are fifteen times higher than normal and usually represent pneumonia or urinary tract infections. 57 response to vaccination has also been shown to be suppressed in humans with mm. 57 immunoglobulin levels are often severely depressed in affected animals. 6 in addition, leukopenias may be present secondary to myelophthisis. variable cytopenias may be observed in association with mm. a normocytic, normochromic, nonregenerative anemia is encountered in approximately two-thirds of dogs with mm. 1, 5, 6 this can result from marrow infiltration (myelophthisis), blood loss from coagulation disorders, anemia of chronic disease, or increased erythrocyte destruction secondary to high serum viscosity. rare erythrophagocytic forms of mm have also been reported in both dogs and cats and may contribute to anemia. 58, 59 similar factors lead to thrombocytopenia and leukopenia in nearly one-third and onequarter of dogs with mm, respectively. in cats, approximately twothirds, half, and one-third will be anemic, thrombocytopenic, and neutropenic, respectively. 9, 11, 12 cardiac disease if present is usually a result of excessive cardiac workload and myocardial hypoxia secondary to hyperviscosity. 43, 45, 53 myocardial infiltration with amyloid and anemia may be complicating factors. nearly half of cats with mm in one report presented with a cardiac murmur, the etiology of which was not established. 9 three cats with hvs presented with congestive heart failure, murmurs, and echocardiographic signs consistent with hypertrophic cardiomyopathy. 53 clinical signs of mm may be present up to a year prior to diagnosis with a median duration of one month reported in dogs. 1, 9 in one cat, m-component elevations were detected 9 years prior to clinical presentation. in this latter case, the m-component elevation was consistent with monoclonal gammopathy of unknown significance (mgus). mgus (i.e., benign, essential, or idiopathic monoclonal gammopathy) is a benign monoclonal gammopathy that is not associated with osteolysis, bone marrow infiltration, or bence jones proteinuria. mgus has also been reported in dogs. 60, 61 signs of mm can be variable based on the wide range of pathologic effects one-quarter of cats have clinical evidence of hemorrhage. 1, 9, 11, 12 in dogs, nearly half have abnormal prothrombin (pt) and partial thromboplastin (ptt) times. thrombocytopenia may also play a role if bone marrow infiltration is significant (i.e., myelophthisis). hyperviscosity syndrome (hvs) represents one of a constellation of clinicopathologic abnormalities resulting from greatly increased serum viscosity. the magnitude of viscosity changes is related to the type, size, shape, and concentration of the m component in the blood. hvs is more common with igm macroglobulinemias due to the high molecular weight of this class of immunoglobulin. iga-secreting myelomas (usually present as a dimer in the dog), may undergo polymerization resulting in increased serum viscosity. 1, 6, 45 igg-associated hvs can also occur, albeit less frequently. high serum viscosity occurs in approximately 20% of dogs with mm and can result in bleeding diathesis, neurologic signs (e.g., dementia, depression, seizure activity, coma), ophthalmic abnormalities (e.g., dilated and tortuous retinal vessels, retinal hemorrhage [ figure 32 -24], retinal detachment), and increased cardiac workload with the potential for subsequent development of cardiomyopathy.* these consequences are thought to be a result of sludging of blood in small vessels, ineffective delivery of oxygen and nutrients, and coagulation abnormalities. hvs has been reported in cats with igg-, iga-, and igm-secreting tumors. 6, 8, [49] [50] [51] [52] [53] in several of these cases, relative serum viscosity was increased above control ranges. renal disease is present in approximately one-third to one-half of dogs with mm, and azotemia was observed in one-third of cats in one report. 1, 5, 9, 11 the pathogenesis of renal failure is often multifactorial and can ensue as a result of bence jones (light-chain) proteinuria, tumor infiltration into renal tissue, hypercalcemia, amyloidosis, diminished perfusion secondary to hyperviscosity syndrome, dehydration, or ascending urinary tract infections. 1, 6, 43, 44 normally, heavy-and light-chain synthesis is well balanced in serum biochemistry profile, and urinalysis. particular attention should be paid to renal function and serum calcium levels. if clinical hemorrhage is present, a coagulation assessment (e.g., platelet count, pt, ptt) and serum viscosity measurements are indicated. all animals should undergo a careful funduscopic examination. serum electrophoresis and immunoelectrophoresis are performed to determine the presence of a monoclonal m component (see and to categorize the immunoglobulin class involved. heat precipitation and electrophoresis of urine may be performed to determine presence of bence jones proteinuria possible. tables 32-14 and 32-15 list the relative frequencies of clinical signs observed in the dog and cat, respectively, based on a compilation of several reports.* bleeding diathesis is usually represented by epistaxis and gingival bleeding. funduscopic abnormalities may include retinal hemorrhage (see figure 32 -24), venous dilatation with sacculation and tortuosity, retinal detachment, and blindness. † cns signs may include dementia, seizure activity, tremors, and deficiencies in midbrain or brain-stem localizing reflexes secondary to hvs or extreme hypercalcemia. signs reflective of transverse myelopathies secondary to vertebral column infiltration, pathologic fracture, or extradural mass compression can also occur. 1, 6, 35, 62, 63 one case of ataxia and seizure activity in a dog with emp secondary to tumor-associated hypoglycemia has been reported. 64 additionally, paraneoplastic polyneuropathy has been reported in a dog with mm. 65 a history of chronic respiratory infections and persistent fever may also be present in cats. hepatosplenomegaly and renomegaly can occur due to organ infiltration. bleeding diathesis due to hvs is less common in the cat; however, epistaxis, pleural and peritoneal hemorrhagic effusions, retinal hemorrhage, and central neurologic signs have been reported. 6, 8, [49] [50] [51] [52] [53] polydipsia and polyuria can occur secondary to renal disease or hypercalcemia, and dehydration may develop. hindlimb paresis secondary to osteolysis of lumbar vertebral bodies or extradural compression has been reported in cats. 12, 66 diagnosis and staging the diagnosis of mm in dogs usually follows the demonstration of bone marrow plasmacytosis (see , the presence of osteolytic bone lesions (see , and the demonstration of serum or urine myeloma proteins (m component) (see . in the absence of osteolytic bone lesions, a diagnosis can also be made if marrow plasmacytosis is associated with a progressive increase in the m component. in the cat, because the degree of bone marrow infiltration may not be as marked, it has been suggested that consideration of plasma cell morphology and visceral organ infiltration (figure 32 -25) be given in cases with demonstrable m-component disease in the absence of marked (<20%) marrow plasmacytosis. 9, 11, 24 all animals suspected of plasma cell tumors should receive a minimal diagnostic evaluation including a cbc, platelet count, • because commercial urine dipstick methods are not capable of this determination. definitive diagnosis usually follows the performance of a bone marrow aspiration in the dog. a bone marrow core biopsy or multiple aspirations may be necessary due to the possibility of uneven clustering or infiltration of plasma cells in the bone marrow. normal marrow contains less than 5% plasma cells, whereas myelomatous marrow often greatly exceeds this level. current recommendations require more than 20% marrow plasmacytosis to be present, although a 10% cutoff in cats has been recently recommended with special attention to cellular atypia. 9 even the 10% threshold is problematic in cats, and cellular atypia and visceral organ involvement (assessed through needle aspiration cytology or tissue biopsy) should be considered equally important in the species. 9, 11, 24 rarely, biopsy of osteolytic lesions (i.e., jamshidi core biopsy; see chapter 24) is necessary for diagnosis in the dog. in one case of mm in a dog, splenic aspirates were diagnostically helpful. 67 overall frequencies of clinical diagnostic abnormalities for dogs and cats with mm are compiled from published series having at least five cases each and are listed in table 32 -16. histochemical and immunohistochemical analyses of cells or tissues suspected of mrd are more often applied in the case of solitary plasmacytomas or where emp is suspected in the absence of marrow involvement and will be discussed in subsequent sections; however, they have been occasionally useful in the diagnosis of mm. molecular diagnostic techniques for mm have received limited use thus far in veterinary oncology; however, determining clonality of the immunoglobulin heavy chain variable region gene has been performed in feline plasmacytoma and myeloma using parr techniques (see chapter 8), 68 and use of this technology in cases where diagnosis is not straightforward awaits further investigation. the author has used parr analysis both before treatment and after clinical remission in a small number of dogs with mm involved in clinical trials and documented its utility (1) for initial diagnosis and (2) to characterize molecular remission. routine thoracic and abdominal radiographs are recommended in suspected cases. occasionally, bony lesions can be observed in skeletal areas on these standard films, and organomegaly (liver, spleen, kidney) is observed in the majority of cats. 9, 11 abdominal ultrasound is recommended in all cats suspected of mm because this modality reveals involvement of one or more abdominal organs in the majority of cases. 9,11 these include splenomegaly with or without nodules, diffuse hyperechoic hepatomegaly with or without nodules, renomegaly, and iliac lymph node enlargement. in one case series in cats, 85% of organs with ultrasonographic abnormalities were subsequently confirmed to have plasma cell infiltration. 11 skeletal survey radiographs are recommended to determine presence and extent of osteolytic lesions, which may have diagnostic, prognostic, and therapeutic implications. although nuclear scintigraphy (bone scan) for clinical staging of dogs with mm has been performed, due to the predominant osteolytic activity with osteoblastic inactivity present, scans seldom give positive results and are therefore not useful for routine diagnosis. 69 in physician-based oncology, bone mineral density analysis (dual-energy x-ray absorptiometry [dexa] scan) to document osteoporosis, mri scan of bone marrow, and pet/ct are commonly used for staging; however, these modalities have not been applied consistently in the veterinary literature. a clinical staging system for canine mm has been suggested 1 ; however, at present, no prognostic significance has been attributed to it. • disease syndromes other than plasma cell tumors can be associated with monoclonal gammopathies and should be considered in any list of differentials. these include other lymphoreticular tumors (b-cell lymphoma, extramedullary plasmacytoma, chronic and acute b-lymphocytic leukemia), chronic infections (e.g., ehrlichiosis, leishmaniasis, fip), and mgus.* agent may more quickly alleviate systemic effects of the disease. cyclophosphamide is initiated at a dosage of 200 mg/m 2 iv, once, at the same time oral melphalan therapy is started. because cyclophosphamide is less likely to affect thrombocytes, it may be substituted in those patients in which thrombocytopenia has developed secondary to long-term melphalan use. chlorambucil, another alkylating agent, has been used successfully for the treatment of igm macroglobulinemia in dogs at a dosage of 0.2 mg/kg po, once daily. 6, 40 little or no clinical signs of toxicity result from this dosing schedule. chlorambucil has also been used in cats with mrd. 11 lomustine (ccnu), yet another alkylating agent, has been used in a limited number of cats with mm and a partial response has been reported following dosing at 50 mg/m 2 po, every 21 days. 74 evaluation of response to systemic therapy for multiple myeloma is based on improvement in clinical signs, clinicopathologic parameters, and radiographic improvement of skeletal lesions or ultrasonographic improvement of organ involvement. 1, 6, 11 subjective improvement in clinical signs of bone pain, lameness, lethargy, and anorexia should be evident within 3 to 4 weeks following initiation of therapy. objective laboratory improvement, including reduction in serum globulin, immunoglobulin, and calcium, along with normalization of the hemogram, is usually noted within 3 to 6 weeks ( figure 32-26) . radiographic improvement in osteolytic bone lesions may take months and resolution may only be partial. ophthalmic complications (including long-standing retinal detachments) and paraneoplastic neuropathies can be expected to resolve along with tumor mass. 48, 65 in cats responding to chemotherapy, clinical improvement is noted in 2 to 4 weeks and serum protein and radiographic bone abnormalities were greatly improved by 8 weeks. 11, 12 as previously discussed, complete resolution of mm does not generally occur and a good response is defined as a reduction in measured m component (i.e., immunoglobulin or bence jones proteins) of at least 50% of pretreatment values. 6 reduction in serum immunoglobulin levels may lag behind reductions in bence jones proteinuria because the half-lives are 15 to 20 days and 8 to 12 hours, respectively. 75 for routine follow-up, quantification of the increased serum globulin, immunoglobulin, or urine bence jones protein is performed monthly until a good response is noted and then every 2 to 3 months thereafter. repeat bone marrow aspiration or imaging (in the case of visceral disease) for evaluation of plasma cell infiltration may be occasionally necessary. bone marrow reevaluation is particularly prudent when cytopenias develop during chemotherapy, and drug-induced myelosuppression must be differentiated from myelophthisis due to neoplastic marrow recurrence. the long-term control of complications, including hypercalcemia, hvs, bleeding diathesis, renal disease, immunosuppression, ophthalmic complications, and pathologic skeletal fractures, depend on controlling the primary tumor mass. therapy directed more specifically at these complications may, however, be indicated in the short term. if hypercalcemia is marked and significant clinical signs exist, standard therapies, including fluid diureses, with or without pharmacologic agents (e.g., calcitonin), may be indicated (see chapter 5) . moderate hypercalcemia will typically resolve within 2 to 3 days following initiation of melphalan/prednisone chemotherapy. therapy for mm is directed at both the tumor cell mass and the secondary systemic effects they elicit. all diagnostic procedures should be completed before initiating primary therapy to ensure a diagnosis is complete and baseline values are procured for monitoring response. chemotherapy is effective at reducing myeloma cell burden, relieving bone pain, allowing for skeletal healing, and reducing levels of serum immunoglobulins in the majority of dogs with mm and will greatly extend both the quality and quantity of most patients' lives. mm in dogs is initially a gratifying disease to treat for both the clinician and the companion animal owner, although complete elimination of neoplastic myeloma cells is rarely achieved and eventual relapse is to be expected. unlike dogs, only one-half of cats with mm will respond to chemotherapy and most responses will be short-lived; however, several long-term responses (i.e., >1 year) have been reported and treatment should be attempted when educated clients decide on a therapeutic option.* melphalan, an alkylating agent, is the chemotherapeutic of choice for the treatment of multiple myeloma. 1, 6 in the dog, an initial starting dose of 0.1 mg/kg po, once daily for 10 days, is then reduced to 0.05 mg/kg po, once daily continuously. the addition of prednisone therapy is thought to increase the efficacy of melphalan therapy. prednisone is initiated at a dosage of 0.5 mg/kg po, once daily for 10 days, then reduced to 0.5 mg/kg every other day prior to discontinuation after 60 days of therapy. melphalan, however, is continued at 0.05 mg/kg/day until clinical relapse occurs or myelosuppression necessitates a dose reduction. the vast majority of dogs on melphalan and prednisone combination therapy tolerate the regimen well. the most clinically significant toxicity of melphalan is myelosuppression, in particular a delayed thrombocytopenia. cbcs, including platelet counts, should be performed biweekly for 2 months of therapy and monthly thereafter. if significant myelosuppression occurs (usually thrombocytopenia or neutropenia), reduction of the dosage or treatment frequency may be necessary. an alternative pulse-dosing regimen for melphalan (7 mg/m 2 po, daily for 5 consecutive days every 3 weeks) has been used successfully by the author in a small number of cases in which myelosuppression was limiting more conventional continuous low-dose therapy. this pulse-dose regimen is now being used first-line by the author with the caveat that long-term response data are currently lacking. melphalan and prednisone therapy can also be used in cats with multiple myeloma; however, it appears this protocol is more myelosuppressive than in the dog and careful monitoring is required. in the cat, a dosing schedule similar to the dog has been reported 12,26 ; 0.1 mg/kg (approximately 0.5 mg, or one-quarter of a 2 mg tablet) once daily for 10 to 14 days, then every other day until clinical improvement or leukopenia develop. long-term continuous maintenance (0.1 mg/kg, once every 7 days) has been advocated. 12 an alternative protocol advocated in the cat uses melphalan at 2 mg/ m 2 , once every 4 days continuously, and appears to be well tolerated. 11 cyclophosphamide has been used as an alternative alkylating agent or in combination with melphalan in dogs and cats with mm. 1, 6, 11 there is no evidence to suggest it is superior to melphalan therapy. in the author's practice, cyclophosphamide is limited to those cases presenting with severe hypercalcemia or with widespread systemic involvement in which a faster acting alkylating *references 6, 9, 11, 12, 31, 26. water intake at home is important, and occasionally, educating owners in subcutaneous fluid administration is indicated. continued monitoring of renal function is recommended along with follow-up directed at tumor response. patients with mm can be thought of as immunologically impaired. some have recommended prophylactic antibiotic therapy in dogs with mm 6 ; however, in humans, no benefit for this approach over diligent monitoring and aggressive antimicrobial management when indicated has been observed. 43 cidal antimicrobials are preferred over static drugs, and avoidance of nephrotoxic antimicrobials is recommended. pathologic fractures of weight-bearing long bones and vertebrae resulting in spinal cord compression may require immediate surgical intervention in conjunction with systemic chemotherapy. hvs is best treated in the short term by plasmapheresis.* whole blood is collected from the patient and centrifuged to separate plasma from packed cells. packed red cells are resuspended in normal saline or other crystalloid and reinfused into the patient. bleeding diathesis will usually resolve along with hvs; however, platelet-rich plasma transfusions may be necessary in the face of thrombocytopenia. renal impairment may necessitate aggressive fluid therapy in the short term and maintenance of adequate hydration in the long term. careful attention to secondary urinary tract infections and appropriate antimicrobial therapy is indicated. ensuring adequate the prognosis for dogs with mm is good for initial control of tumor and a return to good quality of life. in a group of 60 dogs with mm, approximately 43% achieved a complete remission (i.e., serum immunoglobulins normalized), 49% achieved a partial remission (i.e., immunoglobulins <50% pretreatment values), and only 8% did not respond to melphalan and prednisone chemotherapy. 1 longterm survival is the norm, with a median of 540 days reported (figure 32-27) . the presence of hypercalcemia, bence jones proteinuria, and extensive bony lysis are known negative prognostic indices in the dog. 1 the long-term prognosis for dogs with mm is poor because recurrence of tumor mass and associated clinical signs is expected. eventually, the tumor is no longer responsive to available chemotherapeutics and death follows from renal failure, sepsis, or euthanasia for intractable bone or spinal pain. 1, 6 the prognosis for mm in the cat is not as favorable in the short term as it is in the dog. 6, 9, 11, 12, 26 whereas most cats (approximately 60%) transiently respond to melphalan/prednisone or cop-based protocols, most responses are partial and not durable. typically, cats with mm succumb to their disease within 4 months. 8, 9, 12, 26 however, long-term survivors (>1 year) have been occasionally reported.* in one european case series, seven cats undergoing melphalan or cop-based therapy had a median survival of 9.5 months. 11 one investigator grouped mm in cats into two prognostic categories (table 32 -17) based on criteria known to predict behavior in dogs. 12 although no rigorous statistical analysis was performed on this small group of nine cats, the median survival for cats in "aggressive" and "nonaggressive" categories was 5 days and 387 days, respectively. experience in dogs with igm macroglobulinemia is limited. 6, [40] [41] [42] response to chlorambucil is to be expected, and in nine treated dogs, 77% achieved remission with a median survival of 11 months. 6 orthopedic stabilization of fractures should be undertaken and may be followed with external-beam rt (see . recently, inhibition of osteoclast activity by bisphosphonate drugs has been shown to reduce the incidence and severity of skeletal complications of mm in humans. 69 this class of drugs may hold promise for use in dogs and cats with various skeletal tumors; however, they have not been adequately evaluated in mrd. 78 when mm eventually relapses in dogs and cats undergoing melphalan therapy or in the uncommon case that is initially resistant to alkylating agents, rescue therapy may be attempted. the author has had success with vad, which is a combination of doxorubicin (30 mg/m 2 iv, every 21 days), vincristine (0.7 mg/m 2 iv, days 8 and 15), and dexamethasone sodium phosphate (1.0 mg/kg iv, once a week on days 1, 8, and 15), given in 21-day cycles. whereas most dogs initially respond to this rescue protocol, the duration of response tends to be short, lasting only a few months. high-dose cyclophosphamide (300 mg/m 2 iv, every 7 days) has also been used with limited success as a rescue agent. liposomal doxorubicin has produced a long-term remission in a dog with mm previously resistant to native doxorubicin. 79 mm is ultimately a uniformly fatal disease in most species, including humans, and thus significant effort is being placed on investigational therapies for this disease. currently, bone marrow ablative therapy and marrow or stem cell rescue, thalidomide (and other antiangiogenic therapies), bortezomib (a proteasome inhibitor), arsenic trioxide, the bisphosphonates, and several molecular targeting therapies are under investigation; however, their use in veterinary species is limited or completely absent at present. the promise of molecular targeted therapies is, however, foreshadowed by a case of a dog with mm that was resistant to melphalan, prednisone, and doxorubicin that subsequently achieved a partial response to tyrosine kinase inhibitor therapy (toceranib; see chapter 14, section b) that was maintained for 6 months. 18 rights were not granted to include this figure in electronic media. please refer to the printed publication. to be of low biologic aggressiveness, and most do not recur following surgical excision. 100 conversely, the majority of sops eventually progress to systemic mm; however, the time course from local tumor development to systemic mm may be many months to years. 33, 102 sops have been reported in the dog involving the appendicular skeleton, as well as the zygomatic arch, and ribs. 33 sops are less common in cats, and fewer reports exist in the literature. 11, 22, [103] [104] [105] [106] [107] they occur in older cats (mean ages 9 to 14 years), with no significant sex predilection. the skin is the most common site; however, other sites include the oral cavity, eye, gi tract, liver, subcutaneous tissues, and brain. reports exist of cutaneous emp in cats that progressed to systemic mrd. 11, 22, 105 clinical signs associated with sops relate to the location of involvement, or in those rare cases with high levels of m component, hvs may occur. most cutaneous plasmacytomas are solitary, smooth, raised pink nodules from 1 to 2 cm in diameter (see figure 32 -28), although tumors as large as 10 cm have been reported. combining large series, greater than 95% occur as solitary masses and less than 1% occur as part of a systemic mm process. 17, [80] [81] [82] [83] [84] [85] [86] 95, 96 cutaneous and oral emps usually have a benign course with no related clinical signs. gi emp, however, typically presents with relatively nonspecific signs, which may suggest alimentary involvement. colorectal plasmacytomas usually present with rectal bleeding, hematochezia, tenesmus, and rectal prolapse. 100 one case of ataxia and seizure activity in a dog with emp secondary to tumor-associated hypoglycemia has been reported. 64 sop is usually associated with pain and lameness if the appendicular skeleton is affected or neurologic signs if vertebral bodies are involved. the diagnosis of sop and emp usually requires tissue biopsy or fna for diagnosis. cells making up solitary plasmacytic tumors in both cats and dogs have been histologically classified into mature, hyaline, cleaved, asynchronous, monomorphous blastic and solitary collections of monoclonal plasmacytic tumors can originate in soft tissues or bone and are referred to as extramedullary plasmacytoma (emp) and solitary osseous plasmacytoma (sop), respectively. the systemic, multicentric, biologically aggressive emp syndrome encountered in cats 11, 24 has been discussed in the mm section and will not be included in this discussion. a number of large case compilations of cutaneous plasmacytoma have been reported in the dog. 17, [80] [81] [82] [83] [84] [85] [86] the most common locations for emp in the dog are cutaneous (86%; figure 32 -28), the mucous membranes of the oral cavity and lips (9%; figure 32 -29) , and the rectum and colon (4%). the skin of the limbs and head (including the ears) are most frequently reported cutaneous sites. other sites accounted for only 1% of the remaining cases and can include stomach, spleen, genitalia, eye, uterus, liver, larynx, trachea, third eyelid, sinonasal cavity, and intracranial sites. [87] [88] [89] [90] [91] [92] [93] [94] the american cocker spaniel, english cocker spaniel, and west highland white terrier (and perhaps yorkshire terriers, boxers, german shepherds, and airedale terriers) are at increased risk for developing plasmacytomas and the median age of affected dogs is 9 to 10 years of age. 86 cutaneous and oral emp in dogs are typically benign tumors that are highly amenable to local therapy. there exists, however, a rare form of multiple cutaneous plasmacytomas in dogs that is part of a more generalized biologically aggressive mm process. 95, 96 the natural behavior of noncutaneous/nonoral emp appears to be somewhat more aggressive in the dog. gi emp have been reported on a number of occasions in the veterinary literature, including the esophagus, 91 stomach, 97, 98 and small 99 and large intestine. [98] [99] [100] [101] metastasis to associated lymph nodes is more common in these cases; however, bone marrow involvement and monoclonal gammopathies are less commonly encountered. colorectal emps tend • • figure 32 -28 a cutaneous plasmacytoma on the limb of a dog. cases of sop in cats were recently reported; one was treated with external-beam rt and one managed with melphalan chemotherapy and both enjoyed durable remissions of greater than 4 years. 114 similarly, emp of the gi tract in humans are treated most commonly by surgical excision and thorough staging of disease. systemic therapy is not initiated unless systemic involvement is documented. systemic chemotherapy has been used following gastric emp in a cat; however, the utility of adjuvant therapy in the species is unknown. 115 long-term follow-up of patients with sop is indicated in order to recognize both recurrence of disease and systemic spread. careful attention is given to serum globulin levels, bone pain, and radiographic appearance of bone healing in cases of sop. restaging of disease, including bone marrow evaluation, is indicated if systemic spread is suspected. prognosis for solitary plasma cell tumors is generally good. cutaneous and mucocutaneous plasmacytomas are usually cured following surgical excision. 17, 86, 112 in large compilations of cases in dogs, the local recurrence rate was approximately 5%, and nodal or distant metastasis occurred in only 7 of 349 cases (2%). 17, [80] [81] [82] 86 new cutaneous plasmacytomas at sites distant from the primary developed in less than 2% of cases. neither tumor cell proliferation rate (as measured by ki67 immunohistochemistry) in the dog nor histopathologic grading in dogs and cats were prognostic in large compilations of cases, although it has been suggested that the polymorphous-blastic and plasmablastic type may act more aggressively in the dog and cat. 17, 24, 86 the presence of amyloid and overexpression of cyclin d1 (prognostic in human plasmacytomas) were not shown to be of prognostic value in dogs. 17 dogs with emp of the alimentary tract and other abdominal organs (e.g., liver, uterus) treated by surgical excision alone or in combination with systemic chemotherapy (if metastasis is present) can enjoy longterm survival in the majority of cases.* in a compilation of nine dogs with colorectal plasmacytoma, two dogs had local recurrence at 5 and 8 months following surgery, and the overall median survival was 15 months following surgery alone. 100 dna ploidy and c-myc oncoprotein expression in biopsy samples were determined to be prognostic for emps in dogs; however, those that were malignant were all from noncutaneous sites (i.e., lymph node, colon, spleen). therefore location appears to be as predictive. 116 as previously discussed, the majority of cases of sop will eventually develop systemic disease; however, long disease-free periods usually precede the event. the prognosis in cats is less well-defined 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electrophoresis in 155 cats myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up multiple myelomas in cats a resume of the current status of the development of plasma cell tumors in mice induction of plasmacytomas with silicone gel in genetically susceptible strains of mice multiple myeloma and prolonged stimulation of res the development of myeloma-like condition in mink with aleutian disease clinico-pathological aspects of canine cutaneous and mucocutaneous plasmacytomas phase i doseescalating study of su11654, a small molecule receptor tyrosine kinase inhibitor, in dogs with spontaneous malignancies multiple myeloma and family history of cancer multiple myeloma: a case control study a case-control study of multiple myeloma in whites: chronic antigenic stimulation, occupation and drug use progression of a solitary, malignant cutaneous plasma-cell tumour to multiple myeloma in a cat evolution of a b-cell lymphoma to multiple myeloma after chemotherapy histopathologic, immunohistochemical, and cytologic analysis of feline myelomarelated disorders: further evidence for primary extramedullary development in the cat histological classification of hematopoietic tumors of domestic animals multiple myeloma in the cat biclonal gammopathy in a dog with myeloma and cutaneous lymphoma immunoglobulin a and immunoglobulin g biclonal gammopathy in a dog with multiple myeloma prognostic factors for multiple myeloma in the dog primary and secondary bone tumors in the dog a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital treatment of three cats with hyperviscosity syndrome and congestive heart failure using plasmapheresis bone destruction and hypercalcemia in plasma cell myeloma parathyroid hormone (pth)-related protein, pth, and 1,25-dihydroxyvitamin d in dogs with cancer associated hypercalcemia hypercalcemia in two cats with multiple myeloma infections complicating multiple myeloma and chronic lymphocytic leukemia erythrophagocytic multiple myeloma in a cat phagocytic plasmacytoma in a dog a benign hypergammaglobulinemia mimicking plasma cell myeloma idiopathic monoclonal (iga) gammopathy in a dog cervical cord compression as a neurologic complication in an igg multiple myeloma in a dog vertebral plasma cell tumors in 8 dogs hypoglycemia and polyclonal gammopathy in a dog with plasma cell dyscrasia multiple myeloma with associated polyneuropathy in a german shepherd dog plasma cell sarcoma in a cat fine-needle aspiration of the spleen as an aid in the diagnosis of splenomegaly characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of b-cell neoplasia plasma cell neoplasms use of plasmapheresis and chemotherapy for treatment of monoclonal gammopathy associated with ehrlichia canis infection in a dog monoclonal gammopathy associated with naturally occurring canine ehrlichiosis hyperviscosity syndrome associated with lymphocytic leukemia in three dogs monoclonal gammopathy in a dog with visceral leishmaniasis hematological toxicity and therapeutic efficacy of lomustine in 20 tumor-bearing cats: critical assessment of a practical dosing regimen the treatment of multiple myeloma therapeutic plasmapheresis multiple myeloma in a cat bisphosphonates and cancer biclonal gammopathy associated with immunoglobulin a in a dog with multiple myeloma monoclonal gammopathies in the dog: a retrospective study of 18 cases (1986-1999) and literature review multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats detection of biclonal gammopathy by capillary zone electrophoresis in a cat and a dog with plasma cell neoplasia nonsecretory multiple myeloma in two dogs monoclonal gammopathy without hyperglobulinemia in 2 dogs with iga secretory neoplasm neurologic complications of iga multiple myeloma associated with cryoglobulinemia in a dog monoclonal cryoglobulinemia with macroglobulinemia in a dog monoclonal immunoglobulin g cryoglobulinemia and multiple myeloma in a domestic shorthair cat light-chain myeloma in a dog light-chain multiple myeloma in a cat different biological behaviour of waldenstrom macroglobulinemia in two dogs macroglobulinemia in the dog, the canine analogue of gamma m monoclonal gammopathy macroglobulinemia with hyperviscosity syndrome in a dog plasma cell tumors plasma cell neoplasms serum hyperviscosity syndrome associated with iga multiple myeloma in two dogs ocular lesions in a dog with hyperviscosity secondary to an iga myeloma blindness in a dog with iga-forming myeloma ophthalmic disease as the presenting complaint in five dogs with multiple myeloma immunoglobulin a myeloma in a cat with pleural effusion and serum hyperviscosity hyperviscosity syndrome with igm monoclonal gammopathy and hepatic plasmacytoid lymphosarcoma in a cat serum hyperviscosity syndrome associated with multiple myeloma in two cats serum hyperviscosity syndrome associated with igg myeloma in a cat primary igg secreting plasma cell tumor in the gastrointestinal tract of a dog colorectal plasmacytomas: a retrospective study of nine dogs metastatic extramedullary plasmacytoma of the colon and rectum in a dog solitary plasmacytomas of bone and extramedullary plasmacytomas histopathologic and immunophenotypic characterization of extramedullary plasmacytomas in nine cats intraocular extramedullary plasmacytoma in a cat extramedullary plasmacytoma and immunoglobulin-associated amyloidosis in a cat immunohistochemical staining of neoplastic and inflammatory plasma cell lesions in feline tissues immunoglobulin-producing tumours in dogs and cats identification of immunoglobulin light chains in canine extramedullary plasmacytomas by thioflavine t and immunohistochemistry immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors immunohistochemical detection of multiple myeloma 1/interferon regulatory factor 4 (mum1/irf-4) in canine plasmacytoma: comparison with cd79a and cd20 imaging diagnosis-fdg-pet/ct of a canine splenic plasma cell tumor survival data for canine oral extramedullary plasmacytoma: a retrospective analysis use of strontium-90 plesiotherapy for the treatment of a lingual plasmacytoma in a dog solitary plasmacytoma of bone in two successfully treated cats gastric extramedullary plasmacytoma in a cat analysis of dna aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry response to liposomeencapsulated doxorubicin (tlc d-99) in a dog with myeloma extramedullary plasmacytomas in dogs: results of surgical excision in 131 cases mucocutaneous plasmacytomas in dogs: 75 cases cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study cutaneous plasmacytomas with amyloid in six dogs primary cutaneous plasmacytomas in the dog and cat an immunohistochemical study of canine extramedullary plasma cell tumours prognostic value of histopathological grading in canine extramedullary plasmacytomas extramedullary laryngeal plasmacytoma in a dog solitary extramedullary plasmacytoma of the canine larynx extramedullary plasmacytoma of the third eyelid gland in a dog sinonasal plasmacytoma in a cat solitary intracerebral plasmacytoma in a dog: microscopic, immunohistochemical, and molecular features extramedullary plasmacytoma in the trachea of a dog uterine extramedullary plasmacytoma in a dog a primary hepatic plasma cell tumor in a dog anaplastic atypical myeloma with extensive cutaneous involvement in a dog immunoglobulin a multiple myeloma with cutaneous involvement in a dog esophageal plasmacytoma in a dog extramedullary plasmacytoma of the gastrointestinal tract in two dogs for gi emp (including colorectal emp), endoscopic evaluation of the entire gi tract is recommended. a single case report of the use of pet/ct imaging for extramedullary splenic plasmacytoma in a dog exists; however, its utility remains unknown. 111 cutaneous and oral plasma cell tumors in the dog are almost always benign and carry an excellent prognosis following conservative surgical excision. 17, [81] [82] [83] [84] [85] [86] 100, 112 emps of the trachea, liver, and uterus have also been reported in dogs, and all had a benign course following local resection. [92] [93] [94] successful therapy with melphalan and prednisone has been rarely applied for a local recurrence or incomplete margins in dogs and cats. rt has been used infrequently for cases that are nonsurgical, including the application of strontium-90 plesiotherapy for lingual plasmacytoma in a dog. 113 surgery is recommended in combination with radiotherapy for those cases of sop in which the lesion results in an unstable, long bone fracture (see , or the patient is nonambulatory from neurologic compromise resulting from a vertebral body sop. in the latter case, spinal cord decompression, mass excision, and possibly spinal stabilization may be necessary. 63 radiotherapy can be used alone (i.e., without surgery) in those cases where fractures are stable, as a palliative measure for bone pain, or in the case of vertebral sop if the patient is ambulatory and stable. good local control is usually achieved; however, most go on to develop systemic multiple myeloma. 33, 63, 102 sop of the axial skeleton can be managed by excision or radiotherapy alone. there is controversy as to whether systemic chemotherapy should be initiated at the time of local therapy for sop when systemic involvement is not documented. systemic spread may not occur for many months to even years beyond primary sop diagnosis in humans and dogs, and studies in humans reveal no benefit derived from initiation of systemic chemotherapy prior to documentation of subsequent systemic spread. 44, 63 two polymorphous blastic cell types; however, no prognostic significance has been observed following classification, although it has been suggested that the polymorphous-blastic type may act more aggressively in the dog. 17,86,103 a different classification was proposed for emp in cats based on percentage of plasmablasts (see previous section), and some prognostic importance has been documented. 24 in the case of poorly differentiated plasmacytic tumors, immunohistochemical studies, directed at detecting immunoglobulin, light-and heavy-chains, mm-1/interferon regulatory factor-4 (mum1/irf4), and thioflavin t, may be helpful in differentiation from other round cell tumors. 33, 85, 86, [106] [107] [108] [109] [110] immunoreactivity has been demonstrated for canine igg f(ab) 2 and vimentin. 82 a variant characterized by an igg-reactive amyloid interspersed with the neoplastic cells has also been described. 83 a panel of mabs (recognizing tryptase, chymase, serotonin, cd1a, cd3, cd79a, cd18, mhc class ii) in association with a histochemical stain (naphthol as-d chloroacetate) has been advocated for use on formalin-fixed, paraffin-embedded sections of cutaneous round cell tumors to help classify poorly differentiated round cell tumors (mast cell tumors, histiocytomas, lymphomas, and plasmacytomas). 109 additionally, clonality of the immunoglobulin heavy chain variable region gene can be performed in plasmacytomas and myelomas using pcr technology, and this may have some diagnostic utility in difficult cases.it is important to thoroughly stage dogs and cats with plasmacytomas that are at higher risk for systemic spread if contemplating local or locoregional therapy without systemic therapy. this should include bone marrow aspiration, serum electrophoresis, abdominal ultrasound, and skeletal survey radiographs to ensure the disease is confined to a local site prior to initiation of therapy. this is most important in cases of sop and gi emp due to their relatively high metastatic rate and less important for cutaneous, oral, and colorectal plasmacytomas because of their more typical benign behavior. key: cord-344131-e7phs0jd authors: ford, richard b.; mazzaferro, elisa m. title: section 4 diagnostic and therapeutic procedures date: 2012-12-31 journal: kirk & bistner's handbook of veterinary procedures and emergency treatment doi: 10.1016/b978-1-4377-0798-4.00004-9 sha: doc_id: 344131 cord_uid: e7phs0jd nan for anorexic dogs or when pills must be given without food, give medications quickly and decisively so that the process of administering the medication is accomplished before the dog realizes what has happened. with cooperative dogs, insert the thumb of one hand through the interdental space, and gently touch the hard palate. this will induce the cooperative dog to open its mouth (figure 4-1) . using the opposite hand (the one holding the medication), gently press down on the mandible to open the mouth further (figure 4 -2). note: oral medication frequently is dispensed to owners without regard for the client's knowledge of how to administer a pill or tablet or without asking whether the client is even physically able to administer medications. clear instructions that include having the client perform the technique in the hospital significantly improve compliance. quickly place the tablet or capsule onto the caudal aspect of the tongue. quickly withdraw the hand and close the dog's mouth. when the dog licks its nose, the medication likely has been swallowed. dogs that offer more resistance can be induced to open their mouths by compressing their upper lips against their teeth. as they open the mouth, roll their lips medially so that if they attempt to close the mouth, they will pinch their own lips. alternatively, dripping water onto the nostrils or blowing into the patient's nose sometimes encourages the patient to accept and swallow oral medications (tablets or capsules). pilling syringes are also available and in some dogs seem to work well. critical to the oral administration of medication is the ability of the owner to effectively administer the medication at home. animals that aggressively resist oral medication should be treated by alternative methods-for example, parenteral administration of medication. it is inappropriate, and unsafe, to delegate treatment responsibilities to the owner of a dog (or cat) that might injure the individual who is attempting to treat the patient. none required. caution: only experienced individuals should attempt this technique of administering tablets or capsules to cats. even cooperative cats that become intolerant will bite. therefore, this is not a technique recommended for most owners to try at home, even if specific instructions have been given. two methods of pill administration are used in cats. in both methods the cat's head is elevated slightly with the nose pointed upward. success in administering pills and tablets to a cat entails a delicate balance between what works well and what works safely. in cooperative cats, it may be possible to use one hand to hold and position the head (figure 4 -3) while using the opposite hand (the one holding the medication) to open the mouth gently by depressing the proximal aspect of the mandible (figure 4-4) . press the skin adjacent to the maxillary teeth gently between the teeth as the mouth opens, thereby discouraging the alternatively, some cats will tolerate a specially designed "pilling syringe" in an attempt to administer a tablet or capsule. the pilling syringe works well as long as it is inserted cautiously and atraumatically into the cat's mouth. however, if resistance ensues, the rigid pilling syringe may injure the hard palate during the ensuing struggle. subsequent attempts to use the syringe may be met with increasing resistance and increasing risk of injury. success with a pilling syringe depends largely on the cat. pill pockets treats are also available for use in cats and are manufactured in chicken and fish flavors. in addition, as is the case in dogs, some cats will respond to the application of water drops on the nostrils or blowing into the nostrils to encourage swallowing. when dispensing oral medications for home administration to cats, do not expect clients to force a tablet or capsule into a cat's mouth. although some clients are remarkably capable and confident with their ability to administer oral medications to cats, the risk of injury to the client can be significant. whenever feasible, liquid medications or pulverized tablets should be mixed with the diet or an oral treat readily accepted and consumed (see the following discussion). none required. technique is appropriate for owners to perform at home. small amounts of liquid medicine can be given successfully to dogs and cats by pulling the commissure of the lip out to form a pocket . deposit the liquid medication into the "cheek pouch, " where it subsequently flows between the teeth as the head is held slightly upward. patience and gentleness, along with a reasonably flavored medication, contribute to the success. spoons are ineffective, as fluids are easily spilled. a disposable syringe can be used to measure and administer liquids orally. depending on the liquid administered, disposable syringes can be reused several times, assuming they are rinsed after each administration. in addition, disposable syringes can be dispensed legally to clients for home administration of liquid medication. mixing of medications in the same syringe is not recommended. however, dispensing of a separate, clearly marked syringe for each type of liquid medication prescribed for home administration is recommended. compounding pharmacies are also available and can mix many medications into palatable flavors to help facilitate the oral administration of medications. dogs with swallowing disorders should not be treated at home with liquid medications because this could cause complications associated with aspiration. none required. administration of medications, contrast material, and rehydrating fluids can be accomplished with the use of a well lubricated feeding tube passed through the nostrils into the stomach or distal esophagus. when a feeding tube is placed for long-term use (multiple days) and repeated use (described under gastrointestinal procedures later), it is generally recommended to avoid passing the tip of the tube beyond the distal esophagus. the reason for recommending nasoesophageal intubation over nasogastric intubation is based on the fact that reflex peristalsis of the esophagus against a tube passing through the cardia can result in significant mucosal ulceration within 72 hours. this is not a factor in patients receiving a single dose of medication or contrast material. note: this procedure is reserved for in-hospital use only. the technique should be performed only by individuals trained to perform this procedure. the narrow lumen of tubes passed through the nostril of small dogs and cats limits the viscosity of solutions that can be administered through a tube directly into the gastrointestinal tract. nasoesophageal intubation can be done with a variety of tube types and sizes (table 4 -1). newer polyurethane tubes, when coated with a lidocaine lubricating jelly, are nonirritating and may be left in place with the tip at the level of the distal esophagus. when placing the nasogastric tube, instill 4 to 5 drops of 0.5% proparacaine in the nostril of the cat or small dog; 0.5 to 1.0 ml of 2% lidocaine instilled into the nostril of a larger-breed dog may be required to achieve the level of topical anesthesia needed to pass a tube through the nostril. with the head elevated, direct the tube dorsomedially toward the alar fold (figure 4-6) . pushing dorsally on the nasal philtrum and pushing the nostril from lateral to medially will help facilitate passage of the tube into the ventromedial nasal meatus. caution: the tip of the feeding tube can be inadvertently introduced through the glottis and into the trachea. topical anesthetic instilled into the nose can anesthetize the arytenoid cartilages, thereby blocking a cough or gag reflex. after inserting the tip 1 to 2 cm into the nostril, continue to advance the tube until it reaches the desired length. if the turbinates obstruct the passage of the tube, withdraw the tube by a few centimeters. then readvance the tube, taking care to direct the tube ventrally through the nasal cavity. occasionally it will be necessary to withdraw the tube completely t a b l e 4 -1 the french catheter scale equivalents * millimeters inches from the nostril and repeat the procedure. in particularly small patients or patients with obstructive lesions (e.g., tumor) in the nasal cavity, it may not be possible to pass a tube. do not force the tube against significant resistance through the nostril. gavage, or gastric lavage and feeding, in puppies and kittens can be accomplished by passing a soft rubber catheter or feeding tube into the mouth, tilting the puppy's or kitten's head, and watching it swallow the tube. most puppies or kittens will struggle and vocalize. they usually will not vocalize if the tube has been placed into the trachea. a 12f catheter is of an adequate diameter to pass freely, but it is too large for dogs and cats less than 2 to 3 weeks of age. mark the tube with tape or a pen at a point equal to the distance from the mouth to the last rib. merely push the tube into the pharynx and down the esophagus to the caudal thoracic level (into the stomach). verify the placement of the tube using the same dry syringe aspiration technique to ensure that the tube is positioned in the esophagus or stomach rather than the trachea. attach a syringe to the flared end, and slowly inject medication or food. depending on the feeding tube type, the end of the tube may or may not accommodate a syringe. for example, soft, rubber urinary catheters are excellent tubes for single administration use. however, the flared end may not accommodate a syringe. to affix a syringe to the outside end of a tapered feeding tube or catheter, insert a plastic adapter (figure 4-7) into the open end of the tube. none required. intranasal administration of liquids in dogs and cats is usually limited to a single dose of a vaccine specifically labeled for intranasal administration. there is little indication for routine instillation of liquids into the nostrils of dogs and cats. rarely, administration of isotonic solutions directly into the nostrils is indicated. in contrast to singledose vaccines, lavage solutions applied intranasally are usually multiple-dose containers. therefore the tip of the administration device should not be allowed to directly contact the patient's skin or nose. doing so may result in contamination of the entire bottle. oily drops are not advised because they may damage the nasal mucosa or may be inhaled. the technique for intranasal administration of vaccine is straightforward and usually works quite well…the first time. some animals, dogs more than cats, will aggressively resist intranasal administration of vaccine. attempts to overcome this resistance include covering the eyes with a towel or otherwise distracting the patient with noise or other visual cues. concerns expressed over the loss of vaccine immediately after intranasal administration are generally unfounded. manufacturers of intranasal vaccines typically include a greater antigen (virus or bacteria) titer per dose than is necessary to induce a protective immune response. if the patient resists aggressively and the vaccine is indicated, parenteral preparations are available for all intranasal vaccines and should be considered. several objectives should be considered when treating dermatologic disorders with topical medication: (1) eradication of causative agents; (2) alleviation of symptoms, such as reduction of inflammation; (3) cleansing and debridement; (4) protection; (5) restoration of hydration; and (6) reduction of scaling and callus. many different forms of skin medications are available, but the vehicle in which they are applied is a critical factor (box 4-1). technique in all cases, apply topical medications to a clean skin surface in a very thin film, because only the medication in contact with the skin is effective. in most cases, clipping hair from an affected area enhances the effect of medication. when dispensing medications to owners for home administration, the owner should be instructed to wear disposable examination gloves if using fingers and hands to apply the medication. with the widespread availability of compounding pharmacies, prescribing compounded medications for topical and oral administration recently has become a popular dispensing technique for dogs and cats requiring long-term, daily medication. caution is warranted. some compounding pharmacies that serve the veterinary profession are using inappropriate or ineffective vehicles in which the drug has been compounded, or the drug itself, purchased in bulk, is of a lower grade and possibly an ineffective product once compounded. studies on the quality and efficacy of compounded drugs for use in veterinary patients are limited. however, of those studies that have been performed, serious questions are being raised over the bioavailability of the drug administered. it would be admirable to prepare the skin surgically before making needle punctures to administer medications. because such preparation is not practical, carefully part the hair and apply a high-quality skin antiseptic such as isopropyl alcohol. place the needle directly on the prepared area, and thrust the needle through the skin. although the use of antiseptics on the vial and skin is not highly effective, the procedure removes gross contamination and projects an image of professionalism. before aspirating medications from multiple-dose vials, carefully wipe the rubber diaphragm stopper with the same antiseptic used on the skin. observe this basic rule with all medication vials, even with modified live virus vaccines. technique dogs and cats have abundant loose alveolar tissue and easily can accommodate large volumes of material in this subcutaneous space. the dorsal neck is seldom used for subcutaneous injections because the skin is somewhat more sensitive, causing some patients to move abruptly during administration. a wide surface area of skin and subcutaneous tissue over the dorsum from the shoulders to the lumbar region makes an ideal site for subcutaneous injections. administration of drugs, vaccines, and fluids by the subcutaneous route represents the most commonly used route of parenteral administration in dogs and cats. for small volumes (<2 ml total), such as vaccines, a 22-to 25-gauge needle generally is used. the site most often used is the wide area of skin over the shoulders. the large subcutaneous space and the relative lack of sensitivity of skin at this location make it an ideal injection site. cleaning of the skin with alcohol or other disinfectant generally is performed before injection. several injection techniques are used. a common technique entails grasping a fold of skin with two fingers and the thumb of one hand. gently lift the skin upward. using the opposite hand, place the needle, with syringe attached, through the skin at a point below the opposite thumb. aspiration before injection is not typically necessary when using this route of administration. after administration and on removal of the needle from the skin, gently pinch the injection site and hold it for a few seconds to prevent backflow of medication or vaccine onto the skin. when larger volumes are to be administered-fluids in dehydrated dogs and cats-the skin directly over the shoulders is the injection site most commonly selected. generally, only isotonic fluids are administered by the subcutaneous route. depending on the patient's 4 size, needles ranging from 16 to 22 gauge can be used. because of the larger volumes of fluid involved, warming of the fluids before administration is recommended. doing so can enhance significantly the patient's tolerance for the displacement of skin during the period of administration and, in small patients, prevent hypothermia. depending on the rate of administration and breed of dog, relatively large volumes of fluid generally can be given in one location. cats typically tolerate 10 to 20 ml/kg body mass in a single location. large dogs can tolerate volumes greater than 200 ml of fluid in a single location. when administering large volumes, it is usually not necessary to use multiple injection sites for purposes of distributing the total fluid volume. doing so actually may increase the risk of introducing cutaneous bacteria under the skin. because the administration time required to deliver larger volumes is longer, and the injection needle will be placed in the skin for extended periods, it is appropriate to cleanse and rinse the skin carefully before actually inserting the needle. isotonic, warmed fluids may be administered by large syringe or through an administration tube attached to a bag. monitor skin tension and the patient's comfort tolerance throughout the procedure. although fluid absorption begins almost immediately on subcutaneous administration of fluids, significant pressure caused by the bolus of fluid delivered can develop within the fluid pocket. on removal of the needle, firmly grasp the injection site with the thumb and forefinger for several seconds. the procedure is not complete until one has verified that back-leakage of fluid from the subcutaneous space onto the skin is not occurring. depending on the patient's hydration status and physical condition, fluid absorption may take from 6 to 8 hours. the rate of absorption of fluid administered by the subcutaneous route largely depends on the patient's hydration state and vascular and cardiac integrity. for that reason, the subcutaneous route is not recommended to manage patients in hypovolemic shock. exceptions to this do exist-for example, when in a life-or-death situation access to a vein is simply not possible. subcutaneous or intraosseous (see the following discussion) fluid administration may be the only option available. recently, implantable subcutaneous ports* have been introduced for use in patients requiring regular administration of subcutaneous fluids at home. a 9-inch silicon tube is preplaced under the skin and is sutured in place by a veterinarian. objectively, this offers easy access to the subcutaneous space without the need for needle penetration. owners simply attach a syringe or extension tube tip to the port and administer the appropriate volume of fluids at an appropriate rate and frequency. because of the usual requirement for long-term placement of an implantable fluid administration tube, there is some risk of infection under the skin and around the incision site. some cats do not tolerate the device. *gif-tube single implant kit for subcutaneous fluid administration, various models available. phoenix, arizona, www.practivet.com (owner instruction guide is also available). note: not all parenteral medications can be administered safely by the subcutaneous route. when administering any compound by the subcutaneous route, verify that the product to be administered is approved for subcutaneous administration. serious reactions, including abscess formation and tissue necrosis, can occur. none required. because the tightly packed muscular tissue cannot expand and accommodate large volumes of injectables without trauma, medications given by the intramuscular route should be small in volume. these medications are often depot materials that are poorly soluble, and some may be mildly irritating. unless the animal is extremely thin, give injections into the lumbodorsal muscles on either side of the dorsal processes of the vertebral column. after proper preparation of the skin, insert the needle through the skin at a slight angle (if the animal is thin) or perpendicularly (if the animal is obese). when injecting any medication by a route other than the intravenous one, it is imperative to retract the plunger of the syringe before injecting to be certain that a vein was not entered by mistake. this is especially crucial with oil suspension, microcrystalline suspension, or potent-dose medications. never give intramuscular injections in the neck because of the fibrous sheaths there and the complications that may occur. also, intramuscular injections into muscles of the rear legs can cause severe pain, lameness, and occasionally peroneal nerve paralysis because of local nerve involvement. intracutaneous (or intradermal) injections are used for diagnostic testing purposes. prepare the skin by carefully clipping the hair with a no. 40 clipper blade. if the skin surface is dirty, gently clean it with a moist towel. scrubbing and disinfection are contraindicated because they may produce iatrogenic trauma and inflammation, which interfere with the test. stretch the skin by lifting a fold, and use a 25-to 27-gauge intradermal needle attached to a 1-ml tuberculin syringe. insert the point of the needle, bevel up, in a forward lifting motion as if to pick up the skin with the needle tip. advance the needle while pushing the syringe (levered) downward until the bevel is completely within the skin. inject a bleb of 0.05 to 0.10 ml of fluid. if the procedure is done correctly, the small bleb will appear translucent. intradermal injections generally are used in patients subjected to intradermal skin testing for allergenic antigens. administration of compounds by the intradermal technique is not necessarily simple. inadvertent administration of medications into the subcutaneous tissues is easy when attempting intradermal injection. for that reason, specific training and experience are recommended before attempting intradermal skin testing of allergic patients. none required. intradermal administration of vaccine and drugs in veterinary and human medicine largely has been limited to the complexities of accurately delivering the desired dose into, and not under, the skin. in 2004 a transdermal administration system* was introduced for cats (recombinant feline leukemia virus [felv] vaccine) that was designed after a similar device *vet-jet transdermal administration system, merial, duluth, georgia. used in human (pediatric) medicine. recently the transdermal administration system used for administration of the recombinant felv vaccine has been re-designed. this same administration system is now used for the transdermal administration of the oral melanoma vaccine. the transdermal administration system consistently delivers a precise volume of vaccine into the skin, subcutaneous tissues, and muscle. use of the transdermal administration system should only be used to administer those vaccines approved for this method of delivery. administration of vaccine using the transdermal administration system requires training to understand proper procedure for loading and administering vaccine. at this writing, sale of the transdermal administration system for delivery of the canine oral melanoma vaccine is limited to select specialists in veterinary medicine. see section 1. none required. generally, two techniques are used. oscillometric blood pressure (bp) measurement entails use of an automated recording system. a cuff is applied to the base of the tail or a distal limb for access to an artery. this technique generally is regarded as being most accurate in dogs. when oscillometric bp measurements are performed in dogs, the patient should be in lateral recumbency. this places the cuff at approximately the same level as the heart. in cats the patient generally remains in sternal recumbency (and minimally restrained). most patients experience a brief acclimation period to the cuff placement. for this reason, at least three to five separate readings are obtained at 1-to 2-minute intervals. this technique can be used on awake or anesthetized patients (figure 4-8) . the doppler-ultrasonic flow detection system is most accurate in cats for measuring systolic bp. again, the ventral tail base or a dorsal pedal artery (hindlimb) or the superficial palmar arterial arch (forelimb) can be used. apply and inflate an occluding cuff. the readings are obtained by a transducer as the pressure on the cuff is reduced. caution is recommended in interpreting results from dogs that are reported as hypertensive but have no overt clinical disease. the higher reported occurrence of falsely elevated bp in normotensive dogs measured by this method justifies additional scrutiny when interpreting doppler bp results in dogs. clinically, the most common use of indirect bp measurement is in assessing cats for the presence (or absence) of systemic hypertension caused by renal insufficiency or hyperthyroidism (thyrotoxicosis). a common finding among untreated hypertensive cats is retinal detachment and blindness. early detection and therapeutic intervention (e.g., enalapril and or amlodipine) is critical. in dogs, bp measurement is indicated in patients with chronic renal insufficiency and/or protein-losing nephropathy, hyperadrenocorticism, and diabetes mellitus. in veterinary medicine, interpretation of bp centers on the systolic bp reading, not the diastolic reading (table 4-2) . stepien rl: blood pressure assessment. results dictate that even routine bacterial cultures and identification are best reserved for the commercial laboratory equipped to carry out these increasingly complex procedures and experienced in doing so. what follows are fundamental methods and techniques used to properly collect diagnostic specimens and the appropriate methods for transporting samples to a laboratory in order for the best possible diagnostic result to be obtained. before actually collecting and submitting a sample to a laboratory for bacterial culture, it is appropriate (whenever feasible to do so) to prepare, stain, and examine, under direct microscopy, exudates or fluid from the suspect material or tissue. staining the air-dried sample with a rapid romanowsky-type stain (e.g., diff-quik stain) or a gram stain may reveal evidence of neutrophilic inflammation (neutrophilia, especially with a left shift) and occasionally degenerative neutrophils with intracellular bacteria visible. these findings greatly facilitate patient management by documenting the immediate need for interventive empiric antimicrobial therapy until definitive culture and antimicrobial susceptibility results are obtained. the absence of cytologic evidence of bacterial infection does not rule out the possibility that the patient is infected or bacteremic (table 4-3) . collecting diagnostic samples for bacterial culture should be attempted as early in the disease process as possible. it is also critical to accomplish the sample collection under aseptic conditions. it is appropriate, therefore, to perform a surgical scrub of the skin or tissue from which the sample is to be collected in advance. this is especially true for tissue biopsies and fluid samples collected by needle aspiration through intact skin. failing to adequately prepare the collection site can result in significant contamination and complicate diagnostic interpretation of results. in addition, it is recommended to collect the diagnostic sample before the administration of antibiotics in order to minimize the risk of false-negative culture results. in the event antimicrobials have been administered to a patient with a suspected infection, and that is not responding to treatment, discontinuing treatment for 48 hours before attempting sample collection is generally recommended. collection of an adequate amount, or volume (fluid), is equally important in obtaining meaningful result. for example, a single sterile cotton-tipped swab of contaminated tissue should be considered inadequate sampling and inappropriate for any patient. multiple specimens are always recommended when feasible. also, biopsy material, surgically removed tissue, and several milliliters of fluid (e.g., urine) should be collected and placed in a sterile container that can be appropriately sealed (leak-proof container) before transport. a "clean catch" of urine in a "clean cup" is not appropriate. inexpensive commercial containers specifically designed for the transport of infectious material are readily available today and should be used. many containers designed to hold bacterial samples contain buffered, nonnutritive transport media to sustain the growth of pathogenic bacteria yet minimize overgrowth of bacterial contaminants during the time required to transport the sample. most commercial laboratories provide appropriate containment devices for the transport of bacterial samples. because most diagnostic specimens collected for bacterial culture are submitted to commercial laboratories for bacterial isolation, identification, and antimicrobial susceptibility testing, it is important to prepare the sample properly for shipping. special transport media are generally not required for routine aerobic culture specimens as long as the sample can remain moist and relatively cool and the sample can t a b l e 4 -3 common bacterial culture results-cont'd be inoculated onto culture medium within 3 to 4 hours only. for samples that must be shipped overnight to a laboratory, it is imperative that the specimen be kept cool (not frozen) and moist. elevated temperatures during shipping contribute to bacterial overgrowth of nonpathogenic bacteria, making isolation and identification of diseaseproducing organisms difficult. special transport media may be required. contact the individual laboratory regarding information pertaining to shipping of specimens for bacterial culture. specimens submitted for anaerobic culture need to be inoculated onto culture media within minutes after collection. although special anaerobic transport media are available, they may not be well suited for extended shipping times (>4 hours). among the most frequently tested fluids for bacteria, urine supports the growth of several types of bacteria. therefore it is necessary that the genitalia be cleaned before collection of urine (free-catch specimen) or cystocentesis (preferred). use of a urinary catheter to collect urine is likely to introduce urethral bacteria and may result in false-positive culture results. bacteria will survive for only a limited time in urine. samples collected must be sealed, and unless processed within 2 hours the sample must be refrigerated. samples held for longer than 8 hours may not contain viable bacteria. if extended transportation times are required to reach a laboratory, a urine reservation tube (vacutainer brand urine transportation system, bd, franklin lakes, new jersey) will allow storage for up to 48 hours at room temperature (table 4-4) . collection from fluid filled compartments (e.g., abscesses, seromas) requires collection with a needle and syringe. the maximum quantity possible should be collected and submitted. the skin or tissue overlying the area from which the sample is to be collected should be surgically prepared. if it becomes necessary to flush an open lesion (or perform tracheatranstracheal aspiration or bronchoalveolar lavage [bal]), it is recommended that a buffered solution of sterile ringer's lactate be used. use of fluids that contain preservative may actually inhibit the growth of bacteria. if it is necessary to submit fecal material for specific bacterial isolation, at least 2 to 3 g of feces should be submitted. a single cotton-tipped swab inserted rectally is unlikely to yield meaningful results. multiple (up to three) samples are recommended when attempting to isolate specific pathogens (e.g., salmonella). samples should be submitted in a sealed, leakproof container (always appreciated by the lab). the containerized sample should refrigerated if there is a significant delay (several hours) involved in submission to the laboratory. confirmation of the presence of bacteria in the blood (bacteremia) can be difficult and requires some patient preparation before collection of a series of samples. in addition, samples should be collected only in vials clearly marked for the collection of blood. furthermore, there are several reasons for an infected patient to have a negative blood culture result-for example, prior or concurrent antimicrobial therapy, chronic (low-grade) infection, and intermittent shedding of bacteria into blood. sample volume, numbers of samples submitted, skin preparation, and timing of collections are variables that can directly affect results. clip and surgically prepare the skin over the cephalic, recurrent tarsal, and/or jugular veins. do not draw blood for culture through an indwelling intravenous or intraarterial catheter. collection vials are available for aerobic and anaerobic bacterial culture. it is generally recommended that three blood samples be collected from separate veins over a 24-hour period. there is no advantage to collecting arterial blood. it has been suggested that samples collected during times when the patient is febrile may improve the likelihood of isolating bacteria. the volume of blood collected is determined by the size of the patient, the collection vials (adult, pediatric, infant) used, as well as the laboratory equipment used to propagate the culture. in addition to adult human blood culture collection vials (10 ml), pediatric blood collection vials (5 to 10 ml) and infant collection vials (0.5 to 1.0 ml) are available. it is appropriate that sterile technique be adhered to during collection of all samples. this includes the use of sterile gloves by the individual collecting the sample. once blood has been collected, air should not be allowed to enter the collection vial. the vial should be gently inverted (never shaken) two to four times. vials may be maintained at room temperature (the laboratory maintains samples at 37° c). the opportunity to submit complementary cultures (e.g., from urine) from patients in which blood cultures are being collected can help to confirm the infecting bacteria and may lead to identification of a likely source (boxes 4-2 and 4-3). samples collected from patients suspected of having fungal infections of the nasal cavity (e.g., aspergillosis) or systemic (also called "deep") mycotic infections (e.g., histoplasmosis, cryptococcosis) are usually assessed by cytopathology or serology (see section 5) or with tissue biopsy and histopathology involving special stains. direct cytologic assessment of samples from patients suspected of having fungal infections is always indicated. you certainly get credit for trying! however, experience in recognizing diagnostic elements of individual fungi and spores is essential, as is the availability of special stains for wet mount (10% potassium hydroxide) cytopathology. scrapings of skin and plucked hair shafts are commonly selected for fungal culture. the area of skin and hair to be sampled should be cleaned with 70% alcohol. iodine-based soaps and solutions should not be used. hair shafts, particularly those immediately adjacent to the lesion, are removed from skin with a sterile hemostat. skin scrapings can be collected with a sterile surgical blade or the edge of a clean (unused) microscope slide. scrapings from healthy, normal-appearing skin as well as abnormal skin should be collected. skin biopsy may be required if results of attempts to culture hair and skin scrapings are negative. sterile cotton-tipped swabs should not be used to collect samples for fungal culture. hair and skin scrapings can be placed directly into a sterile, dry container without need for any type of media as long as the sample can be processed within hours. refrigeration is generally not required. if transport times are extended, it is reasonable to place samples in a vial containing bacterial transport medium and refrigerate for up to 15 hours. samples should never be frozen. skin and hair samples from patients suspected of having superficial fungal infections can be inoculated directly on a commercially available substrate called dermatophyte test medium (dtm). because samples can remain at room temperature and do not require special handling, the use of dtm is ideal for in-hospital use. the medium contains phenol red as a ph indicator. if a dermatophyte is present, characteristic colony morphology will be observed and the medium underlying the colonies will turn red. vials are unreliable after 2 weeks; color change noted 2 weeks or more after inoculation of the dtm should be disregarded. ultraviolet light filtered through nickel oxide produces a beam called wood light. if an animal is taken into a dark room and its hair and skin are exposed to a wood lamp, fluorescence may occur for several reasons. hair shafts affected by some species of microsporum fluoresce a bright yellow-green (like the color of a fluorescing watch face). however, iodide medications, petroleum, soap, dyes, bacteria, and even keratin may produce purple-, blue-, or yellow-colored fluorescence. the positive fungal fluorescence is a valuable aid in selecting affected hairs for culture inoculation. however, a negative fluorescence does not preclude a possible diagnosis of fungal infection. false-negative and false-positive interpretations are common. various laboratory techniques are currently available for the identification of viral infections in dogs and cats. excellent qualitative testing platforms are commercially available for in-practice use. molecular diagnostic tests, viral culture, histopathology, and serology, all of which are routinely available to veterinarians, require that samples be submitted to commercial laboratories for assessment. among the in-hospital test systems used to identify virus, the enzyme-linked immunosorbent assay (elisa) is the most common testing platform used. elisa testing can be performed quickly (minutes) with little or no patient preparation and with relatively high sensitivity and specificity. virus (antigen) detection tests are available as point-of-care tests for felv antigen in blood or serum and canine parvovirus (cpv) antigen in feces. in addition, these point-of-care tests for viral infections are capable of identifying patients that have not been exposed, enabling the clinician to rule out infection and viral shedding. test sensitivity refers to the likelihood that a patient with known infection will have a positive test result (a test with high sensitivity is expected to have few false-positive results). test specificity refers to the likelihood that a patient that is free of the infection will have a negative test result (a test with high specificity is expected to have few false-negative results). in addition, many commercial and point-of-care nonquantitative serologic assays are available that detect antibody to many of the viruses that affect dogs and cats. however, the positive predictive value of antibody tests is typically lower than that of antigen tests. for example, a positive antibody test result to a particular viral pathogen typically does not constitute a diagnosis of infection, especially in the absence of clinical signs. it may merely reflect recent vaccination (e.g., feline immunodeficiency virus). on the other hand, a negative antibody test result generally does indicate that the patient has had no prior exposure to the virus (or vaccine). serology refers to the use of serum to detect the concentration of antibody and is widely used in veterinary medicine. the value of antibody titers in diagnosing a viral infection is dependent on a number of factors, including the infecting virus, vaccination history, and time since exposure. use of acute and convalescent antibody titers in a patient suspected of having an acute viral infection can be a reliable diagnostic tool if a fourfold or greater increase in titer can be demonstrated over 2 to 4 weeks. acute and convalescent viral titers in individual patients are rarely performed in veterinary medicine. virus isolation, however, is a valuable diagnostic tool that is underused in veterinary medicine, perhaps because of the limited number of commercial and university laboratories that provide viral isolation services and the increased availability of molecular diagnostic testing services. diagnosis of viral upper respiratory infection in cats (herpesvirus 1 and/or calicivirus) is perhaps among the situations for which virus isolation can be most useful, especially in cluster households where many shedding carrier cats exist and kittens may be at risk. to obtain a sample for viral isolation from the oral cavity of a cat, quickly insert a sterile cotton swab into the oral cavity to the level of the tonsil or oropharynx. by rolling the swab across the epithelium, it is possible to harvest cells and virus from infected cats. immediately place the swab into a virus transport medium (usually provided by the laboratory). antibiotics added to the solution prevent bacterial overgrowth of the sample. for short-term transit (5 days or less), hold specimens for viral isolation at 4° c rather than frozen. on reaching the laboratory, the specimen will be inoculated into a suitable tissue culture. within a few days it is usually possible to establish, based on the cytopathic effect on the tissue culture, whether a virus infection is present. fluorescent antibody testing can be done subsequently to confirm the isolate. although availability is limited, direct assessment of specimens (e.g., feces for cpv or canine or feline coronavirus) can be accomplished by electron microscopy. these methods can be useful for infections in which the virus concentration in the specimen reaches 10 6 to 10 7 organisms per milliliter. specimens such as feces, vesicle fluid, brain tissue, urine, or serum can be submitted for electron microscopy. tissue specimens and exfoliative cytologic preparations can be submitted for viral identification by histopathology, immunohistochemistry, and direct fluorescent antibody testing. such testing has limited application in patients with active disease because of the limited availability of these types of services and the time required for samples to be processed and reviewed by a pathologist. these tests can be particularly useful in postmortem diagnostics when multiple animals are potentially at risk. molecular diagnostics refers to the use of nucleic acid-based tests for the detection of viral dna or rna. polymerase chain reaction (pcr) is a laboratory technology that offers exceptional test sensitivity. through its ability to amplify trace amounts of dna or rna from pathogenic organisms millions of times, pcr facilitates identification of the "target" sequence of nucleic acid and therefore the infecting organism. this technology is also available commercially for the detection of dna from selected bacteria and rickettsiae. pcr technology is particularly useful in the very early stages of a viral infection, when the level of antibody has not yet reached levels that are detectable with conventional antibody tests. in addition, pcr testing may detect healthy virus carrier animals that pose a risk to a larger population of susceptible animals yet cannot be identified by conventional virus isolation or identification technologies. it should be noted, however, that pcr technology is still subject to false-positive and false-negative test results. therefore such testing is not necessarily indicated as a primary or exclusive test method for an individual patient. serology serum, plasma, or other fluids (e.g., cerebrospinal fluid [csf]) can be tested for the presence of antibodies to selected pathogenic viruses. whole blood samples should be allowed to thoroughly clot and retract (or the sample should be centrifuged) before serum is collected. samples are submitted in a leak-proof vial. refrigeration is appropriate for samples that must be held for several hours before testing. samples are limited to tissue obtained during surgical biopsy. as with conventional histopathology, samples (no more than 5.0 mm thick) should be placed in 10% buffered formalin and submitted in a leak-proof vial. it is recommended that the volume of formalin used be at least 10 times greater than the tissue sample submitted. testing can be performed on tissues collected during surgical biopsy or from tissue impressions (exfoliative) made from tissue imprints on a clean microscope slide. it is recommended that tissue impressions on slides be fixed in alcohol or acetone before submission. fresh tissue is submitted on wet (not dry) ice and is not subjected to formalin fixation. small amounts of tissue suitable for electron microscopy should be no larger than 1 × 2 mm thick. fixation in 2% to 4% glutaraldehyde for 24 hours at 20° c is required. feces and body fluids collected for electron microscopy should be submitted fresh, not frozen or fixed in preservative. if shipping is required, feces and body fluids may be refrigerated or shipped on wet ice. samples should be viable for 48 to 72 hours. sterile swabs may be used to collect samples for viral culture and isolation. samples should be inoculated into a sealed vial containing viral transport medium (usually provided by the laboratory). samples should not be frozen or fixed in preservative. laboratories offering pcr testing typically accept serum or anticoagulated (ethylenediaminetetraacetic acid [edta]) whole blood in leak-proof vials. samples should be refrigerated and shipped on wet ice. samples should not be frozen. in most instances, a 3-to 5-ml sample of anticoagulated whole blood is adequate for routine hematology; some laboratories will accept as little as 1 ml. for routine biochemical analyses, the volume of serum requested can vary from 1 to 2 ml, depending on the number and type of tests requested. plan ahead which samples are required to prevent the need for further venipuncture at a later time. in small dogs and cats, using the jugular veins facilitates collection of an adequate volume of blood. if smaller samples are required, the cephalic, lateral saphenous, or medial saphenous vein can be used for sample collection. do not use the jugular vein if a coagulopathy is suspected, as hemorrhage may be difficult to control after venipuncture. for successful venipuncture, proper restraint of the animal is important. details for the proper restraint for various venipuncture locations are discussed with each specific topic throughout this text. the patient must remain comfortable yet relatively motionless to avoid iatrogenic vessel laceration. stretch the skin tightly over the selected vessel without causing vascular occlusion to help anchor the vessel in place during penetration by the needle. the specific venipuncture will vary somewhat depending on the specific vein selected. the following sections describe venipuncture technique for each of four commonly accessed veins: the cephalic vein, jugular vein, lateral saphenous vein, and medial saphenous vein. to restrain a dog or cat for venipuncture of the cephalic vein, place the dog or cat on the table, sitting or in sternal recumbency. if the right vein is to be tapped or catheterized, the assistant should stand on the left side of the animal and place the left arm or hand under the animal's chin to immobilize the head and neck. the assistant should reach across the animal and grasp the leg just behind and distal to the right elbow joint. the assistant should use the thumb to occlude and rotate the cephalic vein laterally while the palm of the hand holds the elbow in an immobilized and extended position. make sure that the animal stays on the table if struggling occurs. the person performing the venipuncture then grasps the leg at the metacarpal region and begins the venipuncture on the medial aspect of the leg, just adjacent to the cephalic vein proximal to the carpus. for a jugular venipuncture in the dog, place the patient in sternal recumbency, with the hands of the assistant placed around the patient's muzzle to extend the neck and nose dorsally toward the ceiling. in short-coated dogs, the jugular vein usually can be seen coursing 4 from the ramus of the mandible to the thoracic inlet in the jugular furrow. the vessel may be more difficult to visualize in dogs with long hair coats or if excessive subcutaneous fat or skin is present. the person performing the venipuncture should place the thumb of the nondominant hand across the jugular vein in the thoracic inlet or proximal to the thoracic inlet to occlude venous drainage from the vessel and allow it to fill. with the dominant hand, the person performing the venipuncture should insert the needle and syringe or vacutainer (bd, franklin lakes, new jersey) into the vessel at a 15-to 30-degree angle to perform the venipuncture. for smaller and very large animals, the jugular vein also can be tapped by placing the patient in lateral recumbency. the assistant should pull the animal's front legs caudally and extend the head and neck so that the jugular vein can be visualized. the venipuncture then can be performed as previously described. a jugular venipuncture is contraindicated in patients with thrombocytopenia or vitamin k-antagonist rodenticide intoxication. place cats in sternal recumbency. the assistant should stand behind the patient so that the patient cannot back away from the needle during the venipuncture. the assistant should extend the cat's head and neck dorsally while restraining the cat's front legs with the other hand. the cat's fur can be clipped or moistened with isopropyl alcohol to aid in visualization of the jugular vein as it stands up in the jugular furrow. the person performing the venipuncture should occlude the vessel at the thoracic inlet and insert the needle or vacutainer apparatus into the vessel as previously described to withdraw the blood sample. alternately, place the cat in lateral recumbency as described in the previous paragraph. to perform a lateral saphenous venipuncture, place the patient in lateral recumbency. the lateral saphenous vein can be visualized on the lateral portion of the stifle, just proximal to the tarsus. the assistant should extend the hindlimb and occlude the lateral saphenous vein just proximal and caudal to the tarsus. the person performing the venipuncture should grasp the distal portion of the patient's limb with the nondominant hand and insert the needle or vacutainer apparatus with the dominant hand to withdraw the blood sample. to perform a medial saphenous venipuncture, place the patient in lateral recumbency. move the top hindlimb cranially or caudally to allow visualization of the medial saphenous vein on the medial aspect of the tibia and fibula. the assistant should scruff the patient, if the patient is small, or should place the forearm over the patient's neck to prevent the patient from getting up during the procedure. with the other hand, the assistant should occlude the medial saphenous vein in the inguinal region. the person performing the medial saphenous venipuncture should grasp the paw or hock of the limb and pull the skin taut to prevent the vessel from rolling away from the needle. the fur may be clipped or moistened with isopropyl alcohol to aid in visualization of the vessel. the needle or vacutainer apparatus can be inserted into the vessel at a 15-to 30-degree angle to withdraw the blood sample. incorrect proportions of blood to anticoagulant may result in water shifts between plasma and red blood cells (rbcs). such shifts may alter the packed cell volume (pcv), especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results. refrigerate liquid blood mixed with anticoagulant after collection if the sample is to be held before being transported to a laboratory. white blood cell (wbc) and rbc counts, pcv, and hemoglobin level can be measured within 24 hours of sample 4 collection. platelet counts, however, should be done within 1 hour of collection. dried, unfixed blood smears can be stained with most conventional stains 24 to 48 hours after being made. if a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least 5 minutes. blood smears fixed by this method are stable indefinitely. never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. take care to leave unfixed blood smears face down on a countertop or in a closed box. special stains, such as peroxidase, may require fresh blood films. the anticoagulant of choice for hematologic testing is edta. heparin is especially to be avoided if blood films are to be made from blood mixed with anticoagulant because contact with whole blood will distort the morphology of cells significantly. heparin is acceptable for most procedures requiring blood plasma. the anticoagulant effect of heparin is transitory. specimens still may clot after 2 to 3 days. make blood films immediately after collection because cell morphology rapidly deteriorates after sample collection. although blood films can be made after introducing blood to edta, a better practice is to make blood smears (films) immediately from the collection needle before the blood comes in contact with any anticoagulant. never use blood exposed to heparin to make blood smears. incorrect proportions of blood to anticoagulant may result in water shifts between plasma and rbcs. such shifts may alter the pcv, especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results. refrigerate liquid blood mixed with anticoagulant after collection if there is a delay in making the laboratory determinations. wbc and rbc counts, pcv, and hemoglobin level can be measured within 24 hours of sample collection. platelet counts, however, should be done within 1 hour of collection. dried, unfixed blood smears can be stained with most conventional stains 24 to 48 hours after being made. if a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least 5 minutes. blood smears fixed by this method are stable indefinitely. never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. take care to leave unfixed blood smears face down on a countertop or in a closed box. special stains, such as peroxidase, may require fresh blood films. prepare the selected vein as described earlier. most clinical chemistry procedures are performed on serum. the serum is obtained by collecting blood without any anticoagulant and allowing the blood to clot in a clean, dry tube. separate serum from cells within 45 minutes of sample collection (venipuncture). special vacuum vials are available that produce a gel barrier between the clot and the serum (serum separator tubes) which avoid the need to draw off the serum into a separate vial. clotting of the blood and retraction of the clot occur best and maximum yields of serum are obtained at room or body temperature. refrigeration of the sample delays clot retraction. some samples clot and retract faster than others. if a serum separator tube is not used, it is recommended to free the clot from the walls of the container by rimming with an applicator stick. after the clot is freed, allow clot retraction to occur, and then centrifuge and draw off the clear supernatant serum using a pipette or suction bulb. allow whole blood samples to completely clot before attempting to remove serum. failing to do so may result in a mixture of plasma and serum in the submitted sample. serum yield is usually one third of the whole blood volume. patients that are hypovolemic or dehydrated can have a significantly lower serum yield. many clinical chemistry procedures can be performed on plasma and on serum. the advantage of using plasma is that separation of cells can be accomplished immediately after centrifugation or sedimentation, without the need to wait for clot formation and retraction. the disadvantage of plasma is that the presence of the anticoagulant interferes with many of the chemistry assay procedures. plasma is less clear than serum, which may be an additional disadvantage for colorimetric assays. plasma and serum are virtually identical in chemical composition except that plasma has fibrinogen and the anticoagulant. for many procedures in which plasma or whole blood is to be used, heparin is the anticoagulant of choice. heparinized blood is the only acceptable specimen for blood ph and blood gas analyses. although blood containing edta is acceptable for certain chemical procedures, it cannot be used for determination of plasma electrolytes because it contributes to and sequesters them from the specimen. in addition, edta can interfere with alkaline phosphatase levels, decrease total carbon dioxide, and elevate blood nonprotein nitrogen. refer to the tube selection guide in section 5 to assure use the proper collection tube is used for the appropriate test requested. separate serum or plasma and remove it from the cells as soon as possible after blood is collected, because many of the constituents of plasma exist in higher concentrations in rbcs. with time, these substances leak into the plasma and cause falsely elevated values (positive interference) and falsely lower values (negative interference) ( table 4 -5) . under no circumstances should whole blood be sent via the mail; serum derived from such specimens usually is hemolyzed, and results are often inaccurate. separate serum and transfer it to a clean, dry tube for shipment. bone marrow aspiration collection of bone marrow may prove valuable in diseases of the blood in which examination of the peripheral blood reveals abnormal cells or cell counts. conditions such as leukopenia, thrombocytopenia, nonregenerative anemia, agranulocytosis, pancytopenia, leukemias, other bone marrow cancers, and infectious diseases (e.g., histoplasmosis, ehrlichiosis) may be confirmed only by assessment of bone marrow cytology. bone marrow in the young animal is cellular and exists in the flat bones (sternum, ribs, pelvic bones, and vertebrae) and in the long bones (humerus and femur). as the animal ages, the cellular content of the marrow decreases, especially in the long bones. in older animals, bone marrow cells still exist in the flat bones; however, in conditions of stress in which new blood cells must be produced in large numbers, primitive cells in the bone marrow of the long bones again become active. interpretation of the bone marrow smear may be limited by (1) technique used to obtain a bone marrow specimen or (2) the specialized knowledge necessary to interpret bone marrow cells. bone marrow aspiration is much underused in clinical practice. the procedure does require some degree of skill if high-quality samples are to be obtained, but the procedure is of low risk to the patient and can be highly valuable in establishing a diagnosis or prognosis. a short-acting anesthetic occasionally may be needed, but tranquilization together with infusion of local anesthetic is usually sufficient. the site selected for aspiration or biopsy must be shaved and surgically prepared. bone marrow aspiration or biopsy is a percutaneous procedure conducted using sterile technique. the techniques involved include marrow aspiration and bone marrow core biopsy alone or in combination. when aspiration biopsy fails to produce adequate cytology (as in advanced myelofibrosis, neoplasia, or marrow aplasia), a core biopsy of bone marrow is indicated. the bone marrow aspiration needle may be a 16-gauge rosenthal needle or illinois needle for a medium-sized dog; an 18-gauge rosenthal needle for a small dog or a cat; or a jamshidi (pronounced yam-she-dee) bone marrow biopsy needle, 12 gauge for most adult dogs and 14 gauge for small dogs and cats. the selection of needles for aspiration biopsy of bone marrow is based on the biopsy site, the depth of the biopsy site, and the density of cortical bone. for bone marrow aspiration, the modified disposable illinois sternal-iliac bone marrow aspiration needle works well (figure 4-9) . for a core biopsy of bone marrow, the jamshidi bone marrow biopsyaspiration needle (pediatric, 3.5 inches, 13 gauge) can be used (figure 4 -10). the iliac crest is a commonly used site for marrow aspiration in dogs. place the animal in lateral recumbency, and prepare the aspiration site. to aspirate marrow, have the needle enter the widest part of the iliac crest and stop the needle just after penetration of the bone. remove the stylet, place a 12-ml syringe on the needle, and aspirate 0.2 ml of marrow. alternatively, the head of the humerus offers easy access to abundant bone marrow. sedation may be required. with the patient in lateral recumbency and the humerus flexed (the humerus is positioned parallel to the patient's thorax), instill local anesthetic into the skin and subcutaneous tissues to the level of the head of the humerus. the site of needle insertion is on the most proximal facet of the humoral head ( figure 4 -11). direct the needle into the bone toward the elbow and parallel to the humeral shaft. if the needle is positioned too far medially over the humeral head, it is easy to penetrate the joint capsule. although this is a common occurrence, it does not pose a risk of injury to the patient (assuming the skin was surgically prepared). if joint fluid contaminates the bone marrow aspirate, the sample will be rendered useless. contamination of the bone marrow with peripheral blood results if (1) the marrow is not aspirated immediately after the needle enters the marrow cavity or (2) if aspiration time is sustained and a large volume of blood enters the syringe subsequent to the rupture of small blood vessels in the bone marrow. perhaps the least desired technique is to obtain marrow from the proximal end of the femur by insertion of the bone marrow needle into the trochanteric fossa. make a small skin incision over the trochanteric fossa just medial to the summit of the trochanter major. insert the bone marrow aspiration needle medial to the trochanter major, and place the long axis of the needle parallel to the long axis of the femur. once the site has been selected, grasp the needle firmly. apply steady, slight pressure while alternately rotating the needle tip against the bone (fast, 180-degree clockwise and then counterclockwise movements). begin with gentle pressure until the needle begins to seat into the bone. gradually increase the pressure as the needle penetrates into the bone. insert the bone marrow needle 1 ⁄2 inch into the femoral canal. remove the stylet from the needle, and aspirate using a 12-or 20-ml syringe that contains a small volume (approximately 0.1 ml) of 4% edta. use significant negative pressure, for example, by withdrawing the plunger of a 12-ml syringe to the 8-or 9-ml mark. collection of more than 1 ml of bone marrow is unnecessary. collection of larger volumes may cause greater amounts of peripheral blood to enter the syringe, leading to hemodilution of the sample. once collection is complete, immediately transfer the aspirate to a watch glass containing approximately 0.25 ml of 4% edta. immediately mix the sample well using the end of the syringe. this is also a good time to remove the bone marrow needle from the patient. prepare slides in a manner similar to that used for peripheral blood smears. preparation of five to eight high-quality slides for submission is customary. smears are air-dried. slides may be stained using the same stains used for peripheral blood smears. bone marrow biopsy samples, usually obtained as a core, should be placed directly into 10% buffered formalin. it is generally recommended not to roll the core across a microscope slide (exfoliative cytology), as this may significantly disrupt the architecture of the sample and influence histopathologic interpretation. when submitting a bone marrow aspirate or bone biopsy, a complete blood count (cbc) should also be collected from that patient on the same day. the bone marrow sample and the cbc should be submitted together in order to obtain maximum diagnostic information. a thorough patient history should accompany the submitted samples. depending on the volume of bone marrow aspirate obtained, any additional aspirated bone marrow remaining after slides have been made can be mixed with edta in the same type of tube used to collect whole blood for a cbc. tubes may be refrigerated for short periods but never frozen. prompt shipping and processing of liquid samples of bone marrow is encouraged, as these cells tend to rapidly undergo degeneration. bone marrow biopsy core samples, after fixation in 10% buffered formalin, require decalcification before processing and interpretation. when feasible, a short-acting anesthetic administered to a cat before bone marrow aspiration or biopsy is recommended owing to the difficulty of adequately restraining a cat, even if sedated. the site selected for aspiration or biopsy must be shaved and surgically prepared. infusion of local anesthetic at the aspiration or biopsy site is appropriate. supplemental oxygen may be indicated. accessible sites for bone marrow sampling and biopsy in the cat are the iliac crest, the head of the humerus, and the proximal end of the femur via the trochanteric fossa. the techniques described for the dog can be used. smears of bone marrow are made immediately after aspiration. extrinsic thromboplastin present in bone marrow tissue will cause the marrow to clot within 30 seconds. unstained slides should be submitted. a core of bone marrow can be fixed in 10% buffered formalin before submission for decalcification and histologic preparation. another method is to aspirate the sample of bone marrow into a syringe containing 0.25 ml of 4% edta solution. expel the aspirate, up to 0.5 ml, into a sterile petri dish, from which the marrow particles can be isolated easily by aspirating an aliquot with a glass pipette, placing an appropriate volume onto several glass slides, making the appropriate number of smears. slides prepared from bone marrow aspirates should be allowed to air-dry and then labeled appropriately. slides should never be refrigerated, as moisture from condensation can alter or destroy the appearance of individual cells. cytology collection techniques (see also section 5 for additional information on slide preparation of samples to be submitted for cytopathologic examination.) cytopathology involves a simple, direct, and inexpensive technique that can yield significant diagnostic information within a short time at minimal direct cost. cytologic examination can be made of material obtained from pustules, vesicles, or the raw, ulcerated, or cut surfaces of a lesion. to make the smear, press a clean microscope slide firmly against a raw or ulcerated lesion to transfer cellular material to the slide. exudates may be collected by sterile swab or may be aspirated into a sterile syringe. roll the swab gently across the slide, or place a drop of fluid from the syringe onto the slide and carefully spread the fluid in a uniform film. transfer material from a block of tissue to the slide by gently pressing the tissue onto the slide in several locations. use various stains for different conditions. rapid stains such as new methylene blue or a quick romanowsky-type stain (e.g., diff-quik) are useful and convenient for office procedures. even wright and gram stains for evaluation of bacteria in tissues and fluids are easy to use. the presence of many bacteria, especially mixed types, may mean only surface contamination, whereas single types of bacteria, abundant polymorphonuclear wbcs, and especially phagocytosis support the diagnosis of infection and the host response to it. a few acantholytic cells (loose epidermal cells) in the smear may be compatible with infectious processes, but large numbers, or "rafts, " of acantholytic cells are highly suggestive of pemphigus and imply the need for more complex tests for positive diagnosis. large numbers of eosinophils sometimes are found in stained smears. contrary to popular opinion, they usually do not mean allergy. these cells are seen most commonly with furunculosis and may be associated with the eosinophilic granulomas, eosinophilic plaques, sterile eosinophilic pustulosis, pemphigus complex, and ectoparasites. yeasts (usually malassezia, rarely candida) commonly are found as budding cells in masses of wax and debris from ear smears. tumor cells may be recognized in some impression or aspiration samples where giemsa is a preferred stain. although special expertise is needed, cases of mastocytoma, histiocytoma, and lymphoma are recognized most easily. always prepare formalin-fixed tissues for histologic diagnosis in tumor evaluations (box 4-4). fine-needle aspiration, the use of needle and syringe to remove cells from normal and abnormal tissue, apply them to a glass slide, stain the smear, and review the results immediately is among the most useful, cost-effective procedures available in clinical practice. in most cases there will be no specific requirements for patient preparation. shaving hair over the aspiration site is generally not required. surgical preparation of the site is optional. lymph node aspiration is a procedure that can, and should, be performed routinely in clinical practice. follow proper technique to maximize the diagnostic utility of this procedure. lymph node aspiration typically is indicated (1) in patients with generalized lymphadenomegaly, (2) to evaluate abnormally enlarged solitary lymph nodes, and (3) in suspected instances of tumor metastases to lymph nodes. surgically prepare the skin over the node from which a biopsy specimen is to be taken. with one hand, localize and immobilize the lymph node; with the other hand, guide the aspiration biopsy needle into the affected node. affix a 6-ml syringe onto a 22-to 20-gauge needle (a 25-gauge needle can be used when the site to be aspirated is particularly small), and advance the needle into the lymph node. withdrawal of the syringe to approximately 0.5 ml before inserting it into the tissue is recommended. doing so helps to prevent expelling material when removing the sample from the tissue. when the needle is in position in the approximate center of the node, gradually draw negative pressure on the syringe to a level of 4 to 5 ml. hold the negative pressure in place for a few seconds. release, and then repeat two or three times. before removing the needle from the tissue, release the negative pressure in the syringe (this is why it is recommended to have 0.25 ml of air prepositioned inside). do not remove the syringe from the tissue while maintaining negative pressure, because this can enlargement of nucleus or nuclei larger than 10 nm decreased nuclear/cytoplasmic ratio multinucleation because of abnormal mitosis abnormal or frequent mitosis variations in size and shape of nuclei increase in size and number of nucleoli increased basophilia of cellular cytoplasm; increased rna content anisokaryosis or pleomorphism multinucleated giant cells box 4-4 cytologic features of malignancy 4 result in the aspiration of significant amounts of blood from the skin, thereby significantly diluting the sample with peripheral blood. eject cellular material within the needle onto clean glass slides. handle all aspirates gently. to make slides, place two slides together and pull the slides apart to avoid shearing the cells. do not compress or force slides together. in addition, a biopsy of the lymph node can (and usually should) be performed as a means of confirming or supporting diagnostic decisions made on aspirates. lymph node biopsy samples can be obtained easily and safely by punch (core) techniques (e.g., 4-mm skin biopsy punch) or tru-cut biopsy needle. the most significant limiting factors are (1) the technical ability to prepare high-quality slides and (2) the ability to interpret the cytologic findings. some experience is needed to obtain the skills needed to aspirate cells and make diagnostic preparations. significant training is required to interpret the slides adequately. however, access to cytopathologists affiliated with diagnostic laboratories today makes fine-needle aspiration a highly useful diagnostic tool. the lymph node aspiration technique, described next, illustrates the finer points of the fine-needle aspiration technique. also called "touch impression cytology, " exfoliative cytology entails preparing cytologic slides directly from the cut surface of biopsy samples. requirements for patient preparation depend on the location of the tissue from which the sample is taken. the number and quality of cells collected are best when the procedure involves the freshly cut surface of tissue. attempting to collect samples directly from skin lesions on the patient is much less likely to yield diagnostic cytology. preparation, therefore, depends on the target tissue from which slides are needed. preparation may entail local anesthesia and collection of a tissue biopsy specimen from a lesion or suspect tissue (e.g., lymph node or cutaneous tumor) or general anesthesia and an exploratory abdominal procedure (e.g., liver biopsy). once the tissue has been collected, a scalpel blade is used to make a full-thickness linear cut through the biopsy specimen. a fresh surface of the tissue of interest is exposed. using forceps or a sterile needle, gently lay the tissue on a clean glass slide. do not force the tissue onto the slide, because this can significantly damage cells. several imprints can be made from the same surface. as needed, make new cuts to obtain a fresh surface from which to exfoliate cells. allow the slide to air-dry completely. apply conventional staining, and examine the specimen when it is dry. the remaining tissue, if not significantly damaged, can be submitted for histopathologic examination (recommended). once slides have air-dried and are labeled, they may be stained and reviewed immediately or submitted for review and interpretation by a pathologist. unstained slides should not be refrigerated, as moisture from condensation can alter the cytology of the preparation. several slides should be submitted. any remaining tissue may be placed in 10% buffered formalin and submitted for histopathology. the number of diagnostic cells obtained when making slides by way of exfoliative cytology depends on the tissue. epithelial cells (e.g., carcinoma), mast cells (cutaneous mast cell tumor), and lymph nodes readily exfoliate abundant numbers of cells. excessively thick slides can make interpretation difficult. on the other hand, biopsy specimens of tissue composed predominantly of mesenchymal cells (e.g., granuloma, fibrosarcoma) do not readily exfoliate cells, and slides made from these types of tissues are typically hypocellular. contamination of the cytology specimen with peripheral blood is a common mistake that can make interpretation of the sample difficult. it may be appropriate to gently blot the cut surface of the tissue sample, thereby removing excessive blood, before making slides. depending on the tissue type and lesion, it may be possible to obtain diagnostic cytologic samples from scrapings (e.g., conjunctival epithelium for virus inclusions), brushes (e.g., material obtained during endoscopy), and swabs (e.g., ear and vaginal swabs). the cells, once harvested, can be applied delicately directly to a clean glass slide by carefully rolling or even by just touching the material to the slide to create a thin layer. allow the sample to airdry thoroughly before staining. cytologic examination of fluids obtained with needle and syringe from body cavities, cysts, and urine typically requires additional preparation to obtain adequate cell concentration to make diagnostic decisions. analyze fluid specimens with respect to protein and nucleated cell count and a morphologic description of the cells. if overall cell counts are low, centrifugation will be required to concentrate cellular material for analysis. after centrifugation, remove the supernatant (and save it). resuspend the cells in two or three drops of the supernatant. apply a single drop of the mixture to a glass slide and allow it to air-dry. i prefer not to smear the liquid onto the slide; instead, i allow the liquid to run, by gravity, from one end of the slide to the other. after the liquid is thoroughly air-dried, it can be stained and reviewed. none required. skin scrapings frequently are obtained to find and identify microscopic parasites or fungal elements in the skin. material required includes mineral oil in a small dropper bottle, a dull scalpel blade, glass slides, coverslips, and a microscope. select undisturbed, untreated skin for a scraping site. the best method is to scrape the periphery of skin lesions and avoid the excoriated or traumatized center areas. in scraping for demodectic mange, pinch a small fold of affected skin firmly and collect the surface material for examination. this procedure forces the mites out of the hair follicles and onto or near the skin surface. for sarcoptic mange, scrape large areas. select sites on the elbows, hocks, and ear margins when searching for sarcoptic mange. many or frequent scrapings may be necessary to demonstrate sarcoptic mange mites or their fecal pellets or eggs. place the accumulated material on a microscope slide and mix it with mineral oil. examine the entire area with a ×10 objective thoroughly and carefully. none required. acetate tape preparation is one of the simplest diagnostic procedures to perform when looking for the presence of ectoparasites, especially the nits of cheyletiella. use clear (not frosted) acetate tape. bend the tape into a loop around the fingers with the sticky side facing out. part the animal's hair coat, and press the tape firmly onto the skin and hair around suspect lesions. the sticky tape picks up loose particles with which it makes contact. cut the loop of tape and place the strip of tape sticky side down on a clean microscope slide. use a low-power microscope to look through the tape at the collected particles. this technique is excellent for trapping and identifying biting and sucking lice, otodectes and cheyletiella mites, flea dirt and larvae, fly larvae, and dandruff scales. acetate tape also is useful for studying hair abnormalities. use a strong hemostat to securely clamp and quickly avulse a group of 10 to 20 hair shafts. press the pointed distal ends onto sticky acetate tape (lined up like pickets in a fence), and cut the hair shafts off in the middle with scissors. likewise, press the butt ends with the hair roots onto another piece of tape. then press the tape holding the hair onto a microscope slide to allow low-power examination of the hairs through the clear tape. the tips of the hairs will be well oriented and controlled; thus, it is easy to evaluate whether the hairs are split, broken, or bitten off and whether the hair roots are in the anagen or telogen growth stage. urine can be removed from the bladder by one of four methods: (1) voided (the "free catch"), (2) manual compression of the urinary bladder (expressing the bladder), (3) catheterization, or (4) cystocentesis. for routine urinalysis, collection of urine by voiding (micturition) is satisfactory. the major disadvantage is risk of contamination of the sample with cells, bacteria, and other debris located in the genital tract and the perineal hair coat. the first portion of the stream is discarded, as it is most likely to contain debris. voided urine samples are not recommended when bacterial cystitis is suspected. compressing the urinary bladder is occasionally used to collect urine samples from dogs and cats. critical: do not use excessive pressure; if moderate digital pressure does not induce micturition, discontinue the technique. excessive pressure can culminate in forcing contaminated urine (bladder) into the kidneys, or, worse, in patients with a urethral obstruction the urinary bladder can rupture. the technique is most difficult to accomplish in male dogs and male cats. several types of urinary catheters are currently available for use in dogs and cats. the catheter types most often used today are made of rubber, polypropylene, and latex-free silicone. stainless steel catheters are occasionally used but unless placed with care these can cause damage to the urethra and/or urinary bladder. generally, urinary catheters serve one of four purposes: 1. to relieve urinary retention 2. to test for residual urine 3. to obtain urine directly from the bladder for diagnostic purposes 4. to perform bladder lavage and instillation of medication or contrast material the size of catheters (diameter) usually is calibrated in the french scale; each french unit is equivalent to roughly 0.33 mm. the openings adjacent to the catheter tips are called "eyes. " human urethral catheters are used routinely in male and female dogs; 4f to 10f catheters are satisfactory for most dogs (table 4-6) . polypropylene catheters should be individually packaged and sterilized by ethylene oxide gas. equipment needed to catheterize a male dog includes a sterile catheter (4f to 10f, 18 inches long, with one end adapted to fit a syringe), sterile lubricating jelly, povidone-iodine soap or chlorhexidine, sterile rubber gloves or a sterile hemostat, a 20-ml sterile syringe, and an appropriate receptacle for the collection of urine. proper catheterization of the male dog requires two persons. place the dog in lateral recumbency on either side. pull the rear leg that is on top forward, and then flex it ( figure 4-12) . alternatively, long-legged dogs can be catheterized easily in a standing position. before catheter placement, retract the sheath of the penis and cleanse the glans penis with a solution of povidone-iodine 1% or chlorhexidine. lubricate the distal 2 to 3 cm of the appropriate-size catheter with sterile lubricating jelly. never entirely remove the catheter from its container while it is being passed because the container enables one to hold the catheter without contaminating it. the catheter may be passed with sterile gloved hands or by using a sterile hemostat to grasp the catheter and pass it into the urethra. alternatively, cut a 2-inch "butterfly" section from the end of the thin plastic catheter container. this section can be used as a cover for the sterile catheter, and the clinician can use the cover to grasp and advance the catheter without using gloves. if the catheter cannot be passed into the bladder, the tip of the catheter may be caught in a mucosal fold of the urethra or there may be a stricture or block in the urethra. in smallbreed dogs, the size of the groove in the os penis may limit the size of the catheter that can be passed. one also may experience difficulty in passing the catheter through the urethra where the urethra curves around the ischial arch. occasionally a catheter of small diameter may kink and bend on being passed into the urethra. when the catheter cannot be passed on the first try, reevaluate the size of the catheter and gently rotate the catheter while passing it a second time. never force the catheter through the urethral orifice. effective catheterization is indicated by the flow of urine at the end of the catheter, and a sterile 20-ml syringe is used to aspirate the urine from the bladder. walk the dog immediately after catheterization to encourage urination. equipment needed to catheterize a female dog includes flexible urethral catheters identical to those used in the male dog. the following materials also should be on hand: a small nasal speculum, a 20-ml sterile syringe, lidocaine 0.5%, sterile lubricating jelly, a focal source of light, appropriate receptacles for urine collection, and 5 ml of povidone-iodine or a dilute chlorhexidine solution. use strict asepsis. cleanse the vulva with a solution of povidone-iodine or dilute chlorhexidine. instillation of lidocaine 0.5% into the vaginal vault helps to relieve the discomfort of catheterization. the external urethral orifice is 3 to 5 cm cranial to the ventral commissure of the vulva. in many instances the female dog may be catheterized in the standing position by passing the female catheter into the vaginal vault, despite the fact that the urethral papilla is not visualized directly. in the spayed female dog, in which blind catheterization may be difficult, the use of a sterilized otoscope speculum and light source (figure 4 -13), vaginal speculum, or anal speculum with a light source will help to visualize the urethral tubercle on the floor of the vagina. in difficult catheterizations it may be helpful to place the animal in dorsal recumbency (figure 4-14 and 4-15 ). insertion of a speculum into the vagina almost always permits visualization of the urethral papilla and facilitates passage of the catheter. take care to avoid attempts to pass the catheter into the fossa of the clitoris because this is a blind, possibly contaminated cul-de-sac. before attempting urinary bladder catheterization of the male cat, administer a short-term anesthetic (e.g., ketamine, 25 mg/kg im), but only after a careful assessment of the cat's physical, acid-base, and electrolyte status (see treatment of hyperkalemia in section 1). in some cases, drugs to treat hyperkalemia may be required before anesthetic induction. once the patient's electrolyte status has been evaluated and hyperkalemia, if present, addressed appropriately, anesthesia can be induced with a combination of propofol (4 to 7 mg/kg intravenously [iv]) and diazepam (0.1 mg/kg iv); then the patient is intubated and maintained on gas anesthesia. place the anesthetized patient in dorsal recumbency. gently grasp the ventral aspect of the prepuce and move it caudally in such a manner that the penis is extruded. withdraw the penis from the sheath and gently pull the penis backward. keeping sterile catheters in a freezer will help them become more rigid to facilitate passage into the urethra. pass a sterile, flexible plastic or polyethylene (pe 60 to 90) catheter or 3-to 5-inch, 3.5f urethral catheter into the urethral orifice and gently into the bladder, keeping the catheter parallel to the vertebral column of the cat. caution: never force the catheter through the urethra. the presence of debris within the urethral lumen may require the injection of 3 to 5 ml of sterile saline to back-flush urinary "sand" or concretions so that the catheter can be passed. in some instances the presence of cystic and urethral calculi will prevent the passage of a catheter into the urethra. for this reason a lateral radiograph of the penis, with the patient's hindlimbs pulled caudally, may help document the presence of a urethral stone. urinary bladder catheterization of the female cat is not a simple procedure. when indicated, and after a preanesthetic examination has been performed, attempt the technique only in the anesthetized cat. urinary bladder catheterization can be accomplished with the use of a rubber or plastic, side-hole (blunt-ended) urinary catheter. the same catheter type used in male cats is effective in female cats. instilling lidocaine 0.5% has been recommended as a means of decreasing sensitivity to catheter insertion in sedated (not recommended) cats. cleanse the vulva with an appropriate antiseptic. catheterization can be accomplished with the cat in dorsal or ventral recumbency. experience and size of the cat dictate which technique works best. after cleansing of the perineum and vaginal vault, place the patient in sternal recumbency, and gently pass the catheter along the ventral floor of the vaginal vault. conversely, if the patient is placed in dorsal recumbency, direct the catheter dorsally along the ventral vaginal floor. if a catheter cannot be placed blindly, a small otoscopic speculum can be placed into the vagina, and the catheter pushed into the urethral papilla once it is visualized directly. for continuous urine drainage in the awake, ambulatory patient, use a closed collection system to help prevent urinary tract infection. a soft urethral or foley catheter can be used, and polyvinyl chloride tubing should be connected to the catheter and to the collection bag outside the cage. the collection bag should be below the level of the animal's urinary bladder. place an elizabethan collar on the animal to discourage chewing on the catheter and associated tubing. the urinary bladder is catheterized as described previously. despite the quality of care of the catheter, urinary tract infection still may develop in any patient fitted with an indwelling urinary catheter. ideally, remove the catheter as soon as it is no longer necessary, or if there are clinical signs of a urinary tract infection or previously undiagnosed fever. a urinary catheter is generally changed after it has been in place for more than 48 hours. observe the patient for development of fever, discomfort, pyuria, or other evidence of urinary tract infection. if infection is suspected, remove the catheter and submit urine for culture and sensitivity or determination of minimum inhibitory concentration (mic). previously, culture of the catheter tip was recommended to diagnose a catheterinduced infection. however, culture of the catheter tip is no longer recommended, as it may not accurately reflect the type of microorganisms in a urinary tract infection. the empiric use of antibiotics to help prevent catheter-induced infection is not recommended, as their use can allow colonization of resistant nosocomial bacteria in the patient's urinary tract. cystocentesis is a common clinical technique used to obtain a sample of urine directly from the urinary bladder of dogs and cats when collecting a voided, or free-catch, aliquot is not preferred. the procedure is indicated when necessary to obtain bladder urine for culture purposes. urine that is collected by free catch has passed through the urethra and may be contaminated with bacteria, thereby making interpretation of the culture results difficult. cystocentesis also is performed as a convenience when it is desirable to obtain a small sample of urine but the patient is not ready or cooperative. cystocentesis involves insertion of a needle, with a 6-or 12-ml syringe attached, through the abdominal wall and bladder wall to obtain urine samples for urinalysis or bacterial culture. the technique prevents contamination of urine by urethra, genital tract, or skin and reduces the risk of obtaining a contaminated sample. cystocentesis also may be needed to decompress a severely overdistended bladder temporarily in an animal with urethral obstruction. in these cases, cystocentesis should be performed only if urethral catheterization is impossible. warning: penetration of a distended (obstructed) urinary bladder with a needle could result in rupture of the bladder. to perform cystocentesis, palpate the ventral abdomen just cranial to the junction of the bladder with the urethra, and trap the urinary bladder between the fingers and the palm of the hand. use one hand to hold the bladder steady within the peritoneal cavity while the other guides the needle. next, insert the needle through the ventral abdominal wall into the bladder at a 45-degree angle (figure 4-16) . although this procedure is relatively safe, the bladder must have a reasonable volume of urine, and the procedure should not be performed without first identifying and immobilizing the bladder. for the procedure to be performed safely and quickly, the patient must be cooperative. if collection of a urine sample by cystocentesis is absolutely necessary, sedation may be indicated to restrain the patient adequately for the procedure. generally, cystocentesis is a safe procedure, assuming the patient is cooperative and the bladder can be identified and stabilized throughout the procedure. however, injury and adverse reactions can occur. in addition to laceration of the bladder with the inserted needle (patient moves abruptly), the needle can be passed completely through the bladder and into the colon, causing bacterial contamination of the bladder or peritoneal cavity. there is also risk of penetrating a major abdominal blood vessel, resulting in significant hemorrhage. obtaining a skin biopsy from abnormal skin only to receive a nondiagnostic result as reported from a pathologist suggests that improved biopsy technique may result in collecting a specimen with higher diagnostic value. the following guidelines apply when skin biopsies are performed: ❏ consider obtaining multiple samples from multiple sites, which is especially useful when different stages of similar lesions are identifiable. ❏ do not perform a surgical scrub before collecting the sample; shaving the hair away is fine, but surgically prepared skin removes superficial lesions that, had they been left in place, might have been diagnostic. ❏ biopsy of lesions that are depigmenting should be done before they have turned white; the absence of color usually denotes absence of active skin lesions. biopsies from completely depigmented skin are less likely to demonstrate active lesions. ❏ biopsy of lesions associated with alopecia should be done in the center of the most alopecic area. ❏ also, biopsy of lesions associated with alopecia should be done at junctional (between normal-and abnormal-appearing) skin. ❏ consider submitting biopsy samples from completely unaffected, normal-appearing skin. ❏ avoid biopsies of ulcerated skin areas. biopsy samples may be obtained with a scalpel blade (incisional or excisional) or via a dermatologic punch biopsy. punch biopsy instruments are circular blades available in 4-mm, 6-mm, and 8-mm diameter sizes (figure 4-17) . hold the punch perpendicular to the skin site of interest. a back-and-forth motion that rotates the circular blade cuts through the skin. when the skin no longer moves as the punch is rotated, the biopsy is complete and the skin sample may be removed (from the skin or from the biopsy instrument). avoid grasping the dermis or epidermis of the sample with any instrument to prevent crushing of the sample and causing artifact. if the sample must be lifted, use the attached subcutaneous fat only. if the lesion of interest is deep, the punch biopsy technique may not be effective. in this situation, an incisional or excisional biopsy using a sterile no. 10 or no. 15 surgical blade is indicated. biopsies of ulcerated skin and solitary nodules are best done by removing a wedge of skin (incisional biopsy). in some cases it is possible surgically to remove all visible, palpable parts of the lesion (excisional biopsy). place each sample of skin in buffered formalin, using a volume that is at least 10 times that of the sample size. if particularly large areas of skin are harvested during biopsy, cut these into 1-cm thick pieces before placing into formalin. (note: placing larger tissue samples into formalin may result in inadequate or incomplete fixation of the tissue and the inability to properly prepare the tissue for examination). alternatively, it is possible, and in many cases important, to evaluate a biopsy specimen of skin or subcutaneous tissue at the time of collection. when the lesion of interest is suspected to be neoplastic, quickly differentiating between inflammatory cells and neoplasia may be possible by simply performing an exfoliative cytologic examination (described in this section) on one of the biopsy samples in addition to fixing a separate sample in formalin and submitting that for histopathologic examination. small biopsy samples that have been subjected to the additional handling required to make impressions on a glass slide are not good candidates for subsequent fixation and histopathologic examination. it is generally recommended to perform exfoliative cytologic and histopathologic examinations on separate samples. among the most common diagnostic procedures carried out on the skin of dogs and cats is a routine skin scraping. yet despite the frequency with which this test is used, doing a skin scraping in such a manner that the sample recovered maximizes the opportunity to establish a diagnosis can be anything but routine. a skin scraping, properly done, does require using consistent techniques appropriate to the suspected diagnoses, and as such, superficial or deep scrapings, or both, may be indicated. skin scraping is indicated whenever ectoparasite infestation is suspected. superficial scrapings are appropriate for detecting mites that live on the skin surface, such as cheyletiella species and otodectes cynotis, as well as mites that burrow within the outermost layers of skin (stratum corneum), such as sarcoptes species and notoedres cati. because the area to be scraped is relatively large (≥2 cm 2 ), shave dogs and cats with long hair coats before attempting the procedure, unless cheyletiella infestation is suspected. make the scraping over healthy-appearing skin. do not cleanse the skin of superficial scale or crusts. the technique for superficial skin scraping entails the use of mineral oil or pyrethrin ear drops applied to a clean scalpel blade and directly onto the area of skin to be scraped. scraping begins as a gentle motion made in the direction of the hair coat. gradually increase the pressure of the blade against the skin with repetitive scrapings over the same area. take care not to lacerate the skin, although minor capillary bleeding at the site is common. transpose material collected on the edge of the blade to a clean glass slide, cover it with a coverslip, and thoroughly examine the material under low magnification for evidence of ectoparasites. note that for mites such as cheyletiella or scabies, finding just one mite or one egg is diagnostic and justifies implementing treatment. same as described for superficial skin scraping (earlier). a slightly different technique is indicated in dogs and cats suspected of having an infestation that includes demodex canis mites. the mites are known to live predominantly in sebaceous glands and hair follicles. they can survive in the skin of animals without manifesting lesions. hair loss and skin lesions develop where overgrowth of the mite population occurs. demodex infestations can be localized or generalized; infestations can occur in either dogs or cats but the most severe, generalized infestations are much more likely to occur in young dogs. although both superficial and deep skin scrapings may reveal the presence of mites on the skin, deep scrapings may reveal demodex mites in some patients when superficial scrapings are negative. the technique for deep skin scraping targets a small area of skin (<2 cm 2 ). it may be helpful to apply gentle pressure to the skin or actually to squeeze the area of interest between the thumb and a finger in an attempt to force mites from the deeper to the more superficial skin. in some breeds (e.g., old english sheepdogs and shar-peis) recovering mites on a skin scraping can be particularly difficult. in such cases, when demodex infestation is highly suspected but the results of repeated skin scrapings are negative, a skin biopsy is appropriate. alternatively, a procedure called a trichogram that involves pulling (plucking) a few hairs from the hair follicles using a hemostat may be diagnostic. once the hairs have been plucked from the skin, place them on a glass slide that has been preprepared with a drop of mineral oil, add a coverslip, and examine the hair shaft under low magnification. half of all dogs with demodex infestation will have a positive trichogram. not all dogs and cats with otitis externa require comprehensive ear flushing and debridement before or as part of otic therapy. in many cases, home treatment is sufficient to resolve the problems effectively, assuming the underlying diagnosis has been established. however, in patients with chronic or particularly severe infections, topical treatment administered by the owners at home may not be sufficient. in such cases, the external ear canal requires a careful and comprehensive cleaning before administration of topical medications. properly performed, flushing and cleaning of the external ear canals is not a quick procedure. anesthetize the patient. attempting to perform a thorough ear cleansing under sedation usually will not be successful. once the animal is anesthetized, perform a careful otoscopic (or video otoscopic) examination to establish the integrity of the ear canal, evaluating, for example, for the presence or absence of tumors or parasites. in severe cases the tympanic membrane (ear drum) may not even be visible. with the patient in lateral recumbency, flush the ear canal (figure 4 -18) or lavage it with warm saline initially, and then aspirate the material from the canal. if this procedure is not successful in removing the debris attached to the epithelium of the ear canal, use 4 ceruminolytic ear solutions to facilitate breakdown and removal of this material. a 5-minute instillation and soak is recommended, followed by thorough flushing to remove debris and the ceruminolytic material. remove hair growing inside the ear canal with forceps. a suction apparatus is recommended for removal of debris and liquid remaining. reintroduce an otoscope to examine the integrity of the skin in the ear canal and to look for any evidence of stenosis, foreign body, or tumor. the flushing process is not complete until it is possible to visualize the tympanic membrane. carefully remove any remaining debris with an otologic loop (figure 4-19) , not a cotton-tipped swab. repeat the procedure on the opposite ear as indicated. at the conclusion of the examination, apply appropriate topical medication into the ear canal before allowing the patient to recover from anesthesia. systemic therapy or surgical intervention may be required in some patients for complete resolution of the problem. however, a thorough examination and cleaning is critical before actually making decisions regarding medical versus surgical intervention. use of a cotton-tipped swab to remove debris from the external ear canal, although commonly done, is not generally recommended. repeated attempts may ultimately force debris deeper into the external ear canal. general anesthesia and otic lavage or flushing may be required to effectively correct the problem created by the use of cotton-tipped swabs. in selecting an appropriate endotracheal tube, consider the size of the animal and select a tube that has the largest diameter that can be inserted without force (table 4 -7). the length of tube selected must not extend beyond the bifurcation of the trachea (carina). always check the cuff of the endotracheal tube to ensure there are no leaks and that the cuff is working properly before intubation. lubricate the selected endotracheal tube with sterile lubricating jelly before inducing anesthesia. after induction, place the patient in sternal recumbency and elevate the head. the individual inserting the tube should grasp and extend the tongue with a piece of gauze. the tongue is extended to facilitate visualization of the larynx. avoid excessive downward pressure on the tongue in order to prevent inadvertent laceration or injury from the lower incisors. if a laryngoscope is used, place the tip of scope at the base of the tongue. gently press the tip of the laryngoscope ventrally to move the epiglottis and expose the glottis. contact during an attempt to intubate, one or two drops of 2% lidocaine can be applied to the arytenoid cartilages. once the tube has been inserted into the trachea, never advance it farther than the carina. doing so may result in intubation of either the right or the left main bronchus (endobronchial intubation). once the tube is in place, secure it in place with a loop of 1 ⁄2-inch white tape or muzzle gauze. overinflation of the endotracheal tube cuff can cause tracheal ulceration, tracheitis, hemorrhage, tracheomalacia, fibrosis, stenosis, and subcutaneous emphysema. abdominal paracentesis refers to the aspiration of fluid from the abdominal cavity for both diagnostic and therapeutic purposes. always weigh the animal before and after removing abdominal fluid. any subsequent gain in weight indicates a reaccumulation of abdominal fluid. place the animal in left lateral recumbency and restrain it in this position. clip and surgically prepare a 1-to 3-inch square between the bladder and the umbilicus just lateral to the midline. if the bladder is distended, empty it before performing paracentesis. infiltrate the paracentesis site with lidocaine 0.5% using a 22-to 25-gauge needle. in most cases, local anesthesia is not necessary. abdominal puncture can be made with an 18-to 20-gauge needle (figure 4-20) . gently insert the needle through the skin and external abdominal oblique muscles while simultaneously pushing and twisting, to push viscera away from the tip of the needle. blind abdominocentesis without the use of ultrasound to guide needle placement into a fluid pocket can have negative results if there is less than 5 ml of fluid per kilogram within the peritoneal cavity. when the abdominal puncture has been made, allow the animal to rest quietly to 4 facilitate drainage of the fluid. some clinicians recommend tapping while the patient is in a standing position in the hope of obtaining more complete drainage. changing the patient's position after the tapping may result in needle-tip laceration of intraabdominal organs. aspiration may be easier if a specially adapted needle with multiple holes drilled in the shaft is used because it is less likely to become plugged with omentum. ideally, tap four quadrants of the abdomen. if four-quadrant abdominocentesis has been performed and no fluid has been obtained, and if there is suspicion for peritonitis, diagnostic peritoneal lavage can be performed. the abdomen is clipped and aseptically scrubbed as previously described. next, insert an over-the-needle catheter or hypodermic needle just caudal to the umbilicus, and instill 10 ml of warmed 0.9% sterile saline or lactated ringer's per kilogram. after instillation of the fluid, walk the patient around or gently roll the patient from side to side to distribute the fluid throughout the abdominal cavity. next, perform four-quadrant abdominocentesis as previously described. only a small amount of the fluid infused will be obtained. the fluid will be diluted by the saline or lactated ringer's infused, so biochemical analysis will not be accurate. however, the fluid can be examined microscopically for the presence of plant material, bacteria, wbcs, or bile pigment to help diagnose various forms of peritonitis. hackett numerous biopsy techniques are available, and the selection of the appropriate technique is based on the tissue to be examined, the condition of the patient, and the skill of the examiner. inserted cranial and to the right/left of the umbilicus, and caudal and to the right/left of the umbilicus. when the needle is inserted, it should be twisted while penetrating into the abdominal cavity, to prevent iatrogenic puncture of hollow organs. in some cases it may be necessary to place more than one needle before fluid will flow from the needle hub. appropriate locations are marked on the skin with an "x." excisional biopsy refers to the surgical removal of the entire lesion or organ with subsequent histologic examination. excisional biopsy is used most frequently for skin lesions and cases in which an entire organ may have to be removed (such as an eye or an internal organ that has developed a tumor). incisional biopsy refers to the surgical removal of a portion of a lesion with subsequent histologic examination. choose a representative area of the lesion for biopsy. include lesion margins, if possible. needle aspiration refers to the use of needle and syringe to remove representative cells from the tissue or organ of interest. specialized needles are available that allow removal of very small biopsy specimens that can be submitted for histopathologic examination. (see also fine needle aspiration.) needle biopsy techniques: general considerations needle biopsy or aspiration techniques refer to a variety of techniques used to obtain diagnostic tissue or cells from internal organs, including the lung, liver, spleen, pancreas, abdominal lymph nodes, and mass lesions within the abdomen and thorax. in contrast, fine-needle aspiration is a technique generally used to recover cytopathologic samples (cells only) from skin or subcutaneous tissues (e.g., superficial lymph nodes). the advantage of needle biopsy is related directly to how well the abnormal tissue has been characterized and how easily it can be identified during the procedure. in addition, depending on patient cooperation, most procedures can be performed safely with the patient sedated only. shortterm intravenous anesthesia and general anesthesia eliminate undesired patient movement during the biopsy procedure. potential lesions or abnormal tissues from which aspirate or biopsy samples are to be taken are located using palpation, radiographs, or ultrasound-guided imaging techniques. shave the skin over the site of needle penetration and surgically prepare it. the type of sedation or anesthesia depends on the temperament of the animal and the site on which the biopsy will be performed. attach a 22-gauge needle without stylet to a 12-ml syringe prefilled with 0.5 to 1.0 ml of air. optionally, affix a flexible extension set to the needle and connect it proximally to the syringe. needle length may vary from 1 to 3 1 ⁄2 inches depending on the required depth of penetration and size of the patient. guide the needle into the tissue or organ of interest. stabilize the tip of the needle to avoid random movements through organs, especially highly vascular tissue such as liver and spleen. once the needle has been inserted, the aspiration technique entails withdrawing the plunger of the syringe to the 7-or 8-ml level. hold that position for 1 to 2 seconds, and then release. repeat the procedure. depending on the nature of the lesion, it may not be indicated to thrust the needle into the tissue at multiple and different angles. neutralize the pressure in the syringe, and withdraw the needle rapidly. expel any material within the needle onto glass slides using the air in the syringe. this same procedure can be repeated with a new needle to obtain an additional three to five samples from alternative sites. this technique allows samples to be obtained without applying negative pressure to the syringe, which may damage cells. ultrasound guidance for needle aspirations from abdominal tissues greatly enhance the safety of this technique, especially when obtaining samples from smaller animals. automatictrigger needles such as cook or temno biopsy needles (14 to 18 gauge) are available for use in human beings but are seldom used in veterinary medicine. the risks associated with fine-needle aspiration include rupture of an encapsulated inflammatory process, dissemination of an infectious agent, seeding of neoplastic cells in the needle tract, and hemorrhage. larger volumes of fluid and cells can be placed directly into a vial containing edta to prevent clot formation. prepare and examine direct and sedimentation specimens. needle biopsy of internal organs using the tru-cut needle is particularly useful in patients with subcutaneous ( figure 4 -21) or cutaneous masses and for localized abdominal and thoracic mass lesions and diffuse liver, kidney, and splenic disease. serious complications, usually hemorrhage or laceration of the gallbladder (during liver biopsy), can occur when the procedure is performed blindly. therefore ultrasound-guided needle biopsy is strongly recommended whenever a percutaneous biopsy of internal organs is performed. additional safety factors provided by ultrasound guidance include the ability to image, and avoid, large aberrant blood vessels. risk of complications associated with needle aspiration of the lung is considerably higher than for most abdominal procedures. pneumothorax can occur after a single, "clean" aspiration attempt. see respiratory tract procedures for a detailed description of performing fine-needle aspiration of the lung. histologic examination of diseased skin can serve as a means for diagnosis of cutaneous lesions. the causative agent often is found in acute and chronic skin infections. punch biopsy of the skin is a quick and accurate means of removing a small sample of diseased skin for histopathologic examination. select a site that is well developed but not traumatized or excoriated. the sample should include little or no normal tissue. if the lesion (pustule, vesicle) can be identified early in its development and if the biopsy sample is taken only from the lesion, one may obtain a superior specimen. it is best not to take too large a sample that contains much normal skin; by mistake, the technician might take a section that misses the lesion. proper selection of the biopsy site is crucial to accurate diagnosis. carefully clip the hair from the lesion. lightly blot the skin with 70% alcohol. avoid superficial trauma while cleaning the skin. inject a small subcutaneous bleb of 2.0% lidocaine to deaden the area. special equipment needed for the biopsy includes a 4-mm, 6-mm, or 8-mm biopsy punch and 10% buffered formalin solution. after the area has been anesthetized with lidocaine, press and rotate the biopsy punch through the skin until the subcutaneous tissue is penetrated. remove the biopsy specimen by "spearing" the subcutaneous fat with a fine needle. do not grasp the specimen with a forceps. blot the specimen gently between two paper towels. spread the tissue out gently (like a pancake), place the specimen epidermal side up on a piece of cardboard or tongue depressor, press the specimen gently to cause adhesion, and drop the specimen into the formalin fixative. the skin defect may be closed with one or two simple interrupted sutures. if deep subcutaneous tissue or large biopsy samples are needed, a punch biopsy is inadequate. use a small (no. 15) scalpel blade to obtain an appropriate sample. in all cases in which skin biopsies are made, take multiple samples to increase the odds that at least one will have diagnostic lesions. specimens submitted to laboratories should be accompanied by extensive, detailed clinical information, including a differential diagnosis. skin biopsies routinely are stained with hematoxylin-eosin; however, periodic acid-schiff, gomori methenamine silver, and verhoeff stains are used for special problems. the diagnosis of liver disease is generally confirmed on the basis of the patient's clinical signs coupled with laboratory findings, radiography, and abdominal ultrasound. the development of a more specific diagnosis and prognosis in liver disease may be aided greatly by information obtained in a liver biopsy. percutaneous liver biopsies are of much greater value in generalized liver disease such as cirrhosis, generalized acute hepatic necrosis, or amyloidosis than in focal hepatic disease. the major indications for performing a liver biopsy are (l) to explain an abnormal liver profile, (2) to define reasons for abnormal liver size, (3) to identify a possible liver tumor, (4) to arrive at a prognosis and rational approach to management, and (5) to identify the cause of ascites. the procedures for obtaining liver tissue are numerous; however, needle biopsy of the liver, when performed properly, can be helpful. careful physical and clinicopathologic examination should precede a liver biopsy. a normal coagulation profile should be documented on every patient undergoing liver biopsy. detect and correct abnormalities in normal hemostatic mechanisms, if feasible, before needle biopsy of the liver. liver biopsy should be performed only in the fasted patient and only after removal of ascitic fluid. percutaneous needle biopsies and fine-needle aspirations of the liver can be performed with local anesthesia in the sedated, and cooperative, patient. general anesthesia is a reasonable alternative whenever feasible. biopsy sites in the liver can be selected best when needle biopsy techniques are used along with laparoscopy or ultrasound techniques. blind percutaneous needle biopsies of the liver can be performed with relative safety if the liver is significantly enlarged and easily palpated. however, blind biopsies do carry the risk that the operator will be unable to determine the impact of penetrating the liver if only an abdominal radiograph and impression of abdominal palpation are available. in cases in which the liver is not palpable, blind biopsy carries significantly higher risk and should be performed only when no alternative exits. a modified percutaneous liver biopsy can be performed by the following method. place the animal in dorsal recumbency, and place a local block in the midline of the skin and abdomen at the caudoventral aspect of the left hepatic lobe. the incision into the peritoneal cavity should be large enough to accommodate the gloved index finger. make a separate skin puncture site in the abdominal wall to accommodate the biopsy needle. use the index finger manually to fix the left hepatic lobe (or other desired hepatic lobe) against the diaphragm or other adjacent structures, and insert the outer cannula and stylet through the abdominal wall in the isolated hepatic lobe. remove the stylet, and rapidly insert the cutting prongs. if properly placed, the cutting prongs should not go through the entire hepatic lobe. advance the outer cannula over the blades of the cutting prongs, thus entrapping the hepatic tissue material within the cutting prongs. remove the biopsy needle. using a wooden applicator stick, carefully place the biopsy specimen into fixative. biopsy samples can be used to prepare slides for cytologic examination, and the biopsy needle may be cultured. close the abdominal incision in the routine manner. another liver biopsy technique entails use of a tru-cut biopsy needle. place the dog in dorsal recumbency. clip a 5-cm 2 area over the triangle formed by the xiphoid cartilage and left costal arch, and prepare the area as for aseptic surgery. make a small paramedian incision large enough to accommodate a sterile otoscope head 7 mm in diameter. use a sterilized halogen-illuminated otoscope speculum to visualize the liver. pass a tru-cut biopsy needle through the otoscope cone to directly obtain a biopsy specimen of the liver. the technique for performing diagnostic nasal biopsies is sufficiently complex (and bloody) that it is generally recommended to refer patients in need of this procedure to a specialty or referral hospital that has rhinoscopic and/or computed tomography capabilities. blind biopsies of dogs and cats with chronic nasal disease, especially if associated with bleeding, can be associated with significant risk, including penetration of the cranium. no one likes nasal biopsy results that indicate "normal brain. " renal biopsies can be valuable in confirming or eliminating a diagnosis of renal disease that is based on history, physical examination, and radiographic and laboratory data (box 4-5). in addition, biopsy may be a way of arriving at a prognosis in generalized renal disease and a better means of evaluating the type of treatment to be instituted. ultrasonographic guidance can prove valuable during renal biopsy for placing the needle into the tissue desired and avoiding complications. the liver biopsy, although a critical diagnostic tool in patients with laboratory evidence of liver disease, can be a fatal event, even in the hands of the experienced clinician. abdominal ultrasound imaging of the liver before and during biopsy is strongly recommended. before renal biopsy, the animal should have a baseline coagulation profile that includes, at the very least, an activated coagulation time and platelet count. a buccal mucosal bleeding time may be indicated if there is any history of spontaneous bleeding in a patient with a normal platelet count. obtain biopsies from the renal cortex. administer fluids to patients before and after biopsy. many patients with generalized renal disease are critically ill and debilitated, and general anesthesia is contraindicated. in these cases, a neuroleptanalgesic agent may be used for sedation. if the animal is a good anesthetic risk and renal function will permit it, use inhalation anesthesia. when bilateral renal disease is documented, select the left kidney for biopsy because it is more accessible than the right kidney. with the anesthetized patient in right lateral recumbency, surgically prepare the skin behind and below the junction of the costal arch at the level of the second and third lumbar vertebrae. make a 2-inch paralumbar incision parallel to, but just behind, the costal arch. dissect muscle and fascia until the peritoneum is visible. carefully open the peritoneal cavity. digitally feel for and examine the caudal pole of the left kidney. guide the needle toward the posterior pole of the kidney with the index finger. immobilize the kidney against the body wall and insert the tru-cut biopsy needle, with the biopsy notch exposed into the parenchyma of the kidney. capture the biopsy specimen by sliding the outer sleeve of the needle over the (now embedded in the kidney) biopsy notch. remove the needle and gently lift the biopsy sample from the needle and place it into formalin. evaluate the site for hemorrhage. once bleeding is controlled, a second biopsy specimen may be collected. once bleeding from the biopsy site has stopped, the incision can be closed. in dogs, renal biopsy can be performed under ultrasound guidance using probes with channels for biopsy needle insertion. evaluation of bone marrow is indicated in patients with evidence of persistently diminished cell counts of any or all cell lines (wbcs, rbcs, platelets) or evidence of morphologically abnormal cells in peripheral blood. bone marrow aspiration and bone biopsy are extremely helpful but underused diagnostic procedures. the availability of inexpensive, high-quality biopsy needles makes these procedures safe and easy to perform (once experience is gained). conventional practice today is to obtain a bone marrow aspirate (cytopathologic examination) and a bone biopsy specimen from the same patient during the same procedure when changes in the peripheral blood justify this level of diagnostic testing. bone marrow aspiration technique is described earlier in this section. two types of bone biopsy needles are available. the most commonly described procedure involves use of the jamshidi biopsy needle, an 11-to 13-gauge needle that ranges in length from 5 to 10 cm (see figure 4 -11). the needle contains a stylet that extends beyond the needle tip by 3 to 4 mm. because of the size of the jamshidi needle, its use is limited to medium and large dogs. for bone biopsies in cats and small dogs, the illinois bone marrow aspiration needle is preferred (see figure 4 -10), which is a 15-to 18-gauge needle available in lengths ranging from 2.5 to 5.0 cm. the patient usually is sedated or anesthetized for the procedure. although some patients will tolerate this procedure when performed under local anesthesia only, the additional manipulation required to obtain a high-quality sample justifies sedation. in some cases the patient is sufficiently obtunded that sedation is neither indicated nor required. the technique for bone biopsy is the same regardless of the needle used. once the site has been selected (usually the same sites selected for bone marrow aspiration: head of the humerus, wing of the ileum, ischial tuberosity, proximal femur), clip the hair and surgically prepare the skin. make a small stab incision in the skin over the site selected. pass the needle, with stylet in place, through the incision and subcutaneous tissues until the needle tip makes firm contact with bone. advance the needle using steady, increasing pressure and stable rotation. rotation, in this case, means rotating the needle back and forth to the left 180 degrees and then to the right 180 degrees. once the needle is situated in the bone (about 0.5 cm penetration only), stop. carefully remove the stylet. continue the penetration by gradually applying additional pressure and simultaneously rotating the needle. the usual depth of penetration varies from 1 inch to as much as 3 inches. on reaching the desired depth, remove the needle by continuing to rotate as described but gradually withdrawing the needle from the bone. an obturator is provided to push the sample out of the bone. place the core of bone directly into buffered formalin and submit it for histopathologic examination (decalcification will be required, which takes a little longer). some authors recommend carefully rolling the bone core across a glass slide (for cytopathologic examination) before placing the bone in formalin. most pathologists do not recommend this because additional handling of the biopsy sample can sufficiently disrupt the architecture of the tissue and compromise the quality of the biopsy (besides upsetting the pathologist). note also that the needle can, with a little gentle manipulation, be reinserted into the hole from which the biopsy sample was obtained. because the illinois needle and the jamshidi needle accommodate a syringe, it is possible to obtain (if done quickly, to prevent clotting) a bone marrow aspirate from the same site. place that sample directly onto glass slides or (recommended) into 4% edta and mix it before making slides. there are no specific requirements for postbiopsy care of the patient. clean the blood from the skin using hydrogen peroxide; sutures generally are not required. the femoral and dorsal pedal arteries can be punctured to obtain an arterial blood sample for blood gas and electrolyte analyses (see section 1) for information on indications and interpretation of results). to obtain a sample of arterial blood gas, place the patient in lateral recumbency and restrain the patient in a manner similar to that for a medial saphenous venipuncture. a 25-gauge needle affixed to a tuberculin syringe is preferred for arterial puncture. prepare the tuberculin syringe by coating it with heparin and forcing all the heparin out except for that left in the hub of the needle. pull back on the plunger of the syringe slightly to facilitate visualizing the point at which the artery is entered. arterial blood initially will enter the syringe without the plunger being drawn back. for collection of blood from the femoral artery, and once the patient is sufficiently restrained, the individual collecting the arterial blood sample should palpate the medial aspect of the limb over the proximal medial femur until the femoral pulse is palpated. direct the needle at a 30-to 45-degree angle, inserting the needle slowly, watching for a flash of blood in the hub of the needle (figure 4-22) . gradually withdraw the plunger to facilitate blood entering the syringe. collect 0.4 to 0.5 ml and immediately submit the blood for analysis, or place it on ice until the analysis can be performed. to obtain blood from a dorsal pedal artery, place the patient in lateral recumbency and extend the rear limb as for a medial saphenous blood sample collection. the person obtaining the blood sample should pull the paw of the down leg in the nondominant hand toward his or her body, rotating the limb slightly in a medial direction to palpate the arterial pulse. palpate the pulse in the dorsal pedal artery on the dorsomedial aspect of the tarsus. gently insert the needle at a 30-degree angle into the artery, watching carefully for a flash of blood into the syringe. when the necessary amount of blood has filled the syringe, remove the needle and place pressure over the site of arterial puncture for a minimum of 2 minutes. evacuate excess air from the syringe and needle, and cap the needle with a red rubber stopper to prevent air from entering the needle and syringe. place the sample on ice until analysis, if arterial blood gas analyses cannot be performed immediately. in the event that percutaneous access to a peripheral artery is not possible, the femoral artery can be isolated and prepared for surgical cutdown. after appropriate aseptic skin preparation, make a 4-to 5-cm incision in the skin over the femoral artery. find the caudal edge of the sartorius muscle by blunt dissection and then reflect it anteriorly to expose the underlying femoral artery, vein, and nerve. taking care to avoid tearing any vessel branches, gently isolate up to 2 cm of the femoral artery from the surrounding fascia. visually direct the needle into the artery at this point. alternatively, catheterize the artery in the event repeated arterial samples are required. elevate the femoral artery by preplacing two stay sutures beneath the artery and then elevating the vessel to the level of the skin. insert a long catheter-overthe-needle system into the lumen of the artery without penetrating the deep wall. gently insert the catheter into the vessel, remove the needle, and cap and flush the catheter. close the incision and affix the catheter to the skin via a tape tag sutured to the skin. the collection of csf is an important diagnostic procedure indicated for patients suspected of having significant intracranial or certain spinal diseases. however, it is our opinion that the technique to safely perform this procedure requires hands-on training and, preferably, prior experience before attempting the procedure in a clinical patient. attempting to perform csf collection from a written description in a text is not recommended. although this procedure is generally safe when performed correctly, significant injury and even death are possible, despite the experience of the individual performing the procedure. the electrocardiogram provides a fast, efficient way to obtain considerable data about a patient's cardiovascular status. electrocardiography is a clinical test and must be correlated with clinical findings (box 4-6). keep in mind that an electrocardiogram measures only electrical activity of the heart as seen on the body surface at any one instant. electrical disorders of the myocardium can be transient or intermittent and, as such, can be missed on a single electrocardiogram. if the answer to any of these questions is no, proceed to identify the abnormality. next, determine the rate, rhythm, and wave character-that is, evaluate measurements of the p wave, pr interval, and qrs complex. evaluate the st segment, t wave, and qt interval. use all leads to determine the axis and any miscellaneous criteria. depending on the type of electrocardiographic equipment used, there are several methods for determining heart rate from the electrocardiographic tracing. many electrocardiographs compute the heart rate and print that on the tracing. however, in patients with a significant dysrhythmia, these calculations can be flawed and should be verified manually when a question exists. small linear lines or demarcations at the top of the electrocardiogram paper can be used to determine the heart rate. at a paper speed of 50 mm/second, the time between adjacent marks is 1.5 seconds. counting the number of qrs complexes (or r waves) between just two of these divisions and multiplying by 20 equals the heart rate in beats per minute (figure 4-23) . for those inclined to higher mathematics, the heart rate also may be determined by counting the number of small squares between r waves (at a paper speed of 50 mm/sec) and then dividing into 3000 (box 4-7) . the normal heart rhythm is sinus in origin. for every qrs complex there is a p wave (figure 4 -24). the p waves are related to qrs complexes (p-p interval is constant). sinus arrhythmia, sinus arrest, and wandering pacemaker are normal rhythm variations. in sinus arrhythmia, the p-p interval is irregular. the pauses are never longer than twice the usual p-p interval (figure 4-25) . a wandering pacemaker means that the p waves vary in height and may even be negative temporarily (figure 4-26) . sinus arrest is defined as a prolongation of the p-r interval longer than twice the usual p-p interval. the normal p wave is 0.04 second × 0.4 mv (two boxes wide × four boxes tall) for the dog and 0.04 second × 0.2 mv for the cat. in p mitrale (left atrial enlargement), the p wave is wider than 0.04 second. in p pulmonale (right atrial enlargement), the p wave is taller than 0.4 mv for the dog and 0.2 mv for the cat. the pr interval is measured from the beginning of the p wave to the beginning of the qrs complex. the normal interval is 0.06 to 0.13 second (3 to 6.5 boxes wide) for the dog and 0.06 to 0.08 second for the cat. in first-degree atrioventricular heart block, the pr interval is prolonged. the pr interval is sometimes useful in monitoring the effects of digitalis therapy. the qrs complex duration is measured from the beginning of the q wave to the end of the s wave. normal duration is up to 0.04 second in cats, 0.05 second in small dogs, and 0.06 second in large dogs. a qrs complex that is too wide indicates left ventricular enlargement (figure 4-27 ). an r wave that is too tall indicates left ventricular enlargement. the amplitude is measured from the baseline to the top of the r wave (figure 4-28) . the normal r wave can be up to 0.8 mv tall in cats, 2.5 mv in small dogs, and 3.0 mv in large dogs. figure 4 -26: a, the wandering pacemaker in this recording is suggested by the slightly negative p waves in some of the complexes. negative p waves of this nature result from vagal depression of the sinoatrial node and the development of a junctional atrioventricular nodal rhythm. b, marked sinus arrhythmia and a wandering pacemaker result in a decreased heart rate (increased r-r interval) and negative p waves in the fifth complex. as the pacemaker returns to the sinoatrial node, the rate increases, and positive p waves of varying amplitude result in the sixth and seventh complexes. the st segment is between the end of the s wave and the beginning of the t wave. the qt interval is measured from the beginning of the q wave to the end of the t wave. the normal interval is 0.14 to 0.22 second (7 to 11 boxes wide) in dogs and up to 0.16 second in cats. a lengthened qt interval may be seen with hypokalemia or hypocalcemia. the qt interval varies with heart rate and tends to be prolonged when bradycardia occurs. a decreased qt interval may be seen with hypercalcemia. the mean electrical cardiac axis measures the direction (vector) of the cardiac ventricular impulse during depolarization. therefore the qrs complex is examined in leads i, ii, iii, av r , av l , and av f . these six leads determine the axis. they are arranged in a manner known as bailey's hexaxial lead system (figure 4-29) . the procedure is as follows: 1. find an isoelectric lead-that is, a lead for which the total number of positive (upward) and negative (downward) deflections of the qrs complex is equal to zero (figure 4-30) . when there is no perfectly isoelectric lead, use the one that comes closest. 2. find the lead that is perpendicular to the isoelectric lead: lead i is perpendicular to av f ; lead ii is perpendicular to av l ; and lead iii is perpendicular to av r . 3. determine whether the perpendicular lead is positive or negative on the patient's electrocardiogram. if the perpendicular lead is negative, the axis is at the negative end of that lead (each lead has a plus and a minus pole marked). if the perpendicular lead is positive, the mean electrical axis is at the positive end of the perpendicular lead. for example, if avl is isoelectric (normally it is), lead ii is its perpendicular. if lead ii is positive on the electrocardiogram, the axis is +60 degrees. if lead ii is negative on the electrocardiogram, the axis is −120 degrees. the mean electrical axis in the normal dog is +40 to +100 degrees; for the cat it is more variable, at 0 to ±180 degrees. right axis deviation (axis more than +100) indicates right ventricular enlargement in the dog (figure 4-31) . left axis deviation (axis 0 to +40 degrees) indicates left ventricular enlargement in the dog. when there is biventricular enlargement, the axis usually remains normal. axis determinations are of less value in the cat because the normal range is so wide (boxes 4-8 to 4-10). inappropriate use of endoscopic equipment not only can damage expensive equipment but also can cause serious injury to the patient. endoscopy of the upper respiratory tract is among the most important advanced diagnostic and therapeutic tools used in the evaluation of patients that have stertor (snorting), reverse sneeze, stridor (wheezing), and chronic cough. laryngoscopy is of value in the diagnosis of upper airway obstructions such as eversion of the lateral ventricles, collapsed arytenoid cartilages, hyperplasia of the vocal cords, nodules on the vocal cords, elongated soft palate, collapsed proximal trachea, and traumatic injuries to the neck. note also, however, that a careful visual examination of the larynx in the anesthetized patient (only) can be highly valuable even without the use of endoscopic equipment-for example, for assessment of laryngeal movement in patients with laryngeal paralysis. suspected lesions inside the larynx may be difficult to visualize with or without endoscopic equipment. examination of the trachea and main stem bronchi requires endoscopic evaluation to assess the integrity of the airway for conditions such as collapsed trachea, mediastinal tumors, hilar lymph node enlargement, parasitic nodules (filaroides osleri), and foreign body aspiration. in addition, tracheobronchoscopy is a valuable technique that permits culturing and cytologic examination of material from bronchi involved in chronic respiratory disease. upper airway obstruction that is not responsive to conservative therapy is an indication for more extensive diagnostic procedures, such as bronchoscopy. note: the discussion that follows centers around indications and capabilities of endoscopy in clinical practice. the discussion is not intended to be used as a "how-to" instruction guide on performing endoscopic procedures in dogs and cats. today, numerous types of endoscopes and accessory materials are available for use in clinical practice. specific hands-on training and complete familiarity with the equipment package available is essential before attempting to perform any of the procedures outlined. endoscopes of varying sizes are appropriate for use in examining the larynx and trachea. however, in cats and small dogs, examination of the trachea using equipment as small as a (human) bronchoscope may limit the examination because the endoscope nearly occludes the tracheal diameter. additional training and/or experience is recommended for performing tracheoscopy in small patients. one of the most important endoscopic techniques performed in dogs and cats involves examination of the nasopharynx, the upper respiratory compartment above the soft palate. sometimes called pharyngoscopy, examination entails retroflexion of a small-diameter endoscope (e.g., bronchoscope) 170 to 180 degrees to allow visualization of the space between the posterior nares (choanae) and the larynx (figure 4-32) . this is a common location for foreign body entrapment and occasional tumor development in cats and dogs (figure 4-33) . pharyngoscopy is the only effective means of examining this portion of the upper respiratory tract in patients that have a history of stertor (snorting) and so-called "reverse sneeze." lower respiratory tract: bronchoscopy endoscopic examination of the bronchi and lower airways is a highly diagnostic, occasionally therapeutic procedure indicated in patients presented with persistent cough. as in all endoscopic procedures, the patient is anesthetized for the examination. however, examination of the lower respiratory tract requires considerable attention to patient oxygenation and respiratory status during the examination. the requirement for oxygen to be administered throughout the procedure may be a significant limiting factor unless special accessories are used. in the ideal situation, the patient is a medium-to large-sized dog and the endoscope can be passed through the endoscope using a t adaptor while oxygen and anesthetic are administered simultaneously. however, in cats and small dogs it is usually not possible to pass an endoscope through the endotracheal tube. the procedure must be done by passing the endoscope directly into the trachea to the level of the right and left main bronchi and probably not much farther. supplemental intravenous anesthetic is likely to be required because of the time required to complete the examination. training and/or experience is essential before performing bronchoscopy, particularly in cats and small dogs. the greatest advantage in performing bronchoscopy is to visualize the integrity of the trachea and, to a limited extent, the lower airways. airway collapse, not visible on conventional radiography, can be strikingly apparent. foreign body entrapment, tumors, respiratory parasites, and airway trauma also can be identified with bronchoscopy. in addition, the bronchoscopic examination allows for collection of cytologic samples from discrete areas (airways) within the lower respiratory tract. the ability to perform bal in patients with reactive airway disease, subclinical or clinical infections, and certain types of tumors can be highly diagnostic. flexible fiberoptic endoscopy is a noninvasive, atraumatic means of visualizing the mucosal surfaces of the esophagus, stomach, and colon. flexible endoscopes are available from several companies at a wide range of prices. to minimize the risk of injury to the animal and to reduce the possibility of damage to the endoscope, place animals undergoing endoscopic examination under general anesthesia after routine preanesthetic preparation. a fast of 12 to 24 hours is recommended for most patients undergoing upper gastrointestinal endoscopy. however, for patients with indications of delayed gastric emptying, a longer fast (24 to 48 hours) may be needed to empty the stomach completely. in preparation for colonoscopy, a 24-to 48-hour fast is recommended. give a high warm-water enema the evening before and again 2 to 4 hours before the procedure. give such enemas until the return is clear. the clinical signs indicating esophageal disease and a potential benefit of esophagoscopy include repeated regurgitation, excessive drooling, ballooning of the esophagus, anorexia or dysphagia, and recurrent pneumonia. esophagoscopy allows visualization of the mucosal lining of the esophagus and makes it possible to detect inflammation, ulcerations, dilatations, diverticula, strictures, foreign bodies, tumors, and parasite infestations. endoscopic examination of the mucosal aspect of the stomach is indicated when the clinical signs or physical findings suggest the presence of gastric disease or when there is a need for confirmation or clarification of radiographic findings. in most cases, persistent vomiting is the chief complaint. other clinical signs suggestive of serious gastric disease include hematemesis, melena, weight loss, anemia, and abdominal pain. gastroscopy allows visualization of the mucosal lining of the stomach and enables detection of inflammation, ulceration, foreign bodies, and tumors. in most dogs and cats the endoscope can be passed into the proximal duodenum. depending on the patient size and length of the scope, it may be possible to evaluate as much as 12 inches or more of the proximal duodenum. colonoscopy is endoscopic examination of colon, rectum, and anus. the technique is helpful in the definitive diagnosis of lower bowel lesions, such as granulomatous colitis, foreign bodies, tumors, lacerations, and other mucosal abnormalities. the primary indication for colonoscopy is the presence of signs of large bowel disease, which typically include tenesmus and the passage of small, frequent stools containing fresh blood or excess mucus. endoscopic examination of the colon allows direct visualization of the effects of mucosal inflammation, ulceration, mucosal polyps, malignant neoplasia, and strictures. histologic examination of mucosal biopsy material will confirm the diagnosis of colonic disease. the large bowel must be empty for the colonic mucosa to be visualized. the bowel can be emptied by withholding food for 24 hours and performing a colonic irrigation the evening before and again 2 hours before the examination. the material used for the enema must be nonirritating and nonoily. mildly hypertonic saline solutions such as fleet enemas work well if given 2 hours before examination so that gas and fluid can be passed completely. however, do not use fleet enemas in cats or small dogs. if the general physical condition of the animal is poor and withholding food is not possible, feeding a low-residue diet for 12 to 18 hours before colonoscopy can be helpful. this diet could consist of cooked eggs, small amounts of cooked beef or chicken, and small amounts of carbohydrates, such as a slice of toast or 1 ⁄4 to 1 ⁄2 cup of moist kibble. maintain good hydration. if all food is contraindicated, oral electrolyte solutions such as gatorade (pepsico, purchase, new york) can be used to maintain hydration without moving solids through the intestinal tract. give the animal a short-acting anesthetic and place the animal on a tilted table in lateral recumbency with the hindquarters elevated. perform a digital examination of the rectum and pelvic cavity to ensure that there are no strictures, polyps, or other obstructions. lubricate the proctoscope thoroughly with water-soluble jelly and pass it gently through the anal sphincter. press the proctoscope forward slowly and carefully with a spiral motion. if any resistance is encountered, stop the motion, remove the obturator, and inspect the 4 bowel to determine the cause of the resistance. if possible, replace the obturator and continue forward motion until the instrument is passed its full length. withdraw the obturator, and observe the mucosa. the major portion of the examination is conducted as the instrument is withdrawn. to view the colonic and rectal walls completely, one must move the anterior end of the proctoscope around the circumference of a small circle while withdrawing the proctoscope. occasional insufflation with the inflating bulb is helpful in smoothing out folds of tissue. repeated instrumentation may produce petechiae and minor hemorrhages that are not pathologic. for examination of the terminal rectum and anus, the hirschman anoscope provides adequate, convenient visualization. newer techniques for visualizing the upper and lower gastrointestinal tract are being used in dogs. the flexible fiberoptic endoscope enables one to visualize and photograph the esophagus, colon, and stomach. one is able not only to visualize lesions of the gastrointestinal tract directly but also to assess motility, take biopsies of lesions, and remove foreign bodies. the ability to visualize directly the vestibule, the vagina to the level of the cervix, and the urethral orifice in female dogs is of particular value in evaluating patients with known or suspected congenital urinary tract disorders, such as incontinence or ectopic ureters and vaginal strictures (congenital or traumatic). numerous vaginal malformations and chronic infections cause visual changes that are identified easily during endoscopic examination. frequently the procedure can be conducted in the standing awake patient. sedation or general anesthesia is indicated when extensive manipulation, catheterization of the bladder, or a vaginal biopsy are indicated. position the sedated or anesthetized patient in dorsal or ventral recumbency to facilitate orientation during the procedure. if catheterization of the urinary bladder is required during the procedure, dorsal recumbency seems to facilitate visualization of the urethral papilla and insertion of the catheter. vaginoscopy entails use of a relatively small, flexible endoscope 4 to 6 mm in diameter or a 2-to 3-mm rigid scope. the flexible scope offers the advantage of a larger biopsy channel and the ability to view the lateral vaginal wall easily. vaginoscopy is considered an invasive procedure and should be conducted under sterile conditions. before insertion of the sterilized endoscope, the vulva should be free of obvious debris, should be clipped if necessary, and should be cleaned gently with a surgical soap and rinsed. insert the scope such that initial position of the tip of the scope is directed toward the anus. as insertion proceeds, the tip of the endoscope reaches the horizontal portion of the vestibule and vagina. when feasible, pass the scope to the level of the cervix. slight insufflation of the vagina may be useful in dilating the vagina, greatly facilitating the examination. conducting the examination from the level of the cervix caudally is recommended. this maximizes the ability to visualize critical anatomic features. the relatively recent introduction of very small (2-mm diameter) flexible and rigid endoscopes into veterinary medicine allows visual examination of the urethra, trigone, urinary bladder, and right and left ureterovesicular junctions of female dogs and even cats. such examinations are most useful when obstructive lesions (tumor or calculi) of the urethra or trigone are suspected. visual examination of the interior surface of the bladder and the capability of collecting biopsy samples make this a particularly useful diagnostic tool in the hands of the experienced clinician. numerous techniques are described for administering calories and nutrients to patients that are unable or unwilling to take in, chew, or swallow food. one method, intravenous hyperalimentation, is reserved for patients that are not able to tolerate any food being introduced via the gastrointestinal tract and represents a radical, and ideally transient, departure from normal. however, enteral feeding, which is always preferable to intravenous hyperalimentation, allows the clinician several options for administering food directly into the gastrointestinal tract. consideration of several variables is critical when one is initiating enteral feeding programs, such as the patient's diagnosis and attitude, the status of the gastrointestinal tract, and the ability of the patient to digest and absorb food once introduced. in addition, consideration of the type and constituency of the diet provided is important. although the options available for enteral nutrition are much greater that those for intravenous hyperalimentation, the clinician must consider dietary requirements carefully when planning enteral nutritional support. when evaluating enteral feeding for the individual patient, the clinician has four basic options: nasoesophageal tube, pharyngostomy tube (least recommended), esophagostomy tube, and percutaneous gastroscopy tube (which can be introduced using an endoscope or with the so-called "blind" technique). all techniques involve use of a polyurethane or silicone feeding tube. the nasoesophageal tube placement technique does not require general anesthesia, and the tube may be inserted using a topical anesthetic only. each of the other techniques described requires that the patient be anesthetized to ensure proper and safe placement. for temporary, short-term feeding, nasoesophageal intubation is a simple technique that works well in cats, puppies, and adult dogs. patients that are comatose; have severe, persistent vomiting; have esophageal disease or dysfunction; or are unable to swallow are not candidates for this procedure. the objective of the procedure is to place a small-diameter tube (8f to 10f for dogs weighing more than 15 kg and 5f to 8f for small dogs and cats) through the nasal cavity into the distal esophagus. the tube does not have to enter the stomach. when measuring the tube length, measure from the tip of the nose to the eighth or ninth rib (figure 4-34) . administer 3 to 5 drops of a topical ophthalmic solution (0.5% proparacaine) directly into one nostril. hold the head gently upward for a few seconds to allow the solution to reach the back of the nasal cavity. in most patients, it is desirable to wait 1 to 2 minutes and then to repeat the instillation in the same nostril. for larger dogs, 2% lidocaine solution (0.5 to 2.0 ml) gradually instilled into the nostril is an alternative technique to achieve topical anesthesia. lubricate the tube with a thin coat of a water-soluble lubricant, such a 2% lidocaine lubricating gel. pass the tube into the nasal cavity while directing the tube tip medially and ventrally into the ventral meatus. the anatomic shape of a dog's nostril usually requires directing the tip medially but almost perpendicular to the plane of the nasal cavity to facilitate insertion. initial resistance (pressure, not pain) usually is perceived, and the patient's head as expected quickly retracts, leaving the operator holding the tube tip some inches away from the patient's nose. be persistent. repeat the procedure, as necessary, by quickly inserting the first inch or more of the tube into the nostril. with the other hand, push the nasal philtrum up, and with a finger, push the lateral portion of the nostril medially. this will help facilitate movement of the tube into the ventromedial nasal meatus. once started, the remainder of the technique is relatively straightforward. as the tube reaches the caudal aspect of the nasopharynx, it should pass directly into the esophagus with little or no resistance. affix the tube remaining outside the patient to the head or face using a "butterfly" tape, gauze, suture (figure 4-35) , or skin glue (skin glue [superglue] generally is not recommended because this can result in loss of hair and skin pigment when the glue becomes dislodged). caution: the tip of the tube can be introduced inadvertently through the glottis and into the trachea. topical anesthetic instilled into the nose can anesthetize the arytenoid cartilages, thereby blocking a cough or gag reflex. i prefer to check the tube placement with a dry, empty syringe. attach the test syringe to the end of the feeding tube. rather than inject air or water in an attempt to auscultate borborygmus over the abdomen, simply attempt to aspirate air from the feeding tube (figure 4-36) . if there is no resistance during aspiration and air fills the syringe, it is likely that the tube has been placed in the trachea. completely remove the tube and repeat the procedure. however, if repeated attempts to aspirate are met with immediate resistance and no air enters the syringe, the tube tip is positioned properly within the esophagus. if there is any question regarding placement, a lateral survey radiograph is indicated. less invasive and not requiring endoscopy equipment, esophagostomy tube placement in dogs and cats is an alternative technique to use in patients that have long-term feeding needs. use a 14f to 20f rubber, polyurethane, or silicone feeding tube placed at the level of the middle of the cervical esophagus to the level of the eighth rib. the technique does require general anesthesia or, in the hands of an experienced individual, shortterm intravenous anesthesia. the technique has been described in detail in textbooks (see marks sl; additional reading). to place an esophagostomy tube, first assemble the necessary supplies: large curved rochester-carmalt forceps, clipper and clean blades, antimicrobial scrub, gauze squares, red rubber tube, permanent marker, scalpel blade and handle, needle holder, suture scissors, and nonabsorbable suture (0 nonabsorbable, cutting needle. after placing the patient under general anesthesia and intubating the patient, place the patient in right lateral recumbency and clip the lateral left side of the neck from the ramus of the mandible caudally to the thoracic inlet, and dorsally and ventrally to midline. note that the left side of the neck is preferred because of the normal anatomic location of the esophagus. however, if there is injury, infection, or mass that prevents placement of the esophagostomy tube in the left lateral cervical region, the right lateral side of the neck can alternately be used. next, aseptically scrub the clipped area, and push the rochester-carmalt forceps through the mouth into the esophagus. direct the curved tips of the instrument laterally, so the tips can be visualized under the skin. use care to note where the external jugular vein lies, to avoid laceration of the jugular vein. measure the tube from the proposed site of tube entrance to the mid thorax, then label the tube with a permanent marker. next, open the curved tips of the instrument, and make a stab incision through the skin, through the open tips of the instrument, into the esophagus. push the tips of the instrument through the skin incision. grasp the distal end of the tube with the instrument, and clamp the instrument. pull the tube through the skin incision and rostrally out of the front of the mouth. if the tube does not come easily, usually the hinges of the forceps are caught on tissue within the oropharynx or pieces of the endotracheal tube. once the distal end of the tube is through the front of the mouth, push the distal end of the tube caudally into the esophagus with a finger or the instrument. as the distal end of the tube is pushed into the esophagus, pull the proximal end of the tube, to add tension to the tube. the proximal end of the tube will flip toward the patient's nose when the tube is situated in the esophagus. the tube can be taped in place while radiographs are taken to confirm placement. after radiographs confirm placement in the esophagus, suture the tube in place with two sutures, one purse-string and finger-trap around the tube entrance site, and another deep suture near the atlas, with a second finger-trap. finally, place antimicrobial ointment over the tube entrance site, and a loose bandage around the neck. unlike gastrostomy tubes, esophagostomy tubes can be used immediately, and removed immediately, if the patient chooses to start eating voluntarily after tube placement. it is important that one first observe the technique being performed by someone with experience before attempting to place an esophagostomy tube for the first time. although postplacement complications generally are limited to local irritation or minor infection at the site of the stoma in the midcervical region, tube placement into the mediastinum or subcutaneously can occur. percutaneous gastrostomy tube placement percutaneous gastrostomy tubes are used routinely to administer nutrients and medications orally over days or weeks to cats and dogs that cannot have nutrients administered by mouth or that will not eat (e.g., because of feline hepatic lipidosis, oropharyngeal neoplasms, maxillary or mandibular fractures, oral reconstructive surgery, esophageal masses or foreign bodies, or severe pharyngitis). the percutaneous gastrostomy tube is placed so that it extends through the skin and left cranial abdominal wall of the abdomen into the body of the stomach. catheter preparation for percutaneous gastrostomy tube is as follows: 1. use the french-pezzar mushroom-tipped catheter. stomach tube preparation for percutaneous gastrostomy tube is as follows: 1. use a smooth-ended vinyl stomach tube. 2. measure the length of the tube needed to reach the stomach by laying the tube along the animal's side with the rounded end 1 to 2 cm caudal to the last rib. 3. mark the tube with an indelible marker or adhesive tape at the tip of the muzzle and cut off the excess tube. 4. put the tube in the freezer for 30 minutes to stiffen the tube before beginning the procedure. placement of a percutaneous gastrostomy tube is as follows: 1. clip and surgically prepare the skin over the left abdominal wall. 2. place the mouth speculum between the right canine teeth. 3. place the stomach tube in the esophagus to the level of the cardia. 4. rotate the tube counterclockwise while carefully advancing it through the cardia. 5. turn the tube back clockwise and advance the tube until it can be visualized through the abdominal wall 1 to 2 cm caudal to the last rib (figure 4-37 ). 6. rotate the tube so that the tip lies against the stomach and abdominal wall one third of the distance between the epaxial muscles and the ventral midline. 7. make a 2-to 3-mm skin incision directly over the lumen of the stomach tube. 8. use a sovereign catheter (over the needle) and puncture the abdominal and stomach walls, placing the catheter inside the lumen of the stomach tube. remove the needle (figure 4-38) . 9. thread a long, rigid suture through the catheter and advance it through the stomach tube until the end is observed at the mouth end of the tube (figure 4-39) . 10. carefully remove the plastic catheter from the stomach tube opening and place a hemostat clamp at the end of the suture material. 11. remove the stomach tube over the oral end of the stiff introduction suture line. 12. attach the open, beveled end of the french-pezzar catheter stomach tube to a plastic sovereign catheter using a mattress suture (figure 4-40) . 13. force the tip of the rubber stomach tube into the large end of the sovereign catheter. 14. advance the catheter tube through the mouth and esophagus into the stomach by placing traction on the abdominal end of the introduction line. 15. the catheter will emerge through the skin incision, followed by the rubber tube. grasp the tube with forceps and pull it through the incision opening (figure 4-41, a) . 16. remove the catheter by cutting it off 2 cm below the beveled tip. pull the rubber tube through the abdominal wall until slight resistance is felt (figure 4-41, b) . 17. slide the outer flange over the end of the tube down to the skin level (figure 4-42) . 18. apply antimicrobial ointment and a sterile gauze sponge over the skin incision. 19. bandage the gastrostomy tube in place (figure 4-43) . rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. tear production comes predominantly from the tarsal and conjunctival glands and from the accessory tarsal glands. the reflex tear secretors are the main lacrimal gland and the accessory lacrimal glands. the production of normal lacrimal secretions can be tested by using the schirmer tear test, a standardized filter paper (figure 4 -44) that effectively measures the rate of tear production in millimeters per minute. schirmer tear strips now are impregnated with a blue dye to facilitate visualization of the distance (in millimeters) that the tear migrates during the 1-minute test. none required. technique each eye can be tested independently, or both eyes can be tested simultaneously in the cooperative patient. carefully fold the notched end of the test strip before removing it from the plastic package. insert the folded end into the lower conjunctival cul-de-sac (figure 4 -45) and begin the timing. maintain the schirmer test strip in position by gently holding the eyelids closed but not touching the paper. at the end of 1 minute, note the degree (distance) of wetting that occurred and record it in the medical record. the normal dog and cat should produce wetting over 10 to 25 mm in 1 minute for each eye. amounts less than that are consistent with keratoconjunctivitis sicca. amounts greater than 25 mm may be normal or may be consistent with excessive tear production, or epiphora. the cornea is composed of various layers of specialized avascular epithelium and stroma. the outer layer, the corneal epithelium, is a highly sensitive, thin layer overlying the corneal stroma, the thickest layer. descemet's membrane is a distinct, thin layer of tissue beneath the stroma. the innermost layer of the cornea is the endothelium. damage to the corneal epithelium occurs frequently in dogs and cats. clinical presentation typically is characterized by blepharospasm of the affected eye with or without a visible ocular discharge or conjunctivitis. whenever superficial corneal injury is suspected, assessment of the integrity of the corneal epithelium is indicated. fluorescein dye-impregnated test strips can be used to determine whether the epithelial barrier overlying the corneal stroma has been disrupted and thus can establish the presence or absence of a corneal ulcer (figure 4-46) . none required. the test is simple to accomplish. moisten the dye-impregnated tip of the test strip with a drop of balanced saline solution (or commercial ocular irrigation solution). gently allow the tip of the test paper to touch the cornea, or sclera, of the affected eye. (in patients with particularly painful, sensitive eyes, use a topical anesthetic to moisten the test strip or apply the anesthetic directly to the cornea before testing.) immediately rinse the eye with a sterile irrigation solution to remove the excess dye (the test strip has a lot of dye; be prepared to catch the excess fluid with 2-× 2-inch gauze). promptly examine the eye with a direct, focal light source. evidence of green dye uptake in the stroma indicates that an ulcer is present. the absence of staining generally indicates that the corneal integrity is intact. one exception exists. the descemet membrane will not take up fluorescein dye. a patient with a deep corneal ulcer that penetrates through the corneal stroma and allows herniation of the descemet membrane (descemetocele) will not demonstrate a positive stain. careful visualization of the cornea, however, is likely to reveal the presence of a such a serious, deep ulcer. none required. fluorescein dye can also be used to assess patency of the nasolacrimal duct. to perform this examination, place a drop of fluorescein dye from a sterile fluorescein strip into the eye and add 1 or 2 drops of a sterile eye wash. after 2 to 5 minutes, examine the external nares with the aid of a cobalt blue filter or wood light for the presence or absence of fluorescence. a clean, 2-× 2-inch white gauze square touched against the nasal planum also will pick up the greencolored dye if the duct is patent. if dye is present, the lacrimal excretory system is patent and functioning. if epiphora exists but the primary dye test indicates that the lacrimal excretory system is patent, hypersecretion of tear fluid may be implicated as the cause of the epiphora. irrigation of the nasolacrimal system is indicated if the primary dye test result is negative. in the dog the nasolacrimal puncta are located 1 to 3 mm from the medial canthus on the mucocutaneous border of the upper and lower lids. in the dog, use a 20-to 22-gauge (in the cat, a 23-gauge) nasolacrimal cannula (figure 4-47) . topical anesthesia often is required. fill a 2-ml syringe with saline, and attach the lacrimal cannula and pass it into the lacrimal puncta of the upper lid. several points should be made about evaluating the nasolacrimal system. brachycephalicbreed dogs and cats occasionally may have a negative primary dye test result, although no blockage in the nasolacrimal system exists. in flushing the nasolacrimal system of some animals, fluid may not appear at the nose; however, the animal may gag and exhibit swallowing movements, indicating that the fluid has entered the mouth and the system is patent. none required. this procedure is preferably performed without administration of topical anesthesia. topical anesthetics not only are bacteriostatic but also may distort the cells and compromise the cytologic examination. in performing conjunctival scrapings, use a platinum spatula (kimura spatula), the tip of which has been sterilized. gently scrape the inferior conjunctival cul-de-sac (figure 4-48) . place the material on two glass slides. fix one slide in acetone-free 95% methanol for 5 to 10 minutes, then stain the slide with giemsa stain. heat-fix the other slide, and apply gram stain. to culture the conjunctiva, use sterile cotton-tipped applicators, fluid thioglycolate medium, and blood agar medium. evert the palpebral conjunctiva of the lower lid, and pass one side of a sterile cotton applicator, previously moistened with sterile broth or thioglycolate medium, over the palpebral conjunctival surface. streak the swab onto a sterile blood agar plate, then place the plate in a tube of thioglycolate broth. no topical anesthesia is used before culturing because preservatives present in anesthetics can inhibit the growth of bacteria. glaucoma is an increase in intraocular pressure incompatible with normal ocular and visual functions. one method used to measure intraocular pressure is tonometry, in which the tension of the outer coat of the eye is assessed by measuring the impressibility, or applanability, of the cornea. because the measurements based on tonometry involve calculations that have a wide base of variations, tonometry readings are always approximations. the schiøtz tonometer consists of a corneal footplate, plunger, holding bracket, recording scale, and 5.5-, 7.5-, 10.0-, and 15.0-g weights. the principle of the schiøtz tonometer is that the amount that the plunger protrudes from the footplate is related to the indentability of the cornea, which in turn is related to the intraocular pressure. however, use of applanation tonometry today has virtually replaced use of the schiøtz tonometer. in applanation tonometry, a very small area of the cornea is flattened by a known force, usually a calibrated burst of air. the advantage of this technique over the indentation (schiøtz) method is that the errors resulting from ocular rigidity and corneal curvature are greatly reduced. special equipment is required to perform applanation tonometry (figure 4-49) . the presence or absence of glaucoma, an increase in intraocular pressure, can be determined using applanation tonometry. on the other hand, gonioscopy permits one to visualize and examine the iridocorneal angle and potentially establish the cause of glaucoma. however, specific training and equipment are required not only to perform gonioscopy but also to interpret the result. this procedure is most appropriately performed by an ophthalmologist. barnett kc, crispin sm: feline ophthalmology, philadelphia, 1998, wb saunders. barnett kc, sansom j, heinrich c: canine ophthalmology, philadelphia, 2002, wb saunders. gastrointestinal studies when considering a contrast study of the gastrointestinal tract, it is not unreasonable to question the value of doing the procedure. at issue is the fact that abdominal ultrasound and/or gastrointestinal endoscopy has largely replaced contrast radiography of the gastrointestinal tract and for good reason. diagnostic modalities such as ultrasound (in the hands of an experienced individual) and endoscopy have a much greater diagnostic yield than the less sensitive contrast study. so why even try? endoscopes are not available in every practice, and limited access to ultrasound equipment, much less someone who is qualified to use it, puts routine use of advanced diagnostic modalities out of reach for many practices. however, it must be appreciated that with regard to diagnostic value, a radiographic contrast study of the gastrointestinal tract is a far less sensitive diagnostic modality than abdominal ultrasound or endoscopy. the procedure for the gastrointestinal radiographic contrast study is outlined next. contrast agents available for gastrointestinal studies include barium suspension preparations or micropaque (guerbet, villepinte, france), and water-soluble agents (gastrografin [bracco diagnostics, princeton, new jersey], which is 60% meglumine and 10% sodium diatrizoate). water-soluble agents are used if bowel perforation is suspected. undiluted water-soluble agents are hypertonic and should be diluted at a ratio of one part gastrografin to two parts water. no single procedure is appropriate for all gastrointestinal cases. the clinician must select procedures based on the clinical history and physical findings, apparent location of the lesion within the gastrointestinal tract, endoscopic findings, and results from other imaging studies, such as abdominal ultrasound. the contrast esophagram also is called barium swallow. the decision to perform a contrast esophagram is based on physical evidence of dysphagia (difficulty or pain while attempting to swallow) and/or persistent regurgitation (reflux of swallowed food without effort). the procedure necessitates that the animal fast for 12 hours before radiography. remove all leashes from around the animal's neck, and obtain survey radiographs of the thorax. in esophageal contrast studies, administer barium suspension contrast medium, 2 to 5 ml/kg body mass. administration of barium as a contrast material is contraindicated if a perforation of the esophagus is suspected. when the esophagus has been coated with radiopaque material, take lateral, ventrodorsal, and right ventrodorsal oblique thoracic radiographs to visualize the esophagus. properly prepared, the barium should be relatively thick and of a pastelike consistency. position the patient and cassette, and have the radiographic technique set. give a tablespoonful of barium orally. make the exposure when the animal takes its second swallow after the barium has been given. for esophageal studies and barium swallows, sedation with acepromazine and buprenorphine (iv, im, sq) will produce no adverse alteration in gastrointestinal motility. for cats, ketamine 10 mg iv and midazolam 0.2 mg/kg (combined) can be administered intramuscularly (im) with no significant effect in esophageal motility. caution: patients with significant swallowing disorders have a risk of aspiration if contrast material is regurgitated. sedation can increase that risk. in some cases of incomplete esophageal stricture, barium liquid will pass through the esophagus unobstructed, whereas food will not. veterinarians should mix kibbled food with the barium in this case and allow the patient to eat the mixture just before the radiograph is taken. ideally, contrast esophagrams are performed using fluoroscopy rather than conventional radiographs. in this manner it is possible not only to identify strictures and dilatations, if present, but also to obtain a dynamic study of the esophagus that provides valuable information pertaining to swallowing and esophageal motility and function and an opportunity to evaluate sphincter activity at the level of the cardia. contrast studies of the upper gastrointestinal tract are used to facilitate diagnosis of persistent vomiting, hematemesis, unexplained and chronic diarrhea, suspected enteric foreign bodies, and suspected neoplasms and obstructions and for confirmation of displaced intestinal organs, as may be seen in diaphragmatic hernias. that said, abdominal ultrasound has become sufficiently available to largely replaced the upper gastrointestinal series. with an experienced ultrasonographer, the diagnostic value of abdominal ultrasound far exceeds that derived from evaluating sequential radiographs of a patient after oral administration of a contrast medium such as barium. in the event that ultrasound capability is not available, a contrast study of the upper gastrointestinal tract still can be performed. however, the clinician must appreciate that a barium contrast study of the stomach, duodenum, jejunum, and ileum has a low sensitivity as a diagnostic test. that is, negative findings are not expected to correlate well with the absence of clinical disease. a negative study does not rule out disease. likewise, a contrast study of the upper gastrointestinal tract is not recognized for its ability to confirm a diagnosis of gastrointestinal tract disease, even when disease is present. perhaps the greatest value in performing the upper gastrointestinal series in a dog or cat today centers on the need to identify a displacement of the stomach and/or small intestine because of an extraluminal mass lesion or congenital defect in the patient. in addition, the use of a microfine barium suspension may facilitate identification of intestinal ulcers, irregularities (e.g., intraluminal neoplasia), and radiolucent foreign bodies. however, variable-diameter, solid-phase radiopaque markers called barium-impregnated polyethylene spheres (bips) can be used to assess gastric emptying time, gastrointestinal transit times, and, to some extent, obstructive disorders. if an upper gastrointestinal study is indicated, follow the technique described: 1. ensure that the hair of the animal is free from dirt, paint, and foreign material. bathe the animal if necessary. 2. withhold food for 18 to 24 hours. 3. if the colon is filled with feces, administer a cleansing enema the evening before performing the procedure. in dogs, give a second enema 3 to 5 hours before the start of the gastrointestinal series. 4. at the start of an upper gastrointestinal series, obtain survey radiographs of the abdomen. administer a barium sulfate (micropulverized) preparation by stomach tube, or induce the animal to swallow the fluids. flavored, prepared barium suspensions are available, but they taste bad (personal experience). dosage levels vary, but for barium suspensions, give approximately 10 ml/kg. as an alternative to barium, use an organic iodide liquid preparation. administer 0.5 ml/kg by stomach tube. obtain lateral and dorsoventral radiographs of the abdomen immediately after administration of the contrast material and at 30-minute, 1-hour, and 2-hour intervals. watersoluble contrast material passes through the gastrointestinal tract in 30 to 90 minutes. barium suspensions take 60 to 180 minutes to traverse the intestine. the colon usually is filled with barium 6 hours after oral administration and may contain barium for 2 to 3 days after administration. barium contrast radiography is contraindicated if perforation of the stomach or upper gastrointestinal tract is suspected. in these cases, use water-soluble contrast media such as the oral diatrizoates because leakage into the abdomen will produce no foreign body granuloma. in addition, do not administer barium sulfate when an obstruction of the lower bowel may be present. in these cases, barium may only contribute to the obstipation. the following radiographic views are recommended after administration of radiographic contrast material: 1. immediately after administration of contrast material, obtain ventrodorsal, right lateral, and left lateral views. the right lateral view shows the pylorus of the stomach filled with barium, and the left lateral view shows the cardia and fundic portion filled with barium. the objective is to evaluate the distended stomach and initial gastric emptying. 2. twenty to 30 minutes after administration of contrast material, obtain ventrodorsal and right lateral views to assess the stomach, pyloric emptying, and the proximal duodenum. 3. sixty minutes after administration of contrast material, repeat the ventrodorsal and right lateral recumbency views to assess the small intestine. 4. two hours after administration of contrast material, repeat the ventrodorsal and right lateral views to evaluate passage of contrast material into the colon and complete emptying of the stomach; contrast material should be in the terminal portion of the small intestine. the passage of contrast material through the normal gastrointestinal tract is variable; however, the following guidelines have been suggested: 1. contrast material is in the duodenum within 15 minutes in most patients. excitement can delay gastric emptying time to 20 to 25 minutes. 2. contrast material reaches the jejunum within 30 minutes and is within the jejunum and ileum at 60 minutes. 3. contrast material reaches the ileocecal junction in 90 to 120 minutes. 4. at 3 to 5 hours after administration, contrast material has cleared the upper gastrointestinal tract and is within the ileum and the large intestine. in evaluation of gastrointestinal contrast studies, consider the following criteria: (1) the size of the intestinal mass, (2) the contour of the mucosal surface, (3) the thickness of the bowel wall, (4) the flexibility and motility of the bowel wall, (5) the position of the small intestine, (6) the continuity of the opaque column, and (7) the transit time. clinical disorders for which the barium enema is indicated in dogs include ileocolic intussusception and cecal inversion (intussusception), mechanical and functional large bowel obstruction, invasive lesions of the large bowel, a mass outside the large bowel compressing the bowel, and inflammation of the lower intestinal tract. barium sulfate enemas are contraindicated in suspected obstruction of the colon and rupture or perforation of the colon. however, these same disorders also can be identified by ultrasonic examination or colonoscopy, either of which is the preferred diagnostic modality over a barium enema. twenty-four hours before radiographs, administer a liquid diet only, preferably water. during the 18 to 24 hours before the radiographs, administer a mild high colonic enema or give a saline laxative orally. do not give any irritating enemas within 12 hours of the scheduled radiographic examination; however, administer isotonic saline solution or plain water enemas before the examination to ensure that the bowel is clear. obtain survey radiographs of the abdomen, and examine the colon to ensure that this portion of the bowel is clear. sedation or anesthesia may be indicated. barium may be infused through a catheter into the colon or allowed to flow in by gravity through an enema bag. do not force barium into the colon under pressure. do not elevate the enema bag more than 18 inches above the animal. cuffed rectal catheters (bardex cuffed rectal catheters, 24f to 38f, and the bardex cuffed pediatric rectal catheter, 18f [c.r. bard, murray hill, new jersey]) can be used in dogs (figure 4-50) . for very small dogs and cats, use smaller catheters. a plastic catheter adapter and a three-way stopcock are needed. various barium sulfate preparations can be used; however, the final concentration should be 15% to 20% w/v. a commercially available barium enema kit is helpful. place the cuffed rectal catheter so that the inflated bulb is cranial to the anal sphincter. place the animal in right lateral recumbency and fill the colon with contrast material at a dose of 20 to 30 ml/kg. take the radiographs after infusion of a two-thirds dose of barium. if the colon is not filled, infuse more contrast agent. obtain radiographs in the ventrodorsal and lateral positions, and determine whether the colon is distended adequately. remove as much of the contrast material as possible from the colon, and repeat the radiographs. insertion of room air at 2 ml/kg into the colon facilitates the evaluation of the colonic surface. deflate the cuff on the catheter, and remove the catheter from the rectum. throughout the procedure of filling the colon with contrast material or air, take care not to overdistend the colon, which may lead to rupture. when reviewing individual radiographs, look for the following radiographic lesions: (1) irregularity of the barium-mucosal interface; (2) spasm, stricture, or occlusion of the bowel . the arterial phase demonstrates renal blood flow; the nephrogram demonstrates the accumulation of contrast agent in the renal tubules and is used to evaluate renal parenchyma; the pyelogram phase evaluates the urinary collecting system, including the ureters; and the cystogram reveals the collection of contrast agent in the urinary bladder. excretory urography does not result in any quantitative information about renal function and is not a substitute for renal function tests. the degree of visualization of contrast material within the renal excretory system depends on the concentration of iodine in the contrast medium, the technique of excretory urography performed, the state of hydration of the patient, renal blood flow, and the functional capacity of the kidneys. an intravenous catheter is prepositioned. the contrast medium most commonly used is a diatrizoate or iothalamate compound. administer 850 mg/kg of an iodine compound iv by syringe. continuous iv "push" is indicated. obtain a ventrodorsal radiograph at 10 seconds after injection, and repeat ventrodorsal and lateral radiographs 1, 3, 5, 15, 20, and 40 minutes after injection. this method is the current standard technique. if the patient's blood urea nitrogen level is greater than or equal to 50 mg/dl or the creatinine level is greater than 4 mg/dl, double the dose of contrast material. lesions that can be detected by using intravenous urography are renal mass lesions; neoplasia; renal cysts; renal and ureteral traumatic lesions; pyelonephritis; hydroureter; hydronephrosis; renal agenesis; hypoplasia; pelvic and ureteral obstructions (calculi, blood clots); renal parasites; ectopic ureter; and duplication of the collecting system. retrograde urethrography is a diagnostic tool used to localize diseases of the lower urinary tract of dogs and cats. this method can reveal conditions such as urethral neoplasms, strictures, trauma, calculi, or other anomalies. the procedure involves the injection of an aqueous iodine contrast medium into the urethra through a ureteral or balloon-tipped catheter. the radiopaque contrast material is mixed to a threefold to fivefold dilution with sterile lubricating jelly to increase the viscosity. a dilution of 1:3 contrast medium with sterile distilled water or saline also can be used. before retrograde contrast urethrography is performed, give the animal a cleansing enema. sedation or anesthesia may be necessary. inject 5 to 10 ml of contrast medium. near the end of the injection, while the urethra is still under pressure, obtain a lateral radiograph. if the urinary bladder is to be distended with contrast material or air, remove urine from the bladder. in the male dog, position the catheter so that the tip of the catheter is distal to the os penis. inject lidocaine 1 to 2 ml into the urethral lumen to anesthetize the urethra adjacent to the balloon-tipped catheter. in male cats, retrograde contrast urethrography can aid in defining the extent of urethral damage (stricture) or the presence of urethral calculi. in male cats, use a 4f balloon catheter or a 3.5f tomcat open-ended urethral catheter. insert the catheter 1.5 cm into the penile urethra. if the urethra is patent, 2 to 3 ml of contrast material will enable visualization of the urethra, but increased amounts of contrast material (2 to 3 ml/lb) injected into the bladder are needed for maximum distension of the preprostatic urethra. a voiding positive contrast urethrogram is necessary to visualize the distal (penile) urethra. apply external pressure to the bladder (using a wooden spoon or other external compression device), and radiograph the distal urethra. take extreme care with the amount of fluid placed in the bladder if the urethra is occluded by a balloon catheter. overdistension of the bladder results in hematuria, pyuria, urinary bladder rupture, and mild to severe bladder inflammation. palpate the bladder carefully during distension, and note the backpressure on the syringe used in filling the bladder. cystography refers to contrast radiographic procedures that facilitate visualization of the lumen and/or contents of the urinary bladder and trigone (box 4-12). three procedures can be used to image the urinary bladder: positive contrast cystography, negative contrast cystography (also called pneumocystography), and double-contrast cystography (combination of positive and negative cystography performed in the same patient). note: many of the indications for performing contrast cystography are also indications for ultrasound examination. contrast cystography is most useful for characterizing congenital and acquired alterations in the normal anatomy and function of the ureters and lower urinary tract, such as ectopic ureter. abdominal ultrasound, when available, remains the preferred method for imaging abnormalities within the bladder lumen (e.g., calculi and tumors) and changes within the bladder wall. pneumocystography, also called negative-contrast cystography, involves the insufflation of a soluble gas into the lumen of the urinary bladder to facilitate imaging of any material or tissue within the bladder lumen that otherwise would be obscured by the presence of urine or positive contrast material. prepare the patient as described previously. once a urinary catheter has been placed and the urethra is occluded, use a syringe and a three-way stopcock to inject 4 to 10 ml of carbon dioxide or nitrous oxide per kilogram. palpate the bladder while filling it with gas to avoid overdistension or rupture. inject air until there is pressure on the syringe barrel or leakage of air around the catheter. replace any air that escapes during the procedure. take lateral and ventrodorsal views of the abdomen. caution: room air is the most accessible contrast material for pneumocystography and generally can be found in most practices. however, an increased risk of air emboli is associated with the placement of room air into the bladder under positive pressure, particularly in patients with hematuria. pneumocystography is not an innocuous procedure; fatal venous air emboli have occurred in dogs and cats. this complication is seen most commonly in cases of severe hematuria. ultrasound or positive contrast cystography is preferred over pneumocystography in such cases if a soluble gas is not available. if possible, use a gas that is readily soluble in blood (such as carbon dioxide or nitrous oxide) for bladder insufflation. the injection of radiographic contrast material into the urinary bladder is referred to as contrast cystography or positive contrast cystography. when ultrasound examination is not available or not feasible, the clinical and radiographic findings noted in box 4-13 justify the use of contrast radiography to image the bladder. the same principles of preparation apply as for obtaining a pneumocystogram. use a urethral catheter with a three-way valve or a small foley catheter with an inflatable cuff. organic iodides are the contrast material of choice and should be used in 5% to 10% concentrations. double-contrast cystography also can be performed in patients for which a positive contrast study is not diagnostic, yet there is reasonable indication for an intraluminal lesion. in this case, the same urinary catheter as used for the contrast study, remove all remaining urine and contrast material. if necessary, inject 2 to 5 ml of an aqueous organic iodine contrast material into the bladder. gently roll the patient over in an attempt to coat the bladder with contrast material. then distend the bladder with air in the same manner as described for pneumocystography. some of the lesions routinely diagnosed with the aid of cystography are calculi ( vaginal examination is indicated for collection of material from the mucosal wall for culture and exfoliative cytologic examination and for vaginoscopic examination of vaginal and cervical mucosa (box 4-14). examination of the vagina for culture and cytologic or vaginoscopic examination occasionally can be performed in the cooperative patient without the use of sedation or anesthesia. an assistant is used to restrain the patient on an examination table. bitches that can be restrained for other minor examinations (ears, teeth, toenails, anal sacs, and blood samples) often will tolerate vaginal examinations. those that need further restraint may require sedation or administration of a short-acting barbiturate anesthetic. trim long perivulvar hair and cleanse the perineum with a germicidal or surgical scrub such as povidone-iodine. water and germicidal soap usually will not control surface contamination by pseudomonas and proteus species, which frequently contaminate culture swabs. in dogs with long tail hair, it is appropriate to wrap the tail with gauze before the procedure to prevent bacterial contamination. if vaginal culture is indicated, this procedure should be conducted first to avoid contamination induced by the general examination. pass a sterile, warm vaginal speculum with only a thin coating of lubricating gel into the posterior vagina while an assistant spreads the vulva. guide the speculum into the vagina by placing the speculum into the vulva just at the dorsal commissure of the vulva and applying pressure up and out against the commissure. direct the speculum dorsally toward the rectum until meeting resistance, and then direct it horizontally into the cranial vagina. this procedure bypasses the clitoral fossa and enables visualization of the urethral opening and pelvic arch. take a guarded culture swab (swab covered by a protective plastic pipette) from its individual sterile bag and pass it inside the vaginal speculum to the anterior vagina or cervical area. then expose the swab from the protective plastic tubing and rotate it against the mucosa. retract the swab into the protective plastic tubing and carefully remove it from the vagina. the protected swab then may be placed back in its original sterile bag until it is processed for culture (30 minutes) or placed in amies transport medium with charcoal. amies transport medium with refrigerator packs and a styrofoam-insulated mailing box will retain fastidious organisms for 72 to 96 hours. process bacterial, mycoplasma, and ureaplasma cultures for potential infectious agents. viral transport medium can be used for a separate sterile swab if viral agents such as the genital form of canine herpesvirus are suspected. immediately after the swabbing for culture, while the vaginal speculum is still in place, advance a clean or sterile swab moistened with sterile physiologic saline solution carefully into the anterior vagina to make a smear for cytologic examination. gently scrape vaginal epithelial cells from the ceiling of the vagina at or cranial to the region of the external urethral orifice. collect samples from the region of the clitoral fossa, which is lined by stratified squamous epithelium at all stages of the estrous cycle. gently rub the swab on the vaginal mucosa. remove the swab and roll it smoothly onto two or three clean glass slides. sterile vaginal speculum (e.g., adjustable spreading, stainless steel, or disposable plastic; cylindric; glass, plastic, stainless steel, or nylon) sterile otoscope heads of variable size for small dogs sterile protected culture swabs (teigland type or other) sterile culture swabs (culturettes) amies transport medium with charcoal viral transport media glass slides and coverslips sterile proctoscope (welch allyn, human pediatric type) or other endoscope, flexible or rigid sterile offset biopsy punch 4 the smears may be fixed immediately in 95% alcohol, sprayed with a commercial fixative or hair spray, or left to dry in air. a drop of new methylene blue stain placed on a coverslip and inverted on the smear can be used to examine a wet mount preparation immediately. this stain is not permanent and precipitates when it dries, and new methylene blue-stained smears cannot be used for comparison with other smears made later in the cycle. the diff-quik or leukostat stain is a permanent stain that can be submitted for review by a pathologist. examine the smear for stage of estrous cycle and evidence of active inflammation. compare these findings with culture results and vaginoscopic findings to interpret evidence for an active genital tract infection, a carrier state of a potential infectious agent, or a possible contaminant at culture. a diagnostic laboratory with the ability to isolate specific infectious agents should indicate the number of organisms (few, moderate, many, or heavy) and report whether the isolates are pure or mixed and their significance. the vagina of the bitch is long in comparison with that of other domestic animals, hence digital examination of the cervix, and in many cases the urethral orifice, is not feasible. the mucosa forms longitudinal folds. the clitoris is in a well-developed fossa in the floor of the vestibule. the vagina can be visualized completely with a small, sterile proctoscope or flexible endoscope. lubricate the warmed, sterile instrument, and pass it to the region of the cervix. examine first without insufflation for true color and vaginal fluids or discharge. when insufflation is performed while the vulva is compressed around the sterile proctoscope, the vagina expands and its entire wall can be viewed completely as the instrument is withdrawn. the normal canine vagina has a uniform light pink color and longitudinal folds. during proestrus and estrus, the folds become more prominent and cross-striations give the surface a cobblestone appearance. this cobblestone appearance remains smooth when estrogen levels are high but quickly becomes angular (cobblestone appearance) when estrogen levels drop during the luteinizing hormone peak (ovulation), and progesterone levels increase. this change can be used to indicate ovulation and the ideal time for breeding. the hyperemia causes the vagina to appear reddish and congested. the pressure of air insufflation balances the mucosa. the canine vulva has a large cranial dorsal median fold that may obscure the cervix. in fact, ridges near the dorsal fold may give a false impression that this fold is the cervix. during estrogen stimulation, the cervix may be open and uterine blood may be escaping. in the management of dystocia, the vaginoscope can be used to detect puppies in the birth canal and to diagnose malpositions and aid in the correction of these conditions. during the endoscopic examination, small tumors or polyps can be removed or large masses can be sampled with the biopsy punch. ulcers or erosions can be cauterized, and foreign bodies can be removed. a complete vaginal examination must include careful palpation of the vaginal wall and pelvic canal. this palpation is accomplished by digital examination through the vulva (using a sterile glove) and is assisted by palpation through the posterior abdominal wall. incomplete hymen rings, vaginal fibrous stenotic rings, or pelvic malformation can be diagnosed. a digital rectal examination may be needed for vaginal masses or pelvic deformities. the canine reproductive cycle begins at the age of 6 to 12 months and repeats at intervals of 4 to 12 months. in the average bitch, ovulation occurs spontaneously 1 to 3 days after the onset of estrus; in normal bitches ovulation may occur 3 days before to 11 days after the onset of estrus. sperm live in the uterus of the estrous bitch up to 11 days, and the ovum lives up to 5 days after ovulation. the fertilized ovum takes 4 to 10 days to reach the uterus, and implantation takes place 18 to 20 days after ovulation. the gestation period from the first breeding is 57 to 72 days and from the luteinizing hormone peak is 64 to 66 days. anestrus is characterized by dryness of the mucosa and a thin vaginal wall with stratified squamous epithelial cells a few cells to several layers thick but without cornification. noncornified epithelial cells and wbcs are present in a ratio of 1:5 in the vaginal smear. the wbcs are polymorphonuclear. the noncornified epithelial cells are 15 to 51 nm in diameter and have round free edges, granular cytoplasm, and large nuclei with distinct chromatin granules. the period of anestrus is 2 to 3 months or longer in some breeds. in proestrus the vaginal wall is thicker than in anestrus, and the mucosa shows prominent cornified squamous epithelium (20 to 30 cells thick) with rete pegs. the longitudinal and transverse vaginal folds are thick, smooth, and round. the vaginal wall becomes impervious to wbcs, but there is extravasation of rbcs to the surface epithelium. the rbcs are discharged. vaginal smears show predominantly rbcs and noncornified epithelial cells, which become cornified as proestrus progresses. wbcs are present, but their numbers decrease as estrus approaches. debris and bacteria are abundant for 7 to 10 days. the vagina is thick with longitudinal and transverse folds that become angular as estrogen levels decrease and progesterone levels increase. fluid is abundant, often tinged with blood. noncornified epithelial cells and wbcs are absent. cornified epithelial cells, which are polyhedral and contain pyknotic nuclei or no nuclei, are predominant; their presence seems to be related to the appearance of flirting by the bitch and acceptance of the stud. wbcs reappear about 36 to 96 hours after ovulation. bacteria and debris are absent during estrus, but they are seen again in the smears after ovulation when wbcs reappear 7 to 10 days later. the number of wbcs increases rapidly, the number of cornified epithelial cells decreases, and the number of noncornified epithelial cells increases. after 5 to 7 days, the number of wbcs may decrease to 10 to 30 per field. after parturition, much cellular debris, wbcs, rbcs, and a few epithelial cells are present for several days, until placental sloughing is complete. the presence of masses of degenerate wbcs (and bacteria) indicates metritis or endometritis. the continued presence of blood-tinged fluids containing abundant rbcs, a few noncornified epithelial cells, and occasional wbcs (nontoxic) plus necrotic cells for months postpartum is evidence of subinvolution of placental sites. most of the characteristics just discussed that apply to bitches also pertain to queens. however, the small size of the feline vagina precludes palpation and early vaginoscopy. a sterile, warm, small-animal otoscope speculum enables fairly good visualization of the vaginal mucosa and can be used with a small, 4-mm-diameter sterile swab to obtain smears for culture procedures. use of the speculum is easiest after parturition or during estrus. vaginal cells for cytologic examination can be obtained with a moistened 3-mm cotton swab (calgiswab) inserted 2 cm into the vagina. in some cases, flushing the vagina with sterile saline injected and aspirated with a clean glass eyedropper is more successful. use of an eyedropper may trigger ovulation, as it simulates coitus. unlike the bitch, the queen shows no diapedesis of rbcs during proestrus or throughout the estrous cycle. cytologic examination of feline vaginal smears reveals the following by stage of the estrous cycle. cytologic examination reveals scarce debris and numerous small, round epithelial cells with a high nuclear/cytoplasmic ratio, frequently in groups (seasonal: from september to january in the northern hemisphere). cytologic examination reveals increased debris and fewer but larger nucleated epithelial cells with a low nuclear/cytoplasmic ratio (0 to 2 days). cytologic examination reveals markedly less debris and numerous large polyhedral cornified cells with curled edges and small dark pyknotic nuclei or loss of nuclei (6 to 8 days) after coitus or induced ovulation. cytologic examination reveals hazy, ragged-edged cornified cells and zero to numerous wbcs with numerous bacteria and increased debris. cytologic examination reveals increasing numbers of small basophilic cells with wbcs still present (total period of metestrus, 7 to 21 days). if ovulation does not occur, the smear will return to an anestrous stage with few to no wbcs. the feline estrous cycle is continuous every 14 to 36 days if 12 to 14 hours of light are present daily. ovulation is induced 24 to 30 hours after coitus. sperm require 2 to 24 hours for capacitation in the uterus. implantation is expected 13 to 14 days after coitus. the procedure for artificial insemination in dogs includes the following steps: 1. determine the correct time to inseminate by test-teasing with a stud, by cytologic examination of vaginal smears, or by vaginoscopic examination to determine the day when vaginal folds change from round to angular. breed the day after the bitch first stands staunchly to accept service and "flags" her tail or during cytologic indications of estrus (complete cornification of vaginal epithelial cells) but before wbcs reappear in the smears. breed at 48-hour intervals until the female dog goes out of heat or for three or four inseminations. 2. if the vulva is soiled, clean it thoroughly with alcohol swabs (box 4-15). 3. gently aspirate semen through the inseminating pipette into the warm syringe. applying the artificial vagina to the shaft of the penis, apply pressure with the thumb and forefinger proximal to the exposed glandis. this usually can be done with one motion as the stud is thrusting. if the stud is shy and not interested, massage the penis slightly in the prepuce or in the artificial vagina to cause erection. when erection of the bulbus is felt, reflect the prepuce posteriorly to free the bulbus. apply pressure with the thumb and forefinger behind the bulbus, circling the shaft of the penis. after completion of the most rapid pelvic thrusting and ejaculation of the sperm-rich fractions of semen (1 to 3 ml), twist the penis 180 degrees backward in a horizontal plane, between the hind legs, so that the penis remains in the same plane as in the forward position, with the thumb and forefinger still applying pressure around the circumference of the penis proximal to the bulbus. the penis cannot be twisted unless the prepuce is reflected posterior or proximal to the bulbus glandis. twisting the penis in this position simulates a natural "tie" and allows the person collecting the semen to better visualize the collection (artificial vaginas are widely available now and are much preferred because they simulate the natural pressure of the vagina). the first drops of ejaculate may be discarded, especially if any urine is present. collect the sperm-rich fraction separately. a clear ejaculate is prostatic fluid, which may be collected separately for examination. 6. after semen collection, place the penis in the forward position, straighten out the prepuce to avoid paraphimosis, and remove the bitch from the room. allow the stud to lick the erect penis and lose the erection. check the stud for evidence of paraphimosis before it is released or caged. the ejaculate consists of three fractions: first fraction: urethral secretion (usually clear fluid)-0.1 to 2 ml within 50 seconds, ph 6.3. if evidence of urine is present, discard this fraction and do not add it to the sperm-rich fraction. in most ejaculates collected from dogs, the first and second semen fractions are collected together. second fraction: sperm-containing secretion (milky opaque fluid)-0.5 to 3 ml within 1 to 2 minutes, ph 6.1. third fraction: prostatic secretion (usually clear fluid)-2 to 20 ml within 30 minutes, ph 6.5. the total specimen is 0.3 to 20 ml, ph 6.4. because the first and third fractions are clear, waterlike material and the second fraction is milky-opaque, the clinician can separate them by changing collecting tubes as each fraction is ejaculated. collection of only enough prostatic fluid to rinse the sperm fraction into the test tube is best. too much prostatic fluid may be detrimental to the longevity of sperm in storage. collecting individual fractions may be important in determining the site of an inflammatory reaction, but for artificial insemination only the sperm-rich, low-volume ejaculate is needed for insemination, dilution, or freezing. 7. return the stud to his cage. retain the bitch until the semen is examined, if insemination is to be performed. immediately after semen collection, slowly invert the tube several times to mix the semen gently. determine the motility of sperm by placing one drop of semen on a warmed microscope slide. cover the slide with a coverslip, and observe the specimen under low power for progressive motility. there will be no "waves, " but general vigorous forward motion should be evident. if the sample is too concentrated for individual sperm to be found, mix one drop of semen with one drop of saline at body temperature on a warmed microscope slide. using high power, count 10 different groups of 10 sperm, observing the numbers of motile and nonmotile sperm. total motility for a suitable sample should be 80% or greater. motility less than 60% is not satisfactory. determine the number of sperm in the total ejaculate. sperm concentration may be determined in a hemocytometer with a 1:100 blood cell dilutor kit (unopette), and concentration then is multiplied by volume to determine sperm numbers per ejaculate. remember that more dilute samples will be obtained when prostatic fluid is collected, but total sperm numbers in the ejaculate will be only marginally influenced by dilution with prostatic fluid. total sperm per ejaculate should exceed 300 million in a normal male dog and may approach 2 billion in large dogs. a minimum number of 200 million sperm per insemination is needed on average for conception. determine morphology. make a smear of a drop of semen like a blood smear and allow it to air-dry. then stain the smear with diff-quik stain; dip the slide into the fixative and solutions 1 and 2 for 2 to 3 minutes each. then count 100 sperm at ×1000 magnification, noting normal and abnormal sperm. if there is any question about abnormality, examine 500 sperm cells. normal canine sperm are 63 nm long; the heads are 7 nm long. the percentage of abnormal sperm should be less than 20%. differential abnormality is important, and the following abnormalities should not be exceeded in any sperm count: abnormality of the head, 10% to 12%; midpiece abnormalities, 3% to 4%; tail abnormalities, 3% to 4%; and retained protoplasmic droplets, 3% to 4%. figure 4 -51 shows abnormalities that should be counted and recorded. the presence and location of distal or proximal protoplasmic droplets, which may indicate cell immaturity, are important to note. defects of the cells within the testes are generally more serious than defects that occur in the sperm during epididymal transport or after ejaculation (such as fractured heads, retained protoplasmic droplets, or bent tails). usually a biopsy should not be done on material from testes unless the testes are azoospermic. damage produced after the sperm have left the testes may indicate epididymal disease or may be the result of cold, trauma, or osmotic or urinary contamination. when abnormalities are found, it is wise to obtain two or three semen samples within a few days for baseline evaluation and then repeat the studies in 4 to 6 weeks to determine whether there is a healing or regressing trend. there are usually 64 days from the date of sperm formation to the date of ejaculation: 54 days in the testes and 10 days in transport and maturation in the epididymis. normal male dogs can be used at stud once every other day indefinitely or once every day for 7 to 9 days, after which sperm numbers in the ejaculate will decline but not to less than the numbers needed to achieve conception. although castration is a common first recommendation for any male dog with known or suspected prostatic disease, a number of prostatic disorders are recognized for which cytopathologic and histopathologic examination, rather than castration, is indicated. benign prostatic hyperplasia is recognized as the most common prostatic disorder of male dogs. in half of the dog population, changes consistent with benign prostatic hyperplasia are present by 4 to 5 years of age, especially in older intact dogs. because benign prostatic hyperplasia is androgen dependent, routine castration is the recommended treatment. however, at least three differential diagnoses justify additional diagnostic tests: prostatic neoplasia (usually adenocarcinoma), acute and chronic bacterial prostatitis, and prostatic cysts (septic and nonseptic). in male dogs with prostatomegaly and associated signs (dysuria and/or dyschezia), further evaluation of the prostate is indicated. several techniques have been described. abdominal ultrasonography is the preferred technique for evaluating prostate size, shape, and consistency. distension retrograde contrast urethrocystogram has been described as a means for evaluating the internal integrity of the prostate and is moderately effective in distinguishing normal from abnormal. however, this technique is not known to distinguish among various types of prostate disease. cytologic examination and quantitative bacterial culture of the ejaculate (especially the third fraction) of a male dog is recommended in any patient with prostatomegaly. however, sample collection can be difficult and is frequently not successful. in addition to lumbar radiographs and abdominal ultrasonography, performing a prostatic wash is a simple, noninvasive technique that may yield diagnostic information. using aseptic technique, place a conventional urinary catheter into the bladder and remove all urine. lavage of the urinary bladder with up to 5 ml of sterile saline is recommended. recover the saline and save it (sample no. 1). subsequently, retract the catheter tip, but only to the level of the prostate gland (immediately caudal to the trigone). position of the tip usually can be verified by tactile placement and the detection of increased resistance to catheter movement during retraction. position can be confirmed with a lateral radiograph of the pelvis. with the catheter in place, identify the prostate on a digital rectal examination and gently massage for approximately 1 minute to force prostatic fluids into the urethra. infuse 5 ml of sterile saline through the catheter. the objective is to wash prostatic fluids and cells into the urinary bladder and recover the saline from the bladder (sample no. 2). examine fluid from both samples cytologically by distributing a drop of fluid across a glass slide, air-drying, and staining; submit a small aliquot (0.5 ml) for bacterial culture. cytologic examination is used to detect the presence of inflammatory cells versus neoplastic cells. low numbers of neutrophils (<5 cells per high-power field) are present in ejaculates and prostatic washes from normal dogs. quantitative bacterial culture, with a yield of greater than 2 log 10 , of one or more bacterial species in sample no. 2 confirms bacterial prostatitis. complications from this procedure are unlikely, but conceivably a patient with septic prostatitis and prostatic abscesses could become bacteremic after this procedure, which in some patients could lead to sepsis. ultrasound examination is an important first step, when available, in assessing the size, shape, and internal integrity of the canine prostate gland and for detecting any changes in structures adjacent to the prostate. however, ultrasonography generally will not distinguish among different types of prostatic disease. further diagnostic tests are especially indicated in castrated, middle-aged to older male dogs with evidence of prostatomegaly. percutaneous fine-needle aspiration and/or prostatic biopsy are indicated. fine-needle aspiration of the prostate is performed through a ventral abdominal approach. use aseptic technique, and surgically prepare the skin at the level of needle insertion. because needle movement, once the needle has been inserted, could damage the urethra or 4 adjacent structures, perform the procedure in the sedated or anesthetized patient. use an approach similar to that used for cystocentesis in a male dog with the exception that needle entry is at a point caudal to that used to enter the urinary bladder but is cranial to the pubis. the procedure can be performed with or without ultrasound guidance. in the absence of ultrasound guidance, determine needle position by tactile placement and detection of resistance as the needle enters the prostate. multiple needle penetrations and aspirations are attempted without withdrawing the needle from the skin. relieve negative pressure in the syringe before removing the needle. apply any material collected to a glass slide and allow it to air-dry before staining. any conventional stain used for peripheral blood is appropriate. a transrectal approach to fine-needle aspiration of the prostate has been used in dogs and is performed routinely in men. however, the distance from the anus to the prostate, visualization, and the risk of infection generally are cited as reasons for not performing this technique in dogs. fine-needle aspiration may not be diagnostic, particularly in patients with isolated, discrete lesions (cysts or neoplastic nodules) within the prostatic parenchyma. in such cases, ultrasound-guided needle (tru-cut) biopsy of the prostate is indicated. specific training and experience are indicated for performing this procedure because significant complications can result. complications associated with prostate biopsy and fine-needle aspiration are not insignificant. hematuria and periprostatic hemorrhage are described. postaspiration and postbiopsy abscess also have been described. consider the risk of urethral penetration and subsequent stricture at the site of penetration. kutzler ma, yeager a: prostatic diseases. in ettinger sj, feldman ec, editors: textbook of veterinary internal medicine, ed 6, st louis, 2005, elsevier. upper respiratory tract for purposes of this discussion, the anatomic limits of the upper respiratory tract of the dog and cat extend caudally from the nasal planum to the first tracheal ring. key anatomic structures that principally can cause clinical signs include the anterior (external) nares, nasal cavity, nasal turbinates, frontal sinuses, maxillary recesses, upper dental arcade (especially the roots of the maxillary canine teeth), choanae (posterior nares), nasopharynx, soft palate, arytenoid cartilages, glottis, larynx, and vocal folds (see table 4 -9). clinical signs related to the upper respiratory tract in dogs and cats are among the most common presenting complaints encountered in small animal practice and, interestingly, are frequent reasons for referral to specialty practices and veterinary teaching hospitals. the oral and nasal cavities are important portals of entry for foreign body entrapment and infectious agents. in addition to the occurrence of nasal neoplasia and trauma, it is not surprising that upper respiratory tract diseases in dogs and cats are common presentations. however, upper respiratory signs can be associated with significantly different underlying causes. localizing the problem amid a variety of clinical signs in an anatomically complex area presents significant diagnostic and therapeutic challenges to even the most astute clinician. the presentation addresses upper respiratory disease in the dog, with specific emphasis on clearly defining the presenting clinical signs, localizing the problem, and establishing the diagnosis. the first and most important step in establishing a diagnosis of canine upper respiratory disease is to define the presenting sign. experience has shown that an owner's ability to describe the patient's clinical signs accurately, particularly when signs are not present at the time of examination, is usually inconsistent and inaccurate, although it can be most entertaining. the four localizing clinical signs characteristically associated with upper respiratory diseases are sneezing and/or nasal discharge, stertor, stridor, and cough. each sign, considered independently, will focus the examination to the appropriate anatomic region of the upper respiratory tract. definition of the clinical signs sneezing and nasal discharge may seem intuitive. this is the most common presenting sign in dogs with upper respiratory disease. owners that present a dog with sneezing are likely to be accurate in their description of the problem. however, the presence or absence of a nasal discharge may be more difficult to establish. volume, character, and frequency of the discharge ultimately determine whether the owner will have even observed this sign. the astute owner will report whether the discharge is unilateral or bilateral. in the patient that has a history of sneezing and nasal discharge, instillation of a topical nasal decongestant into each nostril occasionally will provoke sneezing and elicit the nature of any discharge that is present. sneezing and/or nasal discharge localizes the problem to the nose, nasal cavity, and paranasal sinuses. however, thorough examination of the nose and nasal cavity can be difficult, even with the availability of appropriate endoscopy equipment. in addition to careful examination of facial symmetry, the first part of the examination begins in the oral cavity, with emphasis on the maxilla, the hard palate, and the canine teeth. examine the hard palate for evidence of trauma (penetrating or nonpenetrating) and congenital cleft palate (puppies). carefully probe the medial aspect of the maxillary canine teeth for evidence of oronasal fistulas. despite normal-appearing teeth and gingiva, severe, occult periodontal disease with resulting necrosis of bone does result in a septic communication between the oral and nasal cavities. the owner characteristically describes paroxysms of sneezing associated with a sanguineous nasal discharge or spray. if these findings are negative, radiographs of the skull are indicated. three views, obtained in the anesthetized patient, are indicated: lateral, ventrodorsal, and occlusal (open mouth) view. radiographic interpretation of the nasal cavity and sinuses dictates that the clinician have a thorough understanding of the anatomy of the upper respiratory tract. subsequently, with the patient still anesthetized, attempt a visual examination of the nasal cavity. radiographs are always performed before visual examination of the nasal cavity. manipulation of the tissue may result in intranasal bleeding, which will significantly complicate radiographic interpretation. a simple otoscope speculum placed into each nostril allows an adequate examination of the proximal 20% to 25% of the nasal cavity in most dogs. visual examination of the caudal 75% of the nasal cavity can be attempted only with a small-diameter endoscope. flexible and rigid scopes are available; each has advantages and disadvantages that will be discussed. computed tomography and magnetic resonance the second most common clinical sign associated with upper respiratory disease in dogs, stertor is intermittent, yet persistent or continuous snorting, also called stertorous breathing. paroxysms of stertor, typically called reverse sneezing, are characterized by rapid, consecutive inspiratory bursts through the nose. seldom actually seen during examination, reverse sneezing is likely to result from the patient's attempt to displace matter trapped in the nasopharynx and move it into the oropharynx, where it can be swallowed. visualization of the nasopharynx and choanae is essential in the patient that has chronic or persistent stertor. the examination can be accomplished only in the anesthetized patient. sedation is not sufficient to conduct the examination. a flexible endoscope with the ability to flex approximately 170 to 180 degrees is recommended. examination allows visualization of the nasopharynx and associated mucosa, the choanae (posterior nares), and the top of the soft palate (see figure 4 -34). nasopharyngeal foreign bodies are by far the most common finding. sticks, plant material (grass and juniper twigs), peas, cotton balls, and thread are just a few examples. neoplasia is the second most common finding. in cats, lymphoma (felv related) obstructing the choana most commonly is observed (see figure 4 -35). in dogs, neoplasia is uncommon, but (in my experience) sarcomas in young dogs have been seen most frequently. the least commonly encountered of the upper respiratory signs is stridor, or stridulous breathing. stridor is audible wheezing and is associated with restriction to airflow, usually at the level of the larynx. therefore stridor is the most critical and potentially life-threatening upper respiratory sign. this is especially true when stridor is continuous. the patient that has continuous stridor deserves immediate attention. make every effort to discern the cause once the clinical sign is characterized. in obtaining the history, owners generally describe wheezing accurately; however, some patients actually may have severe dyspnea or orthopnea. careful questioning of the client is indicated to determine whether wheezing is associated with the additional effort to breath. the clinician also should make an effort to discern whether the owner has observed any change in the ability of the dog to vocalize or bark. simply listening to the patient breath in a quiet room is the first step in assessing stridor. a stethoscope is not required to hear wheezing but should always be used to examine the cervical trachea, the larynx, and the lungs. any restriction to airflow in the larynx or cervical trachea can cause stridor. however, in the majority of cases the stridor will be significantly louder at the level of the larynx, indicating a restrictive lesion at that level. if any indication of respiratory distress is reported or manifests during the examination, subject the patient to a visual examination under general anesthesia. sedation is not sufficient to conduct the examination. be prepared. these patients are not routine. emergency resuscitation may be required on induction of anesthesia, including the need to perform a tracheostomy. on induction, carefully place an endotracheal tube. if there are no complications associated with inserting the tube, once anesthesia has been effectively induced and the patient's condition is stable, lateral and dorsoventral radiographs of the larynx and cervical trachea are indicated. metallic objects (e.g., fish hooks) can become buried in the mucosa and may not be observed during a visual examination. remove the endotracheal tube in order to conduct a visual examination. a focal, handsfree light source directed into the oropharynx is strongly recommended. carefully examine the epiglottis, arytenoid cartilages, glottis, and vocal folds using a cotton-tipped applicator. careful observation of the symmetry and function of the arytenoid cartilages is essential. the left and right cartilages normally respond to tactile stimuli when the patient is in a light plane of anesthesia; both sides should move to the medial plane rapidly and at the same time. they may not close, depending on the depth of anesthesia. it should be possible to visualize the cartilage on the inside of the tracheal rings while looking through the glottis. in large breed, middle-aged and older dogs, laryngeal paralysis is the most common cause of stridor. associated signs may include exercise intolerance and collapse during exertion. laryngeal paralysis and stridor also may be observed in young breeds as a congenital disorder (dalmatian, rottweiler, bouvier des flandres, siberian husky, and bull terrier). foreign body penetration of the laryngeal tissues can cause serious and lifethreatening obstruction because of infection and swelling. neoplasia may cause obstructive mass lesions involving the larynx, especially squamous cell carcinoma and lymphoma. granulomatous laryngeal disease and fungal mycetoma have been reported. the presence of a mass lesion, assuming there is no foreign body detected, warrants biopsy of the lesion. additional effort to control postbiopsy bleeding is important. i use a cotton-tipped applicator saturated with a 1:10,000 dilution of epinephrine held against the biopsy site for 30 to 60 seconds. this is time well spent. postbiopsy administration of systemically effective dexamethasone has been suggested to control laryngeal swelling, but i have not found this to be effective or important. the following diagnostic procedures are elective and are indicated in patients with chronic disorders of the lower respiratory tract that are not considered life-threatening. transtracheal aspiration is a safe and clinically useful method for obtaining material for cytologic and bacteriologic examination from the lower respiratory tract of medium-sized to large dogs without invading the oval cavity. this procedure is not indicated in cats. the technique can be performed on the unanesthetized animal, although some sedation may be indicated. the hair overlying the larynx is clipped and prepared surgically. in small dogs and cats, tracheal aspirates are collected by passing the catheter through sterile tracheal tubes. light levels of anesthesia are used to accommodate coughing and tracheal intubation. place the animal in sternal recumbency or in the sitting position. elevate and extend the head. locate the cricothyroid membrane by moving the finger along the proximal trachea until the large ventral ridge of the cricoid cartilage is felt. use a 16-gauge, 1 ⁄2-inch intravenous catheter to collect material through the trachea (figure 4-52) . puncture the cricothyroid membrane with the 16-gauge needle, and pass the catheter into the trachea until it reaches the distal trachea or main stem bronchus. (alternatively, in large dogs, insert the catheter between the tracheal rings at the junction of the middle third and distal third of the cervical trachea.) withdraw the needle, and leave the catheter in place. attach a 12-ml syringe containing sterile saline solution to the catheter. expel 1 to 2 ml of saline from the syringe. when the animal coughs, aspirate with the syringe to collect cells and mucus for bacteriologic and cytologic examination. when material has been collected, remove the catheter and bandage the animal's neck. culture material present in the syringe in blood agar and in thioglycolate medium. prepare material from aspiration for cytologic examination. press large plugs of mucus between two clean glass slides, and stain thin smears with wright or giemsa stain. complications of transtracheal aspiration biopsy include catheter trauma to the lower airway or needle trauma to the larynx, resulting in bleeding, subcutaneous emphysema, pneumomediastinum, pneumothorax, or airway obstruction. in cats and small dogs and in dogs for which general anesthesia is not contraindicated, tracheal aspiration (or tracheal wash) is a relatively safe, easy-to-perform procedure that can yield excellent diagnostic cytologic and culture specimens. the procedure has some advantages over transtracheal aspiration in that it allows sample collection from airways beyond note: cats and dogs with acute, severe dyspnea must be regarded as having a life-threatening condition until proved otherwise. immediate therapeutic and diagnostic intervention is indicated. section 1 describes appropriate interventive procedures for the management of these patients. the bifurcation of the trachea (carina) and avoids complications associated with patient discomfort and movement during the procedure. however, cough reflexes are eliminated completely, thereby decreasing potential sample yields from deep in the airway structure. in either case, transtracheal and tracheal aspirations provide the best diagnostic material from large airways, not small airways and alveoli. the anesthetized dog or cat usually is placed in sternal recumbency. lateral recumbency (affected side down) may facilitate recovery of specimens from patients with focal or regional lung disease. use a sterile endotracheal tube to administer the anesthetic and oxygen. introduce a sterile red rubber catheter (long enough to extend beyond the carina) through the endotracheal tube (figure 4 -53). (note: disposable adapters for use with endotracheal tubes are available that allow continuous administration of anesthetic gases while passing the rubber catheter through the tube [ figure 4 -54].) introduce the catheter blindly until resistance is met as the tube attempts to enter smaller airways. use aliquots of warmed, sterile saline in prepared syringes to wash and retrieve samples. aliquots of 3 to 5 ml can be used per collection attempt in small dogs and cats, whereas volumes up to 10 and 20 ml are appropriate for larger dogs. with the catheter positioned as deep as practical in the airway, infuse the entire volume of saline. gentle agitation (intermittent aspiration and injection) may facilitate sample collection. if a 10-ml quantity is infused, retrieval of only 1 to 2 ml as a final volume per collection attempt is not unusual. the remaining fluid is rapidly (seconds) absorbed into the pulmonary vasculature. important: when performing this procedure, do not withdraw the rubber catheter while maintaining a high negative pressure on the syringe. doing so actually may tear mucosa away from the airway and could lead to pneumothorax or pneumomediastinum. the procedure can be repeated safely in the same patient several times. collection of three to five samples is routine. more samples may be indicated depending on the patient's condition and response to the procedure. monitoring of patients undergoing a tracheal wash procedure for oxygen saturation (pulse oximetry) throughout the procedure is recommended. in some patients with reactive airways, infusion of saline may cause significant bronchoconstriction, detected by a rapid decline in oxygen saturation. process collected samples immediately. submit at least one sample of liquid (not a swab of the liquid) for bacterial culture and sensitivity or mic. quantitative cultures are impractical because specimens will be diluted. if the sample appears to be highly cellular (characterized by turbidity), place aliquots into tubes containing edta. syring rs: tracheal washes. in king lg, editor: textbook of respiratory disease in dogs and cats, st louis, 2004, elsevier. bronchoalveolar lavage bal is an alternative diagnostic procedure to transtracheal aspiration and endotracheal wash. bal has the advantage of retrieving fluid samples from distal airways and alveoli. this is a highly diagnostic procedure indicated in patients with generalized lung and regional (interstitial and/or airway) disease that are not in respiratory distress. patients suspected of having allergic or infectious respiratory disease or neoplasia are candidates for bal. although bal is used as a therapeutic procedure in human beings with chronic lung disease associated with accumulations of surfactant in the alveoli, there is no therapeutic indication for bal in dogs or cats. bal must be performed in the anesthetized patient and consequently may be contraindicated in some patients with severe respiratory disease. technique bal entails instilling sufficiently large volumes of fluid into the distal airways to reach, and recover, reasonable cytologic samples representative of small airways and alveoli. several variations on the technique have been described, but all recommend blind or visual placement of a catheter or bronchoscope into an airway of a lung lobe such that the airway is occluded. sterile, nonbacteriostatic 0.9% saline, warmed to approximately body temperature and drawn into prepared syringes, is the fluid of choice. the volume of fluid varies with the size of the patient. defined doses of saline per kilogram of body mass have not been described. in large dogs, two 25-ml aliquots (50 ml total) can be infused into each lobe sampled. in small dogs and cats, total volumes per lobe generally are restricted to 10-ml aliquots. recovery may be as low as 2 to 5 ml with each attempt. ensure that the animal's hair is free of dirt and debris try to limit the animal's fluid intake in the 12 hours preceding radiography empty the animal's bladder immediately before taking radiographs understanding the most commonly diagnosed causes of sneezing and nasal discharge is especially helpful in patient management. in no particular order, the most common differential diagnoses for sneezing and/or nasal discharge include the following: 1. oronasal fistulas: especially common in middle-aged to older dogs, despite a history of recent dental prophylaxis. empiric treatment with an orally administered antibiotic typically results in rapid and complete resolution of clinical signs no breed is predisposed, but the condition is uncommon in brachycephalic breeds. persistent nasal discharge, sneezing, and intermittent epistaxis are common presenting signs. nasal radiographs may demonstrate lytic bone lesions. lysis of the vomer strongly supports neoplasia versus mycotic rhinitis. exposure to tobacco smoke has been associated with 2.5 times greater risk in long-nosed dogs. no or minimal response of the discharge to antibiotics occurs. eighty percent of nasal tumors are malignant. adenocarcinoma is most common, followed by squamous cell carcinoma persistent and voluminous mucoid nasal discharge, with or without sneezing, and nasal pain are reported. erosion of the external nares is an important physical finding. discharge is not responsive to antimicrobial treatment. occlusal view radiographs of the nasal cavity may demonstrate evidence of turbinate destruction and/or increased fluid density on the affected side. forty percent of patients are 3 years of age or younger; 80% are 7 years of age or younger. the diagnosis is uncommon in brachycephalic breeds lymphoplasmacytic rhinitis: poorly described clinical syndrome associated with chronic sneezing and nasal discharge (bilateral or unilateral) nebulization is used (l) in combination with oxygen therapy upper respiratory disease of cats, pneumonia, and postoperative atelectasis and pneumonia; and (4) in chronic respiratory diseases such as chronic bronchitis, bronchopneumonia, collapsed trachea with secondary tracheobronchitis, emphysema, and bronchiectasis. aerosol therapy, however, is a limited-use therapeutic technique used in dogs and cats to administer antimicrobials, bronchodilators (aminophylline, 100 mg), or corticosteroids. the advantage of doing so is to achieve relatively high levels of drug in the respiratory tract in patients with defined lower respiratory tract disease. in addition, administration of potentially toxic antimicrobials (aminoglycosides) by this route has been shown to be associated with minimal or insignificant uptake into the general circulation drugs that can be applied by jet nebulizer (figures 4-56 and 4-57) include the following: 1. bronchodilators: always use bronchodilators when administering drugs that may be irritating and constricting systemic administration of most antibiotics produces adequate pulmonary concentration for antibacterial effect. for bordetella species that are located at the tips of bronchial cilia, topical contact via nebulization may be useful. antibiotics that have been used successfully and safely include kanamycin (250 mg in 5 ml saline twice daily); gentamicin (50 mg in 5 ml saline twice daily) bland solutions: use these in large volume for prolonged mist effect: 0.9% sterile saline (5 to 200 ml as needed); glycerin (5% in saline) detergents and mucolytics: these compounds are irritating and currently are not recommended by most authors antifoaming agents: administer ethyl alcohol (70% solution, 5 to 10 ml twice daily) figure 4-55: disposable jet nebulizer used to administer humidified air and/or medication drugs affecting the respiratory system advanced and expensive techniques recently have been described: laparoscopic-assisted cystotomy, use of the ellik evacuator, use of stone "baskets" (through a cystoscope), and lithotripsy are examples. however, for removal of uroliths from dogs and cats with partial or complete urinary obstruction, urohydropulsion is among the more effective yet inexpensive techniques available. urohydropulsion is a therapeutic procedure for removal of foreign material, namely, uroliths, from the bladder and/or urethra of dogs canine lower urinary tract diseases canine and feline nephrology and urology 4. using a gloved left index finger (not lubricated) as a guide, insert the pipette through the vulva and dorsally into the vagina and forward to the cervix. elevate the bitch's rear quarters to a 45-degree angle by having an assistant pick up the bitch by holding the hock region so that no pressure is applied to the ventral abdomen and uterus. eject the semen gently and slowly. eject a bubble of air to push all the semen through the pipette. deposit the semen in the anterior vagina. 5. remove the pipette, and hold the bitch in an elevated position for 5 minutes. during this time, use the finger encased in a sterile glove to "feather" the ceiling of the vagina to stimulate constrictor activity. this may be important to simulate a "tie" and transport semen into the uterus. 6. lower the bitch to the normal position, and immediately walk her for 5 minutes so that she does not sit down or jump up on a person and allow semen to run back out of the vagina. 7. for best conception, inseminate undiluted fresh semen immediately. 8. refrigerated extended semen is best used within 24 to 48 hours if possible. however, refrigerated semen has been kept viable for up to 9 days with proper care. skim milk has been used as an economical and adequate extender. heat milk to 92° to 94° c for 10 minutes, cool it, and skim it at room temperature. to each milliliter, add 1000 units of crystalline penicillin. if pseudomonas species affect the semen, polymyxin b may be added at 200 units/ml of extender. dilute semen with extender at a semen/extender ratio of 1:1 to 1:4. extend canine semen for freezing with a diluent containing 11% lactose, 4% glycerin, and 20% egg yolk. refrigerate the 1:4 diluted semen; then pipette 0.05-ml portions into depressions in a block of dry ice and hold them for 8 minutes to freeze. store the frozen pellets in liquid nitrogen. frozen semen can be thawed in buffered saline at 30° to 37° c. good semen may be stored in liquid nitrogen for many years without significant loss of motility. conception is best when large numbers of thawed motile sperm are deposited in the cervix or uterine cavity. conception is poor when thawed semen is placed in the anterior vagina, as done in artificial breeding with raw semen. memon ma, sirinarumitr k: semen evaluation, canine male infertility, and common disorders of the male. in ettinger sj, feldman ec, editors: textbook of veterinary internal medicine, ed 6, st louis, 2005, elsevier. semen collection: canine semen is collected for examination for breeding soundness, for investigation of infertility or prostatic disease, and for artificial insemination (box 4-16).the following steps outline the procedure for collecting semen from a male dog: 1. take the stud and an estrous teaser bitch (if available) to a quiet room where there will be no distractions and where there is good traction (rubber mats or rug) for mounting by the stud. 2. hold the bitch, and allow the stud to "flirt" (become aroused) for several minutes. if the bitch is in heat, a brief period of foreplay ("foreplay" may not be the appropriate word to describe the mating behavior of dogs, but it illustrates the point) with both dogs unrestricted will help the process. 3. if necessary, have assistants restrain the muzzled bitch and control the stud by a collar and leash. bring the stud up to the rear end of the bitch, and allow him to mount her or keep his nose in the region of her perineal area. 4. attach the artificial vagina to the semen collection tube, and apply a scant amount of lubricant to the opening of the artificial vagina. 5. if mounting occurs, allow the stud to grasp the bitch and start to thrust his pelvis in an attempt to copulate. gently, from the side of the sheath, grasp the penis by the prepuce and move the prepuce back over the engorged bulbus glandis; while for dogs undergoing bal, particularly when reactive (allergic) airway disease is suspected, pretreatment with a bronchodilator is appropriate and is recommended. aminophylline can be administered at 5 mg/kg (cats) or 11 mg/kg (dogs) orally 1 to 2 hours before the procedure. alternatively, terbutaline, 0.01 mg/kg, can be administered subcutaneously to cats 30 minutes before the procedure.bronchoscopic bal allows direct visualization of the airway or lobe of interest. in medium to large dogs, place the bronchoscope directly through a sterile endotracheal tube. use of an inexpensive, disposable endotracheal tube adaptor permits simultaneous administration of oxygen and anesthetic throughout the procedure. saline can be infused from a syringe directly through the biopsy channel of the endoscope. the bronchoscope serves as the infusion catheter. using this technique, samples can be collected effectively from multiple lobes. blind placement (nonbronchoscopic) bal using a rubber end-hole catheter is required in cats and small dogs. blind placement is also appropriately used in patients with generalized lung or airway disease when discrete placement of the bronchoscope cannot be accomplished reliably. as with the endotracheal wash procedure described before, gentle agitation with the syringe (intermittent aspiration and injection) may facilitate sample collection. do not withdraw the bronchoscope or catheter while maintaining significant negative pressure because this may lacerate the airway, leading to pneumothorax or pneumomediastinum.bal is an invasive diagnostic procedure that is not without risk of injury or death. after completion of bal, administration of 100% oxygen for 5 to 10 minutes via endotracheal tube is recommended for all patients. evaluate the patient carefully for breathing effort and oxygen saturation (pulse oximetry) during recovery. although significant quantities of fluid remain in the airways after bal, most of the volume is absorbed rapidly. residual amounts of fluid, however, can be retained for 24 to 48 hours after the procedure. during this time, some patients will manifest cough. crackles may be auscultated. percutaneous aspiration needle biopsy can be helpful in establishing a diagnosis in conditions such as (l) chronic inflammatory disease of the lung-for example, granulomatous lung disease caused by mycotic organisms; (2) chronic inflammatory disease; (3) metastases to the lung; and (4) primary lung tumors. the biopsy may provide enough diagnostic information to preclude performing an exploratory thoracotomy. lung biopsy is contraindicated in animals with hemorrhagic disease or thoracic disease that produces forceful breathing and coughing. clip and surgically prepare the biopsy site. infiltrate the skin, subcutaneous tissue, muscle, and parietal pleura with 1% to 2% lidocaine. in patients with diffuse parenchymal lung disease, taking biopsy material from the diaphragmatic lobes is recommended. the dorsal portions of the seventh to ninth intercostal spaces are preferred for percutaneous biopsies. in diffuse lesions, take biopsy material from the right or left thorax. caution: understanding of the risks associated with performing fine-needle aspiration of the lung is important, and these risks must be clearly communicated to owners whose pets undergo this procedure. lung aspirates will yield only cells, fluid, and trace amounts of tissue, yet there is a significant risk of inducing pneumothorax with the procedure, even when performed without difficulty or complications. for the procedure, a 22-to 25-gauge disposable needle (such as a 1-inch spinal needle) with stylet is preferred. leave the stylet within the needle until the lung has been penetrated. then quickly remove the stylet and immediately attach a sterile 6-to 12-ml syringe. the amount of air that might enter the lung between the time the stylet is removed and the syringe is attached is negligible. holding the syringe carefully and steadily against the patient's thorax, establish negative pressure in the same manner as when obtaining an aspirate from a lymph node. as much as the patient will permit, attempt three to four aspirations without withdrawing the needle.alternatively, insert a conventional 25-gauge needle attached to a 6-ml syringe subcutaneously over the area of interest. then establish significant negative pressure while the tip of the needle is still positioned in the subcutaneous tissues outside the parietal pleura. while maintaining the same amount of negative pressure in the syringe, direct the needle into the lung, leave it in place for 1 to 2 seconds, and withdraw it completely. apply any material collected directly to glass slide. this procedure is best conducted in patients that are awake. attempting the procedure in anesthetized dogs or cats could result in an unsuccessful aspiration, or, if the lungs were under positive pressure (ventilation or bagging), the risk of causing pneumothorax could be increased. other reported complications include hemothorax (always exciting), lung laceration caused by patient movement during the procedure, pulmonary hemorrhage, and hemoptysis. contraindications to fine-needle aspiration include patients with a known bleeding diathesis and coagulopathy, thrombocytopenia, uncontrolled coughing, pulmonary hypertension, pulmonary cysts, and bullous emphysema.ultrasound-guided techniques for fine-needle aspiration or biopsy of the lung recently have been described and generally are associated with fewer procedural complications. however, additional training and experience, in addition to having access to the proper size and type of ultrasound probe, are critical. cole sg: fine needle aspirates. in king lg, editor: textbook of respiratory disease in dogs and cats, st louis, 2004, elsevier. inhalation therapy can be defined as nebulization (humidification of the inspired air) and aerosol therapy (the process whereby drugs are vaporized in a solution and delivered directly into the respiratory tract). in companion animals, inhalation therapy is most useful for humidifying air in the respiratory tract and moistening the mucous membranes (nebulization). sustained inspiration of dry air or gases causes irritation to the respiratory epithelium, which in turn results in swelling, bronchial gland hypertrophy, goblet cell proliferation, and loss of ciliary epithelium over time. respiratory secretions become thick and tenacious, and efficient bronchial drainage is impaired. the objectives of inhalation therapy include the following: 1. humidification of bronchial mucous membranes 2. deposition of miniscule amounts of potent drugs in smaller airways to achieve optimal topical therapeutic effects with minimal systemic side effects (e.g., bronchodilators) 3. deposition of moderate amounts of potent agents or agents that are effective only topically (e.g., antibiotics and mucolytics) 4. deposition of relatively large quantities of bland substances that promote bronchial drainage with minimal irritation (e.g., saline, propylene glycol, glycerin, and detergents) the objective of voiding urohydropulsion is to induce forceful voiding of urine by manually compressing the bladder to facilitate removal of cystic uroliths in female dogs. caution: do not perform this procedure until it can be confirmed by catheterization or cystoscopy that the urethra is patent.with the bladder filled with urine or saline (via catheterization), lift the patient (preferably sedated or anesthetized, although this procedure can be done in the awake patient) into a position such that the tail and perineum are ventral and the head is upright. the spine should be approximately perpendicular to the working surface. using one or both hands, gradually increase pressure on the bladder to induce and maintain a forceful stream of urine. objectively, small uroliths will be extruded. if the procedure is only partially successful, it can be repeated as necessary. obviously, voiding urohydropulsion has limitations and cannot be used in male dogs or in dogs with urethral obstructions or strictures. this procedure is indicated for male dogs and cats with partial or complete urethral obstruction caused by uroliths or accumulations of "sand. " this procedure should be performed in the anesthetized patient.note: there are discrepancies in the literature regarding whether to empty the urinary bladder of urine before performing this procedure. because patients with urethral obstructions may have a significant volume of urine in the bladder at the time of presentation, some authors recommend performing cystocentesis to relieve the internal pressure before attempting urohydropulsion. however, in patients that have had a profoundly distended bladder for several hours (even days), penetrating the urinary bladder with a needle presents significant risk of rupturing a fragile bladder. the next step, of course, is abdominal surgery. i recommend avoiding cystocentesis whenever possible. the volume of saline required to flush uroliths into the bladder is inconsequential considering the total volume already present.with the patient positioned in lateral recumbency, retract the prepuce and expose the penis as for conventional bladder catheterization technique. use sterile technique to pass an appropriately sized flexible catheter, which is advanced to the point of obstruction. attach a catheter-tipped 60-ml syringe filled with warmed (my preference) sterile saline and a water-soluble lubricant mixture (approximately two parts saline to one part lubricant) to the urinary catheter. an assistant places a gloved (always preferred) finger into the rectum to identify and occlude the lumen of the pelvic urethra at the level of the pubis. subsequently, infuse saline forcefully into the catheter to dilate the urethra proximal to the obstructing urolith. at that point, release the digital pressure on the proximal urethra while the solution continues to be infused through the catheter. objectively, the pressure within the urethra forces small stones retrograde into the urinary bladder, thereby relieving the obstruction. the objective of this procedure is not to push the calculi into the bladder with the catheter, because this can substantially injure the urethral mucosa, nor is it to force the calculi around the catheter and move it antegrade. key: cord-014516-r59usk02 authors: nan title: research communications of the 24th ecvim‐ca congress date: 2015-01-10 journal: j vet intern med doi: 10.1111/jvim.12491 sha: doc_id: 14516 cord_uid: r59usk02 nan saccharomyces boulardii (sb)is a non-pathogenic yeast used in the prevention and treatment of gastrointestinal disorders in human beings and horses. the aim of this study was to evaluate the effect of sb in healthy dogs and dogs with chronic enteropathies (ce). sb was formulated in 10x10 9 cfu capsules. its concentration and viability within the capsules was controlled by yeast culture in subsequent steps until expiration date. four healthy dogs (hd) and 18 dogs with ce (10 inflammatory bowel disease -ibd, 8 protein losing enteropathy -ple) were included. in hd sb was administered for 10 days (1x10 9 cfu/kg bid); daily clinical evaluation was performed to assess possible adverse effects and quantitative stool cultures for yeasts were performed before, during and after the administration. in dogs with ce a randomized double blind placebo-control study was performed, administering sb (1x10 9 cfu/kg bid) or placebo (pl). sb or pl administration was added to standard therapeutic protocols (diet, antibiotics and immunosuppressive drugs), to evaluate its efficacy for the treatment of ibd and ple. complete blood work, abdominal ultrasonography, gastro-duodenal and colon endoscopy and histopathological evaluation of intestinal samples were performed at diagnosis and after 60 days of treatment. validated score system for the clinical signs (ceccai), ultrasonography, endoscopy and histopathology were applied. significance was set for p < 0.05. results in hd showed the absence of sb in the faeces before treatment, its presence after one day, its steady state (10x10 7 cfu/g) after 5 days and its complete elimination 4 days after withdrawal of treatment. no adverse effects were reported. in ce dogs the clinical score improved significantly in dogs receiving sb compared to dogs receiving pl (p = 0.009). in ple dogs the albumin concentration increased significantly (p = 0.034) in the group receiving sb with respect to pl. the daily frequency of defecation in the sb group was significantly lower with respect to pl after 45 (p = 0.032) and 60 (p = 0.004) days of treatment. no statistical differences were found between dogs receiving sb and pl after treatment, based on the endoscopic evaluation of duodenum and colon. no statistical differences were found between the two groups on the duodenal ultrasonographic and histological evaluation after treatment. in conclusion, sb can be safely used in dogs with ce, in addition to standard treatment, to achieve a better control of clinical signs and a significant increase in albumin concentration compared to the standard therapy alone. no conflicts of interest reported. canine inflammatory bowel disease (ibd) is an immune-mediated enteropathy likely triggered by environmental and immunoregulatory factors in genetically susceptible dogs. previous studies suggest a pivotal role for gut bacteria in disease pathogenesis since luminal microbial composition is markedly altered (ie, dysbiosis) at diagnosis. probiotic bacteria appear to be therapeutically effective in some forms of human ibd. controlled studies evaluating the efficacy of probiotic therapy for canine ibd have not been previously reported. the aim of the present study was to characterize the mucosa-associated microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of orally administered vsl#3 probiotics in dogs with ibd. twenty dogs diagnosed with moderate-to-severe ibd (cibdai score > 5) were randomized to receive standard therapy (ie, elimination diet and glucocorticoids) with or without probiotic vsl#3. the mucosal microbiota from endoscopic intestinal biopsies of ibd dogs and controls was evaluated by fluorescence in situ hybridization (fish) targeting the 16s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. epithelial tight junction protein (tjp) expression was studied using immunohistochemistry. clinical signs and changes in mucosal microbiota and tjp expression were assessed before and after probiotic vsl#3 therapy. ibd dogs showed a reduction in gi signs following 8 weeks of probiotic therapy compared with baseline cibdai scores (p < 0.05). adherent and sporadic invasive bacteria (eub) were observed in the small intestines and colon of healthy dogs. the diseased canine duodenum was nearly bacteria-free. ibd dogs given probiotic vsl #3 had altered spatial redistribution of most bacterial groups in the mucus and adherent compartments of the colon. subset analysis showed that lactobacilli were significantly (p < 0.05) increased in the lumen and mucus post-vsl #3, while the number of mucus laden bifidobacteria approached significance (p = 0.08). expression of tjp showed that occludin was significantly lower in control intestines as compared to duodenal and colonic mucosa obtained from ibd dogs that received probiotic (p = 0.008 and p = 0.01, respectively). in contrast, claudin-2 expression in the colon was significantly higher (p < 0.002) in control dogs versus vsl #3 treated ibd dogs. our data demonstrate that probiotic vsl#3 alters some of the mucosa-associated microbiota in dogs with ibd. these probiotic changes in bacterial composition are associated with up-regulated tjp expression indicative of enhanced epithelial barrier integrity, similar to vsl#3-induced disease protection seen in human ibd. the probiotics used in the trial were supplied free of charge by the manufacturer. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb 10415 e1707 (ef) on intestinal microbiome composition. dogs were recruited to receive ef at 1x10e8 cfu in a double-blinded, placebo-controlled manner in addition to an exclusion diet (hydrolysed protein). seven dogs were included in the probiotics group and 5 dogs in the placebo group. all dogs improved clinically after treatment, however, there was no obvious effect on clinical severity in those that received probiotics. fresh naturally voided faecal samples were collected from all dogs before and after treatment, snap-frozen in liquid nitrogen and stored at -80°c until further analysis. genomic dna was extracted from each faecal sample using the mobio power soil dna isolation kit (mobio laboratories), as recommended by the manufacturer. next generation sequencing was performed on the ion-torrent[trademark] (life technologies) platform based upon the v1-v3 region (e. coli position 27-519) of the 16s rrna gene with the following primers: forward 28f: gag-tttgatcntggctcag and reverse 519r: gtnttacn gcggckgctg. raw sequence data were screened, trimmed, filtered, and chimera depleted with default settings using the qiime pipeline version 1.8 and uchime software, in which microbiome composition between treatment groups before and after treatment was compared. microbiota composition was not significantly different between probiotic and placebo treatment groups, and did not change significantly before and after treatment. however, there was large individual variability in the microbiome composition. species richness of faecal samples increased after treatment in both groups, but was only statistically significant in the probiotic treatment group. in conclusion, probiotic treatment in dogs with ibd leads to a significantly increased richness of the faecal bacterial microbiome. a possible additional effect of the change of diet cannot be excluded. further studies should investigate microbiomic changes in healthy dogs fed the same diet to assess if similar changes in the fecal microbiome occur due to dietary changes alone. this study is based on a phd supported by probiotics ltd., somerset, uk (the manufacturer of the probiotic product enterococcus faecium mentioned in this study). the aim of this study was to assess the prevalence and risk factors for faecal carriage of extended-spectrum beta-lactamases (esbl) and plasmidic ampc beta-lactamases (pampc) e. coliproducers in healthy dogs. a 3-month cross-sectional study was conducted at a private hospital in lisbon, portugal and 151 rectal swabs were obtained from healthy dogs. the dogs included in the study were healthy with no history of antimicrobial consumption in the previous month. esbl and pampc genes were detected by pcr and were sequenced. potential risk factors for esbl-and pampc-producing e. coli faecal carriage were obtained through a questionnaire to the owner regarding reason for veterinary visit, hospitalisation and antimicrobial treatment within the last year, habitat (shelter, dog breeders and private owner), cohabitation with other animals, street access, kennel/hotel access, age and gender. data were analysed by sas software (version 9.3; sas institute inc., cary, n.c.) and logistic regression models were used. rectal swabs obtained from 151 healthy dogs yielded 131 positive samples for e. coli. about 15% of the isolates carried esbl genes (bla ctx-m-32 n = 8, bla ctx-m-15 n = 5, bla ctx-m-1 n = 3, bla ctx-m-9-like n = 4) and 20% carried pampc genes (bla cmy-2 n = 23, bla cmy-2-like n = 2, bla dha-1 n = 1). thirteen dogs carried an e. coli isolate with both an esbl and a pampc gene. dogs previously treated with antimicrobials within the last year were at higher risk of carrying at least one ß-lactamase (p = 0.003; or = 7.85; ci 95%: 1.99-30.89) or both ß-lactamases (p = 0.020; or = 5.18; ci 95%: 1.29-20.81) than non-treated dogs. dogs in shelters/breeders tended to show a higher incidence of esbl-producing e. coli (p = 0.069; or = 3.17; ci 95%: 0.91-11.01) or at least one ß-lactamase producing e. coli (p = 0.050; or = 2.65; ci 95%: 1.00-7.03) than dogs from private owners. males tended to be less likely to carry at least one ß-lactamase (esbl or pampc) (p = 0.060; or = 0.42; ci 95%: 0.17-1.04) or a pampc enzyme (p = 0.017; or = 0.28; ci 95%: 0.10-0.80) than females. this study suggests that dogs may act as reservoirs for resistant bacteria, namely for cephalosporin-resistant e. coli. three potential risk factors associated with the carriage of esbl-and/or pampc-producing e. coli by dogs were identified, which is important for the implementation of effective control measures and judicious antimicrobial therapy. conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party there are few reports in the literature reporting long-term relapse rate, owner compliance and clinical severity of dogs with chronic enteropathies. the goal of this study was to compare clinical activity index (ccecai), number of relapses and compliance rates 1-3 years after diagnosis. food-responsive disease (frd) was defined as dogs that responded to elimination diet alone within 2 weeks after initiating therapy, whereas antibiotic-responsive disease (ard) dogs had an unsuccessful dietary trial before and responded to metronidazole within 2 weeks after initiation of therapy, and steroidresponsive disease (srd)dogs had an unsuccessful dietary and antimicrobial trial before, and required immunosuppressive therapy to control their clinical signs. ccecai was extracted from the medical record database at 1-3 years after diagnosis. relapse rate was obtained by requesting the medical records of the referring veterinarians and defined as number of return visits to the referring practice after diagnosis. compliance data was obtained by telephone questionnaire to the owners. the frd group consisted of 21/29 dogs (72%), whereas the ard and srd groups consisted of 4 (10%) and 5 dogs (17%), respectively. there was a significant difference in ccecai at follow-up between frd and ard, and frd and srd (median ccecai 1.8 (range 0-5) for frd, 5 (range 0-8) for ard, and 4.5 (range 0-8) for srd, p = 0.01). for the frd dogs, 43% of owners stated that they deviated from the prescription diet on a daily basis, 24% once a week, and 5% once a month, with a median ccecai at the time of deviation from the diet of 4.8 (range 2-8). relapse rate was highest for the ard group, when compared to frd and srd (10 for ard, 1.7 for frd, and 4.7 for srd, p = 0.004). in the frd group, 17/21 dogs had been kept on the prescribed diets, and 4 dogs had been changed to supermarket brands. all of the ard dogs had been given immunosuppressive treatment in addition to antibiotics at the time of follow-up, while 4/5 srd dogs were still on immunosuppressive treatments, with one dog being in remission with dietary treatment alone. in conclusion, this pilot study indicates that compliance rate for frd dogs is the lowest, with owners willing to tolerate the highest severity of clinical signs related to deviation from the prescription diet. ard dogs had the highest relapse rate in this cohort, indicating poor response to treatment in the long-term. conflicts of interest: dr allenspach has received research funding from bbsrc, american kennel club, comparative gastroenterology society, probiotics ltd uk, laboklin gmbh germany, and bioiberica sp. she has also undertaken paid consultancy work for bioiberica spain and hoffmann-laroche, switzerland. despite the high prevalence of canine pancreatitis in postmortem studies and the introduction of new diagnostic tests, it is believed that the disease, particularly in its chronic form, remains under recognised due to the non-specific nature of presenting signs. histology is considered to be the gold standard for diagnosis of canine pancreatitis, however, most clinicians are reluctant to take pancreatic biopsies due to significant risks to the patient. numerous serum markers have been reported to be elevated in canine pancreatitis, although most lack sensitivity or specificity. consequently, confirmed diagnosis requires results from a range of tests including imaging, serum biochemistry and physical examination. we and others have previously shown in other diseases that performance of individual low specificity markers can be dramatically improved by combining data from multiple markers with clinical information using analytical algorithms. we therefore applied this approach to the detection of pancreatitis in dogs. the activity of two non-specific biomarkers, amylase and lipase, was determined in 132 serum samples from dogs suspected of having pancreatitis by their veterinarian. of these samples 42 were positive by virtue of their pancreatic lipase (cpli) results (cpli > 200 ug/l). the amylase and lipase data was then used to develop a series of algorithms using mathematical data mining and classification techniques. additional algorithms were developed using extra parameters including age, sex, vomiting, diarrhoea and abdominal pain in addition to the two enzyme levels. the performance of the algorithms was assessed using 27 separate blinded serum samples taken from dogs which were scored clinically for acute pancreatitis according to the system described by mccord et al (j vet intern med 2012; 26:888-896) . these cases presented for evaluation with vomiting, diarrhoea, inappetance and abdominal pain and were included if a clinical history, results of routine haematology, biochemistry, cpli assay and abdominal ultrasound were available. the results of the multifactorial analysis and cpli assay results were compared to the clinical scores. using amylase and lipase data alone, the algorithm gave a sensitivity of 81.8% and specificity of 93.3%, compared to cpli results for the same samples of 63.6% and 86.7% respectively when both methods were referred to clinical scoring. when the presence of additional clinic data was also included into the algorithm, the sensitivity increased to 90.9% with specificity of 100%. the data suggests that test performance for canine pancreatitis can be dramatically improved when multiple diagnostic parameters are combined using disease specific algorithms. the author receives a salary as editor of the bsava journal companion, and has undertaken unrelated paid consultancies for bayer and merial. the author also receives a salary from avacta animal health, and duties involved working directly on this project. canine chronic enteropathy (cce) can cause significant long-term morbidity. in some cases this is due to intestinal inflammation, resulting from idiopathic inflammatory bowel disease (ibd). currently, the diagnosis of idiopathic ibd and assessment of disease severity relies on results of subjective clinical indices, laboratory data, diagnostic imaging and intestinal histopathology, whilst ruling out known causes of inflammation. in humans with ibd, a number of faecal biomarkers including lactoferrin, aid with diagnosis and determining disease activity. it may therefore be valuable to develop similar non-invasive objective methods to aid diagnosis and clinical assessment of disease severity in dogs with intestinal inflammation due to idiopathic ibd. this pilot study aimed to measure faecal lactoferrin concentration (flc) in dogs with cce and histologically confirmed intestinal inflammation (hcii) and to compare this with control dogs. in addition, the flc in dogs with hcii would be compared with the canine inflammatory bowel disease activity index (cibdai) and wsava standard histopathological criteria for intestinal inflammation to determine whether there was correlation between these methods when assessing disease severity. faecal samples were obtained from dogs with hcii (n = 13) having undergone investigation for cce (serum biochemistry, complete blood count, full faecal and urinalysis, serum cobalamin, quantitative cpli, abdominal ultrasound and intestinal biopsies). the control population were dogs presented for reasons unrelated to cce (n = 7). analysis was carried out using a faecal lactoferrin elisa previously validated in dogs (techlab, usa). the flc in dogs with hcii (median 9.79 lg/g -range 0.47 to 150.2) was significantly higher than control dogs (median 0.75 lg/g -range 0.12-4.14) (p < 0.003). a cut-off flc of 4.5 lg/g correctly identified 10/13 (77%) of dogs with hcii. using this cut-off, there was no overlap between non-cce dogs flc and the hcii group; giving a sensitivity of 76% and specificity of 100%. neither the presence of neutrophils nor the extent of inflammation on histopathology showed significant correlation with flc. the cibdai showed moderate correlation with flc in dogs with hcii (r = 0.79, p = 0.05). the results of this pilot study suggest that flc is able to discriminate between dogs with cce due to hcii and dogs without cce. it is possible that incorporating flc into a panel of faecal biomarkers will enable non-invasive assessment of hcii and could serve as an adjunct to current measures of disease severity in dogs with idiopathic ibd. ryan bettencourt and james boone are employees of techlab, usa. they provided the elisa kits free of charge for this work. there was no other incentive provided and the results have been openly discussed between all parties. there has been no censorship placed on the results by tech-lab and they have been supportive of the work and submission of this abstract. there are no other conflicts to disclose. fecal s100a12 and fecal calprotectin concentrations have been described as biomarkers in dogs with chronic enteropathies [1]. however, to date there has been no direct comparison of these two markers in dogs with chronic diarrhea. the aim of this study was to evaluate the performance of these two markers in this situation. thirty one dogs presented for a history of chronic diarrhea were prospectively enrolled. the initial diagnostic workup for all patients included a serum biochemistry profile, fecal parasitology, abdominal ultrasound examination, and gastrointestinal endoscopy with collection of endoscopic biopsies. the severity of clinical signs was evaluated using the ccecai scoring index and patients were grouped by having a ccecai of < 12 or ≥ 12. fecal calprotectin and s100a12 were quantified as previously described [1] . correlations were evaluated with the spearman rank correlation test. for both markers a receiver operating characteristics (roc) curve was used to select cut-off value that allowed the best discrimination between dogs with a ccecai<12 and dogs with a ccecai ≥ 12. sensitivity and specificity were calculated. correlation analyses revealed a significant positive correlation between s100a12 and calprotectin (r = 0.77; p < 0.001). the optimal cut-off value for fecal calprotectin concentration was 49.1 lg/g, which was associated with a sensitivity of 53.3% and a specificity of 86.7% (auc=0.698; p = 0.049). the optimal cutoff value for fecal s100a12 concentration was 173.7 ng/g, which was associated with a sensitivity of 93.3% and a specificity of 60% (auc=0.782; p = 0.001). the sensitivity for fecal s100a12 was higher than that for fecal calprotectin (p = 0.019). no significant difference was observed for the specificity of these two markers (p = 0.215). 20 out of the 30 dogs (67%) had concordant results for s100a12 and calprotectin tests. among these 20 dogs, 11 presented with a ccecai <12 and 8 of these 11 dogs had both markers below their cut-off values. among the 9 dogs with a ccecai≥12, 8 dogs had both markers above their cutoff values. 66% of dogs (19/29) presented histologic signs of inflammation. sensitivities for fecal calprotectin and s100a12 concentrations for histopathological intestinal inflammation were 42% and 68%, respectively, and specificities were 80% and 30%, respectively. at least in this group of patients fecal s100a12 concentration was more sensitive (but less specific) to detect dogs with a cce-cai ≥ 12 or histopathologic intestinal inflammation than fecal calprotectin concentration. weight loss and malabsorption of fat, protein, cobalamin and tocopherol in the face of normal exocrine pancreatic function have been reported in up to 30-40% of cats older than 12 years of age fed a variety of nutritionally balanced dry and wet foods (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, focus on gastroenterology, [55] [56] [57] [58] [59] [60] [61] 2010) . the objectives of this study were to determine if serum cobalamin concentrations increased after oral administration of a cobalamin supplement to affected cats, and the duration of any positive response following cessation of supplementation. the study evaluated 14 cats older than 12 years of age with fat malabsorption demonstrated by either increased fecal fat (>20%) or subnormal fat digestibility (<80%), but without exocrine pancreatic insufficiency (epi) as assessed by assay of serum trypsin-like immunoreactivity (ftli). a commercially available solution of cobalamin containing 1 mg mixed with 1 ml of a liquid flavor enhancer was added to the food of each cat in a single dose each day for 2 months, after which supplementation ceased. serum cobalamin (assayed by competitive binding assay performed through the gi laboratory at texas a&m university and evaluated using reference ranges derived by that laboratory) was determined immediately prior to initiation of supplementation, then weekly for 3 weeks, then monthly for 4 months. at the start of the study serum cobalamin was subnormal (<290 ng/l) in 4 of the 14 cats (range <150 to 244 ng/l) and within the reference range (290 to 1499 ng/l) in the remaining 10 cats (334 to 1065 ng/l). serum cobalamin was above the reference range in every cat (1892 to 13109 ng/l) after one week of supplementation and remained above the reference range in every sample collected during the supplementation period, with the exception of two cats with values within the reference range when supplementation was stopped. serum cobalamin 1, 2 and 3 months after cessation of supplementation ranged from <150 to 1783, <150 to 1494, and <150 to 948 ng/l, respectively. at the end of the study serum cobalamin was subnormal in 5 of the 14 cats. it is concluded oral cobalamin supplementation can effectively increase serum cobalamin concentrations in geriatric cats with idiopathic chronic enteropathy, but that following cessation of supplementation concentrations decrease rapidly and can become subnormal again within as few as 4 weeks. the primary author collaborated with nestle-purinaon the work reported in this abstract, and a co-authoris an employee of nestle-purina. the primary author has previously received funding from iams, mars, hills and nestle-purina. the author also acts as a paid consultant for the gi-lab, texas a&m university. left atrial measurements are crucial in assessing severity of cardiac disease in dogs with myxomatous mitral valve disease (mmvd). however, linear and area dimensions might not provide a comprehensive assessment of patient status, and cannot differentiate between severe subclinical (b2) and clinical disease (chf). estimates of left atrial function could provide additional information to help categorize these patients. we examined 87 dogs with mmvd (25 normal, 9 b1, 40 b2 and 13 c) presented for cardiac evaluations by 2d echocardiography. left atrial linear and area dimensions in right parasternal short and long axis views were obtained at 3 time points -early diastole (la max ), just prior to mitral valve opening, at the onset of atrial systole (la p ) and just prior to mitral valve closure (la min ). we calculated 4 indices of la function: total la emptying fraction (la tef ), active la emptying fraction (la act ), passive la emptying fraction (la pas ) and la reservoir function (la res ) for all 4 sets of measurements. we examined the differences in selected la function indices between different disease stages with a kruskal wallis test with post-hoc multiple comparisons. we also examined the diagnostic accuracy of selected indices of la function in differentiating dogs in stage b2 and stage c (chf) using roc analysis. three functional indices consistently differed across the various stages of mmvd -la tef , la act and la res . these differences were most apparent in the rpla view for linear measurements and rpsa view for area measurements. dogs with chf had worse function than all other groups, which differed variably depending on the functional index being examined. laarea act showed the best ability to discriminate between b2 and chf dogs, with a 95% specificity, 69% sensitivity and an auc of 0.84, but this was no better than use of la:ao measurements. our data suggest that la function differs between dogs with differing severities of mmvd, but does not provide a clear distinction between dogs with subclinical disease and chf. no conflicts of interest reported. materials and methods: 3de was used to evaluate 85 consecutive, non-sedated dogs that weighed more than 5 kg. the study population for the morphologic study included 15 normal dogs, and 41 dogs with acvim stages b1 or b2-c mmvd. 3de image data were digitally recorded and then analyzed offline, using commercially available software. results: 3de image acquisition was feasible in 67/85 (78.8%) consecutive dogs. patient anxiety (6), arrhythmias (6) and panting (5) explained failure to obtain a 3de dataset. fortyone of 67 (61.2%) datasets were of analyzable quality. body weight and heart rate were significantly lower in dogs for which it was possible to perform 3de. dogs with analyzable 3de datasets were significantly older and weighed less than dogs in which 3de could not be analyzed. the mitral valve of normal dogs is saddle shaped (annulus height to commissural width ratio (ahcwr): 0.22 ae 0.06 [mean ae sd]) and has an elliptical annulus (sphericity index (si): 0.91 ae 0.07). the following measurements were significantly related to body surface area (bsa): antero-posterior diameter (apd) (r 2 = 0.46, p < 0.01), anterolateral-posteromedial diameter (alpmd) (r 2 = 0.38, p < 0.05), annulus area (aa) (r 2 = 0.39, p < 0.05), anterior leaflet length (all) (r 2 = 0.38, p < 0.05), anterior leaflet area (ala) (r 2 = 0.38, p < 0.05). these variables were indexed (i) to bsa for subsequent statistical analyses. dogs with mmvd had a significantly greater si, non-planar angle, apdi, alpmdi, alai and alli, while having a significantly lower posterior leaflet area (pla), posterior leaflet length (pll), annulus height (ah), tenting height (th), tenting volume (tv), tenting area (ta), and ahcwr compared to normal dogs. ah, tv and ta were significantly greater in normal dogs, compared to dogs with mmvd. si, apdi, al-pmdi, aai, alai and alli were significantly greater in dogs with stages b2-c mmvd, compared to normal dogs and those in stage b1. pll and pla were significantly lower in b2-c dogs, compared to normal dogs. th was significantly different between the three groups; greatest in normal dogs and lowest in dogs in stages b2-c, suggesting that flattening of the mv occurs with disease progression. conclusions: 3de assessment of the canine mv is feasible. morphologic changes associated with mmvd progression are presented. effective regurgitant orifice area (eroa), calculated from a 1dimensional measurement of the width of vena contracta (vc) as the narrowest portion of the proximal regurgitant jet, might be used to estimate severity of mitral regurgitation (mr). however, this simplified assumption only holds when the eroa is circular, which might not be true in dogs with myxomatous mitral valve disease (mmvd). the aim of the study was to compare measured eroa using color doppler real-time 3 dimensional echocardiography (rt3d) with calculated eroa estimated by 2 dimensional echocardiography (2d) in 4 chamber (4ch) and 2 chamber (2ch) views of the left ventricle (lv) in dogs with mmvd. ninety-three privately owned dogs of 32 breeds diagnosed with naturally acquired mmvd were examined using 2d and rt3d. according to the acvim classification of congestive heart failure (chf), 23 dogs were classified with chf (2 in class c1 and 21 in class c2) and 70 dogs without chf (65 dogs in class b1 and 5 dogs in class b2). age ranged from 1 to 15 years (median 10 years), and body weight ranged from 2.5 to 35 kg (median 10 kg). fifty-nine males (63%) and 34 females (37%) were included, and heart rate ranged from 80 to 222 beats/minute (median 126 b/min). eroa was calculated from 2d measurements of vc diameter, in the 4ch view only (assuming a circular regurgitant orifice), and from measurements of vc diameter in both 4ch and 2ch views (assuming an eliptical regurgitant orifice) of lv. bland-altman plots were used to compare eroa measured by rt3d with calculated eroa obtained from 2d 4ch and 4ch/ 2ch lv views. none of the 2d estimations of eroa showed good agreement with the measured rt3d eroa when corrected for bsa, and the difference between methods increased with increasing eroa. the difference between rt3d and 2d methods normalized to the mean eroa value did not increase with increasing eroa, but showed a systematic underestimation of eroa by 60% (4ch) and 40% (4ch/2ch), respectively, compared to rt3d. the beat-to-beat variation of eroa assessed by rt3d (n = 56) had a coefficient of variation ranging from 2.8% to 68% (median 30%). in conclusion, substituting assessment of eroa with a measurement of vc in 1 or 2 dimensions might underestimate the mr severity in dogs with mmvd. in some dogs, the beat-tobeat variation of the eroa was large, thereby necessitating the need for several consecutive measurements. no conflicts of interest reported. micrornas (mirna) are short (19-22 nucleotides), singlestranded, non-coding rnas that specifically anneal with complementary sequences in multiple mrna targets, and they silence mrnas and suppress downstream protein translation. a mirna can act as a fine-tuner of gene expression or an on/off switch. these features highlight the potential of mir-nas as therapeutic targets. the role of mirnas in myocardial fibrosis and hypertrophic cardiomyopathy has been widely studied in human patients. however, there is no data available for canine and human myxomatous mitral valve disease (mmvd). the aim of this study was to investigate mirna transcriptomics in canine mmvd by using global transcriptional profiling, mirna target prediction software (diana tool, targetscan 6.2) and network analysis software (biolayout express 3d ). four myxomatous mitral valves (ckcs) and 4 controls valves were profiled using the affymetrix canine gene 1.0 st array. in total 29 out of 302 mirnas were found to be statistically significantly differentially expressed (down-regulated) based on the false discovery rate, p-value, and fold-change. expression of three mirna (cfa-mir-23b, cfa-mir-29c, cfa-mir-218) were also validated by quantitative polymerase chain reaction (q-pcr, taqman), and the results were in agreement with the microarray findings. for network analysis and visualization, markov clustering algorithms were conducted in bio-layout express 3d , and major clusters of mirnas were exported and uploaded to the diana-mirpath (kegg pathway) web-server. the pathways identified in the main cluster were attributed to the biological functions of focal adhesion, cytoskeleton (actin) regulation, tgf-b signalling, glycosaminoglycan biosynthesis, osteoclast differentiation, notch signalling and vegf signalling. the most significantly down-regulated mirna in mmvd was cfa-mir-218, which is an endothelial specific mirna shown to regulate endothelial migration and vessel patterning. the top predicted target of cfa-mir-218is glucuronic acid epimerase (glce) which is the main enzyme controlling heparan sulphate biosynthesis. other interesting findings were down-regulation of cfa-mir-29 and members of the cfa-mir-23 family. cfa-mir-29 targets multiple extracellular matrix transcripts, such as collagens, elastin, integrin, laminin, mmp (matrix metalloproteinase) and adamts (a disintegrin and metalloproteinase with thrombospondin motifs), whereas cfa-mir-23 targets hyaluronic acid synthase 2 (has2). since the major pathology of mmvd is aberrant turnover of extracellular matrix proteins, this may be linked to mir-na regulation. dysregulation of valve mirnas might be potential therapeutic targets in the treatment of canine mmvd. no conflicts of interest reported. mitral regurgitation (mr) progresses slowly, but dogs living long enough often develop congestive heart failure (chf). however, tools to predict onset of chf are sparse. 225 echocardiographic examinations in 78 dogs were performed in a longitudinal, multicenter study with a surveillance time of up to 4.5 years. client-owned dogswere enrolled at the university hospitals in finland, sweden and denmark (subset to the svep study). left ventricular end diastolic (lvidd) and systolic (lvids) diameters, fractional shortening (fs), left atrial (la) and aortic root (ao) diameters were estimated. values were normalized for body size (nlvidd, nlvids, and nla, respectively) and, for comparison, ratios to aortic root were calculated (lvidd/ao, lvids/ao and la/ao, respectively). a cox's proportional hazard analysis with a counting process approach was used. spline smoothed graphical models were constructed to evaluate linearity of hazards. curves were then used to find cut-off values for interval hazard ratios (hrs). the hr for nlvidd, nlvids and nla (per 0.1 unit, 95% confidence intervals), were 1.5 (1.32-1.70, p = 0.00034), 1.3 (1.05-1.62, p = 0.016), and 1.5 (1.28-1.83, p = 0.0039), respectively. the hrs for lvidd/ao, lvids/ao and la/ao (0.1 unit increase) were 1.3 (1.14-1.55, p = 0.0025), 1.1 (0.99-1.32, p = 0.07), and 1.4 (1.24-1.66, p = 0.0041), respectively. the hr for fs was 1.1 (1.05-1.19, p = 0.00037). the relative hazard plot presented a steep increase for fs values above 31%. hrs for intervals 31 < 36%, 36 < 40%, and ≥40% were 1.0 (0.2-5.1, p = 0.99), 4.8 (1.2-18.4, p = 0.023), and 9.1 (2.4-33.7, p = 0.00098), respectively. the hr for nlvidd increased linearly. hrs for intervals 1.73 < 1.86, 1.86 < 1.92, 1.92 < 2.1 and ≥2.1 were 0.7 (0.1-6.6, p = 0.7), 6.0 (1.3-26.7, p = 0.02), 6.8 (1.9-25.1, p = 0.0037), and 12.5 (3.8-40.1, p = 0.00088), respectively. in contrast, the hazard for nlvids remained stable until 1.4, whereafter it increased. the hrs for nlvids (1 < 1.33, 1.33 < 1.68, 1.68 < 1.86 and ≥1.86) were 1.6 (0.6-4.6, p = 0.35), 3.4 (1.0-11.5, p = 0.048), 78 (9.6-634, p = 0.030), and 47.6 (7.1-316, p = 0.044), respectively. hrs for values normalized to ao diameter behaved in a parallel way. we conclude that fs, left ventricular and atrial size may be used to predict chf. however, because the value of a hr is dependent on the unit used and, more essentially, does not account for nonlinear change in hazard, interpretation of hazards is challenging. in contrast, interval hazards are only dependent on the reference interval used. therefore they are easier to implement in every day clinical work. no conflicts of interest reported. systemic arterial hypertension is not frequently recognized in dogs with mitral valve degeneration (mvd), although borderline hypertension is difficult to assess, mainly because of different measurement techniques, inter-operator variability and, most importantly, examination-related stress. the object of this study was to evaluate systolic arterial blood pressure (sbp) at initial presentation and at regular intervals in dogs with various clinical stages of mvd. fifty six dogs with mvd that had not received any heart medication prior to admission, were included in the study. based on the isachc staging system, 22 were assigned to class i (group a), 18 to class ii (group b) and 16 to class iii. small-breed dogs and miniature poodles, in particular, were overrepresented. comorbidities that could affect sbp were ruled out prior to enrollment. sbp was measured using a commercially available veterinary oscillometric device, by applying the proper cuff on the cephalic artery. dogs were left to acclimate for 10 -15 minutes and measurements were always taken by the same investigator, before any other examination was performed, with the dog sitting on the owner's lap. a total of 5 readings were taken, outlier values were discarded and the mean of the 3 remaining measurements was documented. after initial consultation, treatment was customized according to the clinical stage. sbp was then measured every 2 months, up to 6 months after initial admission. at presentation, all class i dogs had sbp > 140 mm hg, with only 5/22 having spb ≥150 mm hg, whereas all class ii dogs had sbp < 140 mm hg. of class iii dogs, 6 had sbp > 140 mm hg, and 2 had sbp ≥ 150 mm hg. a linear mixed effects model was used to assess the temporal variability of the measured parameters between groups. groups were matched for gender, age and body weight. blood pressure measurements, for the duration of the study, were higher in group a dogs, compared to groups b and c (p < 0,001). at the same time, group c had significantly higher sbp values than group b dogs (p < 0,001). asymptomatic mvd dogs seem to have higher sbp measurements, compared to those with clinical evidence of heart failure. whether this difference is stress-related, a maladaptive mechanism of sympathetic and raas activation to mvd or idiopathic remains to be elucidated. no conflicts of interest reported. sarcoplasmic reticulum (sr) ca 2 + -atpase and its regulatory proteins are pivotal determinants of myocardial active relaxation via calcium uptake against the sr-cytoplasmic gradient. the lowered density of the sr ca 2 + -atpase has been well demonstrated in many species during chronic hemodynamic overload. the genes linked to sr calcium uptake were reported not only being expressed in peripheral blood but serving as potential cardiac biomarkers in dogs with chronic mitral regurgitation, such as sr ca 2+ adenosine triphosphatase isoform 2a (ser-ca2a), phospholamban (pln), and hs-1 associated protein x-1 (hax-1). the aim of this study is to determine whether the target genes expressed in the blood will be translatable to the myocardial setting as cardiac biomarkers. the mrna expression levels of the target genes (serca2a, pln, hax-1) from biopsied left ventricle (lv) and peripheral white blood cells (pwbc) in 129 surgical mitral valve repair cases were estimated with quantitative real-time pcr using comparative ct method with gapdh. the gene expression levels in lv and pwbc were compared and their clinical relationships were evaluated. the diagnostic power of the genetic expressions in pwbc was analyzed by comparing to those of 33 normal dogs. the levels of all target genes expressed in lv and pwbc were highly correlated each other in linear regression analysis (p < 0.0001; serca2a, r = 0.7425, r 2 = 0.5513; pln, r = 0.7720, r 2 = 0.5959; hax-1, r = 0.6598, r 2 = 0.4353), although lv and pwbc showed different expression levels in a paired comparison (p < 0.0001). according to the severity of the heart failure (isachc), the expression levels of all genes were gradually and significantly reduced in both lv and pwbc (p < 0.01). especially, the serca2a and pln expressed in pwbc could clearly discriminate all isachc groups from the control (p < 0.0001). multivariate regression adjusted by age and body weight revealed that serca2a and pln in lv were negatively associated with lv internal systolic dimension (p = 0.0251, adjusted r 2 = 0.524 and p = 0.012, adjusted r 2 = 0.530, respectively). pln was also negatively related with lv internal diastolic dimension (p = 0.037, adjusted r 2 = 0.573). additionally, receiver-operating characteristic analysis using pwbc showed high area under the curve (auc) values for all target genes on overall isachc groups (p < 0.0001; serca2a, auc=0.9212; pln, auc=0.8936; hax-1, auc=0.8797). in conclusion, the transcriptional changes of the calcium uptake related genes in pwbc may be able to reflect myocardial hemodynamic stress as well as to be utilized as promising cardiac biomarkers. no conflicts of interest reported. the aim of this study was to estimate heart-rate normalized pulmonary transit times (nptt) in cardiomyopathic cats with or without congestive heart failure (chf), to assess potential associations of echocardiographic variables and nptt, and to evaluate nptt as a test for presence of chf. 48 privately owned cats were included. nptt was measured using echocardiography and the ultrasound contrast media sonovue â in 3 groups of cats: healthy cats (group 1), cats with cardiomyopathy (cm) but without chf (group 2), and cats with cm and chf (group 3). receiver operating characteristic curves (roc) were created for nptt, left atrial diameter (lad) and the left atrial to aortic root ratio (la:ao) to assess and compare their usefulness as tests for presence of chf. interrelations between pulmonary blood volume (pbv), nptt, stroke volume (sv) and echocardiographic variables were investigated by means of uni-and multivariate analysis. nptt values in group 1, group 2 and group 3 were 3.63 (interquartile range (iqr) 3.20-4.22), 6.09 (iqr 5.0-7.02), and 8.49 (iqr 7.58-11.04), respectively. values were significantly different between all 3 groups. pulmonary blood volumes in group 1, group 2, and group 3 were 27.94 ml (iqr 21.02 ml-33.17 ml), 42.83 ml (iqr 38.46 ml-50.36 ml) and 49.48 ml (iqr 38.84 ml-64.39 ml). sv, pbv and shortening fraction <30% were significant predictors of nptt. nptt and la:ao ratio, not sv were the main predictors of pbv. analyzing roc for nptt as a clinical test for chf yielded an auc of 0.956 which was similar for la:ao ratio. nptt may be useful test for the presence of chf in cats with cm and as a measure of cardiac performance. nptt and la: ao ratios predict chf with equal accuracy. increased pbv is significantly associated with higher nptt and la:ao ratios. both decreased sv and increased pbv explain the increased nptt in cardiomyopathic cats. the author received a travel scholarship from zoetis to attend this congress. acute arterial thromboembolism (aate) occurs commonly in cats, and less frequently in dogs, mostly resulting in limb paresis or paralysis. diagnosis is based typically on physical examination and advanced imaging. diminished affected-limb peripheral blood flow induces changes in several analytes concentrations in affected limb venous samples, compared to their peripheral venous concentration. we hypothesized that in aate, local, affected-limb venous glucose concentration decreases below reference interval, while systemic glucose concentration remains unaffected. the study included 3 groups for each species: paralytic aate cases, non-ambulatory controls with limb paralysis of orthopedic or neurologic disorders, and ambulatory controls diagnosed with various diseases. systemic and peripheral, affected-limb blood glucose concentrations were measured. group absolute (dglu) and relative (% dglu) differences were compared. no procedure-associated complications or pain were noted. peripheral blood glucose concentrations were decreased (p ≤ 0.006) only in cats and dogs with aate. dglu and %dglu were higher in the aate groups in both cats and dogs compared to their respective control groups (p < 0.0001, p < 0.001, respectively), with no differences between the control groups. receiver operator characteristics analysis of dglu and %dglu as predictors of aate in cats had areas under the curve of 0.96 and 1.00, respectively, and 0.99 and 1.00, in dogs, respectively. dglu cutoffs of 30 mg/dl and 16 mg/dl, in cats and dogs, respectively, corresponded to sensitivity and specificity of 100% and 90% in cats, respectively, and 100% in dogs. dglu and %dglu are extremely accurate, readily-available, simple diagnostic markers of aate in cats and dogs. no conflicts of interest reported. glycaemia determination is usually included in routine biochemisty panels. no works are devoted to the evaluation of pheripheral glycaemia in animals suffering from arterial thrombosis. the aim of this study was to document the pheripheral glycaemia variations in hypoperfused limbs of patients affected by mriconfirmed arterial thrombosis. eleven dogs referred for monoparesis or paraparesis were recruited. inclusion criteria were a clinical examination supportive of limb hypoperfusion and availability of blood cell count, biochemical profile and urine analyses. before mri examination, peripheral glycaemia was tested. two blood samples were obtained, one from the affected limbs and one from a healty limb.plasmatic glycaemia was measured using an automated glucose analyser. all the patients underwent a total body mri (mri intera 1.5t, philips medical systems) that provided the final diagnosis. the arterial thrombosis location was documented and the entity was scored. all the eleven patients were diagnosed with a peripheral thrombosis involving an arterial vessel and in some cases the relative branches. the thrombus was located: in the abdominal aorta (7/11), in the subclavian artery (1/11), in the axillary artery (1/11), in the iliac arteries (2/11). of the total amount of abdominal aortic thrombosis, 3/7 involved also the internal iliac arteries, 2/7 the external ones and 2/7 both internal and external. the extent of the thrombosis was classified as grade 1 (g1), when the greatest portion of the thrombus did not reach half of the vessel lumen (1/11 patients); grade 2 (g2), when the greatest portion of the thrombus was between 1/2 and 2/3 of the vessel lumen (7/11); grade 3 (g3), when the thrombus exceded 2/3 of the lumen (3/11). a substantial decrease in pheripheral glycaemia values was found in sampling arising from the thrombosisaffected limbs. comparing thrombosis-affected limbs values with healthy limbs measurements from the same patient, the reduction was found from 17.65% to 34.41%. accounting only the g3 scored patients, the percentage of reduction was found up to the 28.34% suggesting a proportional decrease related to the grade of occlusion. results from this study suggest that peripheral glycaemia values are affected by limb hypoperfusion disorders. if an arterial thrombosis is suspected, samples from the affected limbs and the healthy ones could be used to compare glycaemia values and to support the early stage therapy in anticipation of diagnostic imaging. further studies are needed to confirm the proportional relation of the decrease with thrombus entity. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwt). pulmonary hypertension (ph) may develop secondary to hypoxic vasoconstriction and/or pulmonary parenchymal infiltration. in the absence of measurable tricuspid regurgitation (tr), this co-morbid condition may be difficult to diagnose non-invasively. the degree of cardio-pulmonary impairment in cipf dogs can be evaluated through blood gas analysis (bga) and 6 minute walking test (6mwt). a new echocardiographic index, the right pulmonary vein to pulmonary artery ratio (pv/pa) has been described for the detection of pulmonary venous hypertension. the aim of this study was to investigate pv/pa in cipf in order to determine its utility in the detection of ph and in the assessment of cardio-pulmonary disease severity. this prospective clinical cohort study included 10 whwt with cipf (group a), 9 healthy whwt (group b) and 25 healthy dogs from other breeds (group c). diameters of right pv and pa were measured, in bi-dimensional (bd) and m-modes (mm), in a parasternal right long axis view, at the end of the t wave. other echocardiographic parameters for evaluation of ph were also measured: speed of tr, acceleration time to ejection time ratio of the pulmonary flow (at:et) and pulmonary artery to aorta ratio (pa/ao). bga was performed in 14 dogs (9, 1 and 4 in groups a, b and c) and 6mwt in 17 dogs (8, 6 and 3). values are given as meanaesd. in bd and mm mode, the pv/pa ratio was lower in group a (mm: 0.62 ae 0.25, bd: 0.51 ae 0.20) compared to group b (mm: 0.98 ae 0.17, bd: 0.93 ae 0.12, p ≤ 0.01) and group c (mm: 1.03 ae 0.13, bd: 1.00 ae 0.10, p ≤ 0.0001). the changes in pv/pa were both due to an increase of pa (p ≤ 0.01) and a decrease of pv (p ≤ 0.05). tr was found in 60% of dogs with cipf; mean pressure gradient was 34.03 ae 16.90 mmhg. at:et was lower in group a (0.42 ae 0.08) compared to group c (0.50 ae 0.04, p = 0.002) and tended to be lower compared to group b (0.48 ae 0.07, p = 0.09). pa/ao was not statistically different between groups. pv/pa was correlated with arterial po 2 values (b mode: r = 0.870, p = 0.0001) and results of the 6mwt (b mode: r = 0.791, p = 0.0003). pv/pa was also correlated with at:et and the speed of tr, but not with pa/ao. in conclusion, in whwt affected by cipf, pv/pa is a useful indicator of ph and could serve in the assessment of disease severity. no conflicts of interest reported. ventricular septal defect (vsd) is the fourth most common congenital cardiac defect in dogs and the most common in cats. the aim of this study was to evaluate the long-term outcome in vsd patients. case records of 95 animals were reviewed, 46 of these re-evaluated echocardiographically and 49 followed up by phone interview only. out of 53 dogs pug was the most common breed (11%) followed by border terrier (8%). out of 42 cats domestic short hair was most common (55%) followed by main coon (19%). isolated vsds were present in 29 dogs and 26 cats. complex defects (cds) were present in 40 cases, most frequent anomalies being sub-aortic stenosis (9 dogs, 1 cat), pulmonic stenosis (5 dogs, 3 cats), tetralogy of fallot (3 dogs, 2 cats), cushion defects (5 cats) and double-chambered right ventricle (dcrv) (5 dogs, 4 cats; in 2/5 dogs not present initially supporting the cause-and-effect theory). eisenmenger was observed in 1 dog and 2 cats. aortic insufficiency, not considered a cd, was noted in 10 dogs. in 3 dogs and 6 cats (15% of isolated vsds and 1 cat with a dcrv) the defect closed spontaneously. nine dogs and 9 cats (22%) died of non-cardiac causes with an age at death of 5 to 204 (mean 63.8) months; 7 dogs and 8 cats died due to cardiac causes with an age at death of 0.25 to 204 (mean 37.5) months. cardiac deaths were sudden (2 dogs with cds) or euthanasia for left sided congestive heart (chf) failure associated with cds (3 dogs, 1 cat); right sided chf associated with cds (2 cats); biventricular failure (1 cat with cd); weakness (eisenmenger, 1 dog and 1 cat; fallot 1 dog; cd 1 cat). two cats developed chf due to unrelated hcm. only one dog with an isolated vsd was euthanized for chf. these results indicate that spontaneous vsd closure occurs more often than previously thought, most patients with isolated restrictive vsds live a normal life span without surgical intervention, but non-restrictive vsds or complex defects can be associated with significant morbidity and mortality. echocardiography early in life is crucial to identify the anomaly and cds, as well as useful to prognosticate long-term outcome and to identify patients where a surgical intervention should be considered if available. follow-up echocardiography is indicated to corroborate the prognosis, to detect complications due to the vsd and to detect unassociated acquired cardiac diseases. no conflicts of interest reported. centronuclear myopathy (cnm) is the most prevalent congenital inherited disorder affecting skeletal muscles in labrador retrievers. this disabling condition segregates worldwide and a recessive loss-of-function founder mutation was identified in the protein tyrosine phosphatase-like, member a gene (ptpla/ hacd1). the objectives of this study were 1) to describe ptpla expression pattern in hearts from homozygous wild type (wt), heterozygous (het) and homozygous mutated (cnm) dogs, 2) to assess and compare the left myocardial function in aging wt, het and cnm dogs. for this purpose, seven wt, four het and eleven cnm dogs were included in the study. ptpla mrna levels were assessed by rt-pcr and rt-qpcr. all dogs were examined using conventional echocardiography, 2d color tissue doppler imaging (tdi) and tdi-derived strain imaging. we found that the expression of the two wild type ptpla splice isoforms increased post-natally in wt dogs. their levels were halved in het dogs and drastically reduced in cnm dogs. in both het and cnm dogs, a slight left ventricular hypertrophy was detected using conventional echocardiography. tdi and strain imaging revealed that the left ventricular myocardial function was significantly altered in both het and cnm dogs compared to wt dogs. moreover, these functional defects were associated with significantly higher values of systemic arterial blood pressure, although maintained within normal ranges. in conclusion, subclinical myocardial alterations were detected in both het and cnm aging dogs from our french pedigree, suggesting a role for ptpla in long-term cardiovascular homeostasis. these findings prompt globalized confirmation in additional ptpla"'deficient dogs, which may thus be considered as a new large-size model for human left ventricular sub-clinical myocardial dysfunction. no conflicts of interest reported. mitral regurgitation (mr) secondary to degenerative mitral valve disease (dmvd) is the most common heart disease in dogs. in dogs with mr, mitral valve prolapse caused by degeneration of the mitral valve leaflet, chordae tendinae extension and/or rupture and mitral annulus dilation are observed. however, limited data are available on morphological changes in dogs with mr. currently, there are no studies confirming the anomaly of the mitral complex via direct observation in living dogs with mitral regurgitation. at our institution over the last ten years, approximately 300 dogs have undergone mitral valve repair. during surgery, the anomaly of mitral complex can be observed macroscopically (directly visualized). to our knowledge, this is the first study evaluating the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. animals: dogs that underwent mitral valve repair with cpb at nihon university between february 2010 and june 2013 were included in this study. methods: confirmation of chordae tendineae rupture was visually confirmed during surgery. the sites of chordae tendineae rupture were also recorded at that time. septal chordae and mural chordae were divided three division depend on the site (s1, s2, s3 and m1, m2, m3 respectively). results: ninety eight dogs were included in this study. the mean age and body weight were 8.8 ae 1.8 years and 5.15 ae 3.24 kg, respectively. of the 98 dogs, ruptured chordae was observed in 80 dogs (81.6%). septal leaflet chordae was ruptured in 73 dogs (74.5%) and mural leaflet chordae was ruptured in 25 dogs (25.5%). chordae of both leaflets were ruptured in 18 dogs (18.4%). no chordal rupture was observed in 18 dogs (18.4%). in the dogs with ruptured septal chordae, the chordae between s2 and s3 was most often ruptured (n = 37, 51%). in this study, rupture of the septal chordae tendineae was most commonly observed. this is the first pilot study to visually evaluate the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. future studies comparing pathological changes and molecular biological analysis to gross findings of mitral chordae tendineae in dogs undergoing mitral valve repair may be useful in advancing the understanding of the disease. no conflicts of interest reported. echocardiographic aortic valve (ao) measurements are routinely obtained during cardiac evaluation of patients. cardiologists commonly use diastolic ao measurements to obtain ratiometric weight-independent estimates of dimensions of other cardiac structures, most commonly the left atrium (la). however, no consensus exists about the point in diastole at which ao measurements should be obtained -immediately after closure of the aortic valve, when la size is largest (ao max , but often with least distinct margins), during the p-wave of the ecg (ao p ) and at the onset of ventricular electrical systole, when la size is smallest (ao min ). we examined the linear and area dimensions of the ao (aod and aoa) to determine if clinically significant differences exist at 3 distinct diastolic time-points, or if these measurements could be interchangeable. we examined 130 patients (114 dogs and 16 cats) presented for cardiac evaluations by 2d echocardiography. three replicates of each time-point linear and area measurement (ao max , ao p , ao min ) were obtained in each patient and averaged for analysis. only patients with aortic valve disease and those with atrial fibrillation were excluded from analysis. beat-to-beat variability of the ao measurements was determined. standard and normalized limits of agreement (loa) plots were generated for each pairwise comparison. the frequency of each ao measurement being the largest or smallest within-patient measurement was determined, and compared via repeated measures anova. all pairwise agreement plots of both aod and aoa demonstrated heteroscedasticity; normalized aod plots showed 95% loa to be 15% of the mean aod measurement, with a bias of approximately 6.5% for aod max -aod min , 4% for aod max -aod p , and 3% for aod p -aod min . normalized aoa plots showed 95%loa to be 20% of the mean aoa measurement, with a bias of approximately 12% for aoa max -aoa min , 5% for aoa max -aoa p , and 7% for aoa p -aoa min . aod max was the largest measurement in 93/131 (71%) patients and aod min was the smallest measurement in 90/131(69%) patients; aoa max was the largest measurement in 96/131 (73%) patients and aoa min was the smallest measurement in 95/131 (73%) patients. rmanova confirmed that ao max >ao p >ao min (p < 0.0001). median within-patient within-measurement variability was 5% for aod and 9% for aoa measurements. our data suggest that ao measurements differ throughout diastole, with ao max >ao p >ao min . the disparity is greater for area than linear estimates. the degree of disagreement between ao max and ao p is small and similar to the within-measurement variability. thus, using either ao max or ao p measurements should result in similar ratiometric estimates of cardiac dimensions. no conflicts of interest reported. feline hypertrophic cardiomyopathy (fhcm) is the most common heart disease in cats. hcm is considered an inherited disease of the sarcomere and fhcm has been linked to mutations in one sarcomere protein i.e. mybpc3. however, the pathophysiologic mechanisms behind disease development and progression are largely unknown. in this study we investigate whether mitochondrial morphological changes in the myocardium accompany mitochondrial dysfunction and enhanced oxidative stress formation that we recently found in fhcm. myocardial tissue from the left ventricle (lv) was obtained immediately after euthanasia from 7 cats diagnosed with primary hcm on echocardiography (3 maine coon, 2 british shorthair, 1 exotic shorthair, 1 norwegian forest cat) and 8 age-matched control cats (5 maine coon, 3 norwegian forest cats). ultrastructural examination was performed by the use of transmission electron microscopy. in hcm cats, marked ultrastructural changes of the cardiomyocytes were observed. the population of subsarcolemmal mitochondria (ssm) was absent in large cellular areas in cats with moderate and severe lv hypertrophy. flattening of the sarcolemma was a common finding, causing disorganization of the t-tubular system. interfibrillar mitochondria (ifm) were disorganized but not depleted. additional changes in cardiomyocytes from cats diagnosed with fhcm included remodeling of sarcomeres, disorganization of myofibrils, convolution of gap junctions, accumulation of intracellular z-disc material, perinuclear lipofuscin granula and extensive extracellular deposits of collagen. in healthy mammalian cardiomyocytes, the t-tubular system upholds cellular structure, prevents mitochondrial reticulum formation and provides calcium, oxygen and substrates, necessary for normal functioning muscle. disorganization of the sarcolemma and t-tubular system may cause the depletion of ssm. possible mechanisms are atrophy or disruption of the mitochondria or altered fusion-fission dynamics. calcium cycling and substrate supply are likely to be compromised by the observed structural changes. we propose this to be related to mitochondrial dysfunction and oxidative stress formation that occurs in fhcm, however a causative relationship remains unknown. in conclusion, morphological changes of mitochondria and extra-sarcomeric structures are common in fhcm, regardless of breed, genotype and phenotypic disease expression. moreover, mitochondrial subpopulation-specific changes occur in fhcm with depletion of ssm. ultrastructural and functional changes of cardiac muscle mitochondria are considered important molecular mechanisms, responsible for the development and progression of fhcm and may be relevant future treatment targets. no conflicts of interest reported. patent ductus arteriosus (pda) is one of the most common congenital cardiac defect in the dog. ductal patency is associated with pulmonary overcirculation, left ventricular volume overload and can rapidly determine congestive heart failure if untreated. several devices to close the pda have been used, with amplatzer canine duct occluder (acdo©) being considered the safer device with lowest complication rates. echocardiography represents the cornerstone of pda diagnosis, but its role has been recently expanded to wider field of application: device sizing and intraoperative monitoring, as well as a tool to quantify cardiac morphology and function. speckletracking echocardiography (ste) has been used to evaluate cardiac function in a wide variety of diseases in human and veterinary patients, however no study has evaluated its usefulness in dogs affected by pda both before and after percutaneous closure of pda. the aim of our study was therefore to assess standard m/bmode derived parameter of cardiac function and ste derived longitudinal, radial and circumferential strain and strain rate before and after pda closure. twenty-five dogs of different breeds, age and weight were prospectively recruited and a complete echocardiographic evaluation was performed before and 24 hours after pda closure. end diastolic and systolic diameters indexed for body surface area (edvi/esvi) both derived by m-mode and b-mode views, allometric scaling derived allod and allos, sphericity index (si) and pulmonary to systemic flow ratio (qp/qs) were assessed both pre and postoperatively. ste derived parameters assessed were longitudinal, radial and circumferential strain and strain rate. a statistically significant difference was found in all standard parameters of cardiac function before and after pda closure (p < 0.002), with a general decrease in values 24 hours postoperatively. ste derived parameters of cardiac function showed a trend toward a decrease back to normal values, which was statistically significant (p < 0.01) for circumferential and radial strain and strain rate, while longitudinal strain and strain rate did not reach statistically significance. based on our results, no cardiac dysfunction was identified by the use of ste derived parameters both before and after pda closure, with an increased contractility as identified by higher than normal ste values before pda closure and a decrease back to normal strain and strain rate values for both circumferential and radial immediately after percutaneous closure. longitudinal strain persists on higher than normal values, refusing the hypothesis of systolic dysfunction after pda closure and suggesting a longer reverse remodeling process after pda closure. dr bussadori receives royalties from esaote (florence, italy) related to an european patent (nr 071129712) he developed for xstrain software. the study was not funded by a research grant. cardiac cachexia which is characterized by progressive weight loss and depletion of lean body mass, is an independent predictor of survival in human patients with congestive heart failure. chronic degenerative mitral valve disease (cdmd) is one of the most common cardiac diseases in dogs. the aims of this study were to evaluate the prevalence and the effects of cardiac cachexia in survival of dogs with cdmd. medical records of 114 client-owned dogs with cdmd were reviewed. the mean age at entry was 11.1 ae 3.2 years; 73 were females, and 41 were males. data obtained from the records including breed, sex, body weight, age at diagnosis, complete blood counts, biochemical profiles, urinalysis, systemic blood pressure, thoracic radiographs, electrocardiograms, ultrasonography and echocardiographic examinations at initial visit and survival time. diagnosis of cdmd was based on echocardiographic characteristics and categorized by modified new york heart association (nyha) functional classification. cardiac cachexia was defined as presence unintentional weight loss (> 5% within 12 months after diagnosis) together with anorexia and muscle weakness, anemia (red cell count < 5.5 10 6 /ll, hemoglobin < 12 g/dl, or both), hypoalbuminemia (plasma albumin < 3.0 g/dl), and azotemia (blood urea nitrogen > 26 g/dl, creatinine > 1.6 g/dl, or both). dogs with other cardiac disorders and other systemic disorders those would cause anemia and hypoalbuminemia were excluded from this study. prevalence of cardiac cachexia, anemia and azotemia was 32.5%, 15.8% and 45.6%, respectively. these conditions were the most prevalent in nyha class 4, followed by nyha classes 3 and 2. the prevalence of hypoalbuminemia was not significantly different among classes. the one-year body weight change was found in the nyha classes 2 (increased 2.7 ae 10.7%), 3 (decreased 1.3 ae 12.3%) and 4 (decreased 6.1 ae 9.5%). the difference between classes 4 and 2 was significant. results of the cox proportional hazard model indicated that survival time was significantly positively associated with nyha functional severity at diagnosis (p < 0.001), presence of cardiac cachexia, weight loss, anemia, hypoalbuminemia and azotemia (p < 0.001, p = 0.003, p = 0.0033, p = 0.003 and p = 0.019, respectively). the prevalence of cardiac cachexia was common in advanced cdmd dogs, and the parameters of cardiac cachexia, namely weight loss, anemia, hypoalbuminemia and azotemia were strong prognostic factors associated with survival. no conflicts of interest reported. mitral valve disease (mvd) is the most common cardiovascular disease in dogs. it's characterized by myxomatous degeneration, which causes mitral valve prolapse (mvp), mitral regurgitation (mr) and a left apical systolic murmur (lasm). mvd affects small breed dogs with a very high prevalence in cavalier king charles spaniels (ckcs). the main goal of this study was to determine the prevalence of lasm, mvp and mr in the maltese, the most presented breed among dogs with mvd in taiwan. the correlation between these 3 measurements and the influence of age, gender, reproductive state, and body weight were also investigated. study results were compared to other mvd prevalence studies in europe and north-america. 162 client-owned maltese dogs (75 males and 87 females; body weight 1.35-7.15 kg; age 2-15 yrs) with no signs of heart failure were recruited. the intensity (grade 1-6) of lasm was recorded. grade of mvp (mild/severe) and mr severity (mild/moderate/ severe) were evaluated by echocardiography. logistic regression was used to determine the correlation between age and presence of lasm, mvp and mr. a chi-square test was used to evaluate whether sex and reproductive-status were related to prevalences of lasm, mvp and mr. spearman's correlation coefficient was used to assess the relationships between age, body weight, lasm intensity, grade of mvp and severity of mr. the prevalence of lasm, mvp and mr were 28.4%, 34% and 41.4%, respectively. all have positive correlation with age (p = 0.000). the age at which 50% of the dogs had lasm, mvp and mr was 7.7, 7.5, and 5.6 years, respectively. the lasm intensity, mvp grade and mr severity were all positively correlated to age (all p = 0.000) and had no correlation with bw and reproductive status. females had a significantly higher prevalence of lasm than males (36% vs. 21.8%, p = 0.046). maltese dogs in taiwan have a very high prevalence and an early development of mvd as compared to other small breed dogs, similar to mvd in ckcs in other countries. since we only recruited asymptomatic dogs, this study may underestimate the prevalence of mvd in the whole maltese population. to our knowledge, this is the first report to document the high prevalence of mvd in taiwanese maltese.the maltese may be a new canine model for genetic, pathology, and natural history studies in mvd. boehringer-ingelheim sponsored the author's accommodation costs for this congress. esvc-o-20 cardiorenal syndrome in dogs with chronic valvular heart disease: a retrospective study. e. martinelli 1 , p. scarpa 2 , c. quintavalla 1 , c. locatelli 2 , p. brambilla 2 . 1 university of parma, parma, italy, 2 university of milan, milan, italy in human medicine, primary disorders of the heart often result in secondary dysfunction or injury to the kidneys. the coexistence of the two problems in the same patient is referred as cardiorenal syndrome (crs). just little information about crs is available in veterinary medicine. the aim of this study was to define the prevalence of chronic kidney disease (ckd) complicating chronic valvular heart disease (cvhd) in dogs and to investigate the relationship between class of cardiac insufficiency (acvim) and class of renal insufficiency (iris). medical records of dogs presented at the cardiology service of the department of veterinary science and public health, university of milan, between january 2003 and december 2012 were retrospectively evaluated. dogs with a complete physical examination, thoracic radiographs, a cvhd diagnosis based on echocardiographic examination, and a serum biochemical panel, including assessment of serum creatinine (scr) and serum urea (bun), were included in the study. dogs with other heart disease, neoplasm or systemic diseases were not included in the study. one hundred eighteen dogs of both genders (73 males and 45 females), 5 to 18 years of age (11.64 ae 2.66 years), 3 to 48 kg of bodyweight (11.38 ae 8.84 kg) fulfilled the inclusion criteria. the 20% of males and the 37% of females were neutered. the most represented breeds were mongrel (44%), miniature poodle (9.32%), york shire terrier (8.5%), shih -tzu (4.24%), pinscher (4.24%) and dachshund (3.38%). dogs were classified as follow: 0% acvim a, 23% acvim b1, 9% acvim b2, 59% acvim c and 9% acvim d. while the 73% of the dogs were normoazotemic (scr < 1,4 mg/dl), 16% were staged in iris 2, 11% in iris 3 and 0%in iris 4. statistical analysis was performed using jmp 7.0 (sas institute inc.). a p value <0,05 was considered significant. the prevalence of ckd associated with azotemia in dogs affected by cvhd was 27%. there was a statistically significant direct correlation between acvim and iris class (pearson test p = 0.0114). unexpectedly, the 58% of dogs receiving drugs for medical management of heart failure (acvim class c and d) were normoazotemic. despite a definite conclusion about the role of cvhd on the induction and/or progression of ckd cannot be drawn from this cross-sectional study, these results suggest that there is a direct correlation between the severity of ckd and cvhd. no conflicts of interest reported. there is growing evidence of breed differences in concentrations of several blood variables in dogs. the aim of the study was to investigate breed differences in plasma concentrations of components of the renin-angiotensin-aldosterone system (raas), endothelin-1 (et-1) and serum cortisol concentration in healthy dogs. 535 healthy, privately-owned dogs of nine breeds were examined at five centers as part of the european lupa-project. absence of cardiovascular or other clinically relevant organrelated or systemic disease was ensured by thorough clinical investigations. plasma concentrations of et-1 and aldosterone, renin activity, and serum concentration of cortisol were measured by ria or elisa assays. overall significant breed differences were found (p < 0.0001 for all 4 variables). bonferroni-corrected pair-wise significant differences between breeds were found in 67% of comparisons for et-1, 22% for cortisol, 19% for renin and 11% for aldosterone. for et-1, the highest median concentration was found in newfoundlands with values >3 times higher than most other breeds, while renin was highest in dachshunds, >2 times higher than in newfoundlands and boxers, which had the lowest concentrations. aldosterone was especially low in belgian shepherds with median concentration <10 times than the other breeds. cortisol was highest in finnish lapphunds, almost 3 times higher than boxers with the lowest concentration. in conclusion, considerable inter-breed variation in concentrations of et-1, components of raas and cortisol was found in healthy dogs. these differences are likely influenced by genetic factors and should be taken into account when designing clinical trials and tests. breed-specific reference ranges might be necessary. no conflicts of interest reported. most studies that assess weight management in obese dogs only examine the early stages of weight loss, and this may not properly reflect a complete weight management regime. the aim of the current study was to examine the kinetics of a complete weight management cycle in obese client-owned dogs. dogs referred to the royal canin weight management clinic, university of liverpool, for the management of obesity, were eligible for inclusion. all dogs were followed until they had either completed (i.e. reached target weight) or the programme was discontinued. rate of weight loss, percentage weight lost, and energy were assessed at different time points. a total of 149 dogs were included, with a range of breeds, ages and sexes represented. rate of weight loss steadily decreased throughout the weight loss period (d28: 1.2 ae 0.67%/wk; d56: 0.8 ae 0.6%/wk; d84: 0.7 ae 0.5%/wk; d168: 0.5 ae 0.4%/wk; d252 0.4 ae 0.3%/wk; d672: 0.1 ae 0.1%/wk; p < 0.001). the energy intake required to maintain weight loss also progressively decreased (p < 0.001). by day 84, mean aesd weight loss was 11 ae 4.9%, and compliance was good, but most had not com-pleted (1% completed, 86% ongoing, 13% discontinued). thereafter, more dogs completed, but the number of discontinuing also increased (d252: 20 ae 7.7% weight loss, 32% completed, 41% ongoing, 27% discontinued; d672: 25 ae 14.6% weight loss; 59% completed, 4% ongoing, 37% stopped). initial weight loss is good in obese dogs but, thereafter, steadily worsens. thus, studies examining only the first few months of weight loss are not fully representative of the entire weight loss process. conflicts of interest: the following conflicts of interest apply: the diet used in this study is manufactured by royal canin.whilst vb is employed by royal canin. vb and ss are employed by royal canin. ajg's readership is funded by royal canin. obesity and obesity-related metabolic dysfunctions are increasing in humans as well as in dogs. obese dogs become affected by chronic diseases at young age, have a decreased quality of life and a shorter life-span. the aim of the study was to describe the metabolic and hormonal response to a feed-challenge test in lean and overweight dogs. twenty-eight healthy intact male labrador retrievers aged 5.2 ae 1.5 years with varying body condition score (bcs, scale 1-9) were included. twelve dogs were classified as lean (bcs 4-5), ten as slightly overweight (bcs 6) and six as overweight (bcs 6.5-8). an overnight fasting period and blood sample collection was followed by a high fat meal. after food intake, blood samples were collected hourly for four hours. a glucagon elisa was validated for use in dogs. the assigned bcs was supported by positive association with serum leptin concentrations. postprandial triglyceride concentration was significantly higher in the overweight group. a tendency to higher cholesterol concentration was seen in the overweight group but cholesterol was not affected by food intake. glucagon concentration rose after food intake and resembled the response seen in humans after a mixed meal. glucose and insulin concentrations followed the same pattern while free fatty acids had declined one hour after the meal. in this study, the metabolic and hormonal response to a high fat meal was similar between lean and slightly overweight dogs, whereas the response of overweight dogs differed. studies on the health significance of postprandial hypertriglyceridemia in dogs are warranted. conflicts of interest: the study was financially supported by the swedish veterinarian federation, the companion animal research foundation, and the foundation of thure f. & karin forsberg. feline weight-loss programs are often hindered by compliance issues and sedentary lifestyle. the purpose of this study was to assess the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned cats. the objectives were 1) to evaluate weight loss parameters in cats fed the ndwmf* and 2) to describe the owner's perception of the cat's quality of life. overweight/obese, otherwise healthy, client-owned cats (>3/5 body condition score -bcs) were enrolled in the study (n = 132). initial veterinary evaluation comprised a physical examination, nutritional assess-ment, determination of ideal body weight (ibw), and development of weight loss feeding plan. daily energy requirement (der) for weight loss was calculated as der = 0.8 x (70 x ibw kg 0.75 ). initial and follow-up evaluations (monthly for 6 months) consisted in determination of body weight (bw), bcs, body fat index (bfi), muscle condition score (mcs), and current feeding practices. quality of life assessment by owners included cat's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis encompassed scatterplots, regression analysis, summary statistics as appropriate for the type of analyses (continuous or categorical variables, distribution), a mixed model anova was used to assess changes over time (statistical significance at p < 0.05). eighty three percent of the cats (n = 110) lost weight with an average weight loss of 11% (sem, 1.8%) over 6 months and an average weekly weight loss rate of 0.45% (sem, 0.03%). a significant decrease in bcs from week 12-24 and in bfi from week 8-24 compared to baseline was observed. mcs did not change. average duration of weight loss was 134 days (sem, 4.8 days) with 32 days (sem, 0.5 days) between visits. fourteen percent of cats achieved ibw (0.14, ci: 0.08-0.22). seventy nine percent of cats ate more than the recommended der (median fed above der=6%), and the majority of these cats still lost weight. owners perceived a significant increase in energy and happiness (>week 12) compared to baseline in the cats that lost weight without changes in appetite or begging behavior. no significant changes were seen in scores for flatulence, stool volume, and fecal score. in conclusion, this clinical study showed that feeding the ndwmf* to client-owned, overweight/obese cats resulted in weight loss. owners reported significant improvements in cat's quality of life without negative side effects. * porphyrias are a group of inborn errors of metabolism resulting from accumulation of porphyrins due to deficient activities of specific enzymes in heme biosynthesis. in humans, they are clinically classified as either erythroid with cutaneous involvement or hepatic with acute neurovisceral attacks. here we describe the clinical, biochemical, and molecular genetic studies in porphyric cats from new brunswick, canada. from 2008 to 2014, three separately identified adult domestic shorthair cats from the city of saint john in new brunswick were found to have erythrodontia (brown discolored teeth which fluoresced pink) and pigmenturia. a mild compensated hemolytic disorder with numerous small dark blue irregularly shaped erythrocyte inclusions was noted. there was no evidence of acute lifethreatening neurovisceral attacks or cutaneous lesions. necropsy of one cat revealed massive deposition of porphyrins in all bones and teeth. urine and edta blood samples from one cat were metabolically studied, while molecular genetic studies were performed in all cats either from edta blood or a formalinized splenic tissue block. urinary d-aminolevulinic acid, porphobilinogen, uroporphyrin i, and coproporphyrin i concentrations were increased in the cat studied, suggesting an acute intermittent porphyria (aip). the erythrocytic hydroxymethylbilane synthase (hmbs) activity in erythrocytes was approximately half normal suggesting a dominant enzymopathy, while the erythrocyte uroporphyrinogen iiisynthase activity was normal. sequencing the feline hmbs gene revealed a heterozygous intronic 12 base deletion (c.772-13_-2del) which results in an insertion in the mrna and would predict a truncated protein. in conclusion, these three domestic shorthair cats had the same hmbs mutation causing an autosomal dominantly inherited aip. cats with discolored teeth and normal or mild hemolysis may have either acute intermittent porphyria or congenital erythroid porphyria. interestingly, seven disease-causing mutations have now been found by us in the hmbs gene -more than in any other gene in cats. the biochemical and molecular characterization facilitates clinical screening of affected cats to reach a specific diagnosis. supported in part by nih od 010939. urs giger and raj karthik are also part of the laboratory that offers dna testing for this mutation. fibrinogen decreases when coagulation is activated to form fibrin, while fdps and d-dimers represent the products of fibrinolysis. in humans, activation of coagulation and fibrinolysis develops in all type of ascites and it is also associated with signs of systemic fibrinolysis.these results have lead to the suggestion that ascitic fluid is inherently fibrinolytic. preliminary studies showed similar results also in dogs (javma nov. 2012 , ecvim proceedings 2013 . in addition, in an old experimental study conducted in dogs, inoculation of blood or of a solution containing fibrinogen and thrombin into the pleural cavity resulted in the activation of the coagulation system followed by fibrinolysis. therefore, the objective of the present study was to determine whether the activation of coagulation and fibrinolysis (i.e. low fibrinogen and elevated fdps and ddimer) occurs not only in the ascitic fluid, as alredy been demonstrated, but also in all type of pleural effusions in dogs. thirty-three dogs referred to the san marco veterinary clinic with pleural effusion, but without ascites, were studied. fibrinogen, fdps, and d-dimer concentrations were measured and then compared in both pleural fluid and venous blood via wilcoxon signed ranks test. the dog's pleural effusions were categorized based on pathophysiology of fluid formation into 5 dogs with transudate (4 due to increased hydrostatic pressure and 1 due to decreased osmotic pressure), 23 with an exudate (of which 11 due to septic causes), 4 with a haemorrhagic pleural effusion, and 4 with a chylous effusions. the fibrinogen concentration in the pleural effusion (median: 59 mg/dl; range: 59-59) was significantly lower (p < 0.0001) than the plasma fibrinogen concentration (median: 419 mg/dl; range: 131-1406). in all dogs, the fibrinogen pleural fluid concentration was lower than the plasma concentration. the fdp concentration in the pleural effusion (median: 151 mg/dl; range: 0.69-151) was significantly (p < 0.0001) higher than plasma fdps concentrations (median: 5.55 mg/dl; range: 0.83-108.47). in 1 case, the fdps pleural fluid concentration was lower than the plasma concentration and in 32 cases the pleural fluid concentration was higher. the d-dimer concentrations were significantly(p < 0.0001) higher in the pleural effusion (median: 3.84 lg/ml; range: 0.05-9.61) than in the plasma (median: 0.07 lg/ml; range: 0.01-7.67). in one case, the d-dimer pleural fluid concentration was lower than the plasma concentration and in 32 cases was higher. these findings support the hypothesis that activation of coagulation followed by fibrinolysis occurs in all type of pleural effusions. no conflicts of interest reported. during primary hyperfibrinogenolysis (phf), fdps production is increased but production of d-dimer is not. therefore, elevated fdps and normal d-dimer are considered an indicator of phf. in humans and dogs, activation of coagulation and fibrinolysis develops in all type of ascites and it is associated with systemic phf, suggesting that ascitis is inherently fibrinolytic. preliminary data have shown that activation of coagulation followed by fibrinolysis occurs also in all type of pleural effusions (pe). the objective of this study was to determine if systemic phf occurs also in dogs with pe. thirty-three dogs referred to the san marco veterinary clinic with pe, but without ascites, were studied (group 1). from the electronic data-base of the clinic dogs for inclusion in control groups 2 (healthy dogs) and 3 (sick dogs without pe or ascites) were randomly selected and individually matched to group 1 dogs for age, sex, and breed. fibrinogen, fdps, d-dimers, c-reactive protein (crp), fibrinogen/crp ratio, and prevalence of phf (i.e., dogs with elevated plasma fdps and normal d-dimer) were determined. differences between the 3 groups were analyzed using anova (fibrinogen), chi-square (fdps and prevalence of phf) and kruskal-wallis test (crp, fibrinogen/crp ratio, and d-dimer). post-test analysis were performed by tamhane and mann-whitney test. fibrinogen concentration in group 1 was significantly increased compared to group 2 (p < 0.0001), but not compared to group 3 (p = 0.504). fdps concentration in group 1 was significantly increased compared to groups 2 (p < 0.0001), but not compared to group 3 (p = 0.148). d-dimers concentration in group 1 was significantly increased compared to group 2 (p < 0.0001), but not compared to group 3 (p = 0.964). crp was significantly increased in group 1 compared to group 2 and 3 (p < 0.0001 for both comparison). fibrinogen/crp ratio was significantly decreased in group 1 compared to group 2 and 3 (p < 0.0001 for both comparison). prevalence of phf was significantly higher in group 1 compared to groups 2 (p = 0.004), but not compared to group 3 (p = 0.186). these results support the hypothesis that phf occurs significantly more often in dogs with pe compared to healthy dogs. despite there was a trend of increased phf also in dogs with pe compared to sick dogs, this difference did not reach significance. nevertheless, the decreased in fibrinogen/crp ratio in group 1 compared to group 3, in the face of a similar d-dimer concentration, would suggest that phf is also more prevalent in dogs with pe compared to sick control dogs. no conflicts of interest reported. the systemic inflammatory response syndrome (sirs) refers to clinical signs of systemic inflammation in response to (non-) infectious insults. current diagnosis of sirs is based on clinical and basic laboratory data and is a sensitive screening to identify patients at risk. c-reactive protein (crp) is a major canine acute phase protein with concentrations related to disease severity and underlying cause. crp rises in response to proinflammatory cytokines, mainly interleukin (il)-6 and tumor necrosis factor (tnf)-a, which are considered the main triggers of sirs. we therefore evaluated crp, il-6 and tnf-a kinetics in canine emergency sirs patients hypothesizing that crp is (1) increased in dogs with a clinical sirs-diagnosis, (2) correlated with il-6 and tnf-a concentrations, (3) influenced by the underlying etiology, and (4) a prognostic marker. canine emergencies with clinically diagnosed sirs were prospectively included. serum and plasma were immediately stored at -80°c after sampling at presentation, after 6 (t6), 12 (t12), 24 (t24) and 72 (t72) hours, and at a control visit (t1 m) over one month after discharge. serum crp was measured with a caninespecific immunoturbidimetric crp assay. plasma il-6 and tnfa were measured using a bioassay measuring biologically active cytokine concentrations. disease categories were infection (i), neoplasia (n), trauma (t), gastric-dilation and volvulus (gdv), other gastrointestinal (gi), renal (r) and miscellaneous (m) diseases. statistical analysis was performed with sas. concentrations of inflammatory cytokines were expressed logarithmically, with univariate analysis confirming normal distribution. a correlation procedure, mixed procedure on a linear model and a logistic procedure were performed (p-value < 0.05). sixty seven dogs (i = 12, n = 13, t = 6, gdv=11, gi=4, r = 3, m = 18) were included. forty-three patients survived (seven died, seventeen were euthanized). twenty patients had a control visit. crp was elevated in 71.2% of dogs at presentation, and only remained within reference range (0-14.9 mg/l) throughout hospitalization in four dogs (6.0%). crp concentrations were significantly higher from t0 (92.7 ae 113.7 mg/l) to t24 (95.2 ae 90.2 mg/l) decreasing at t72 (71.1 ae 76.6 mg/l), and returning within reference range at t1 m (2.4 ae 4.5 mg/l) in all but one dog (18.2 mg/l). crp was significantly correlated with logarithmical concentrations of il-6 and tnf-a, however, these did not change significantly over time. none of the evaluated parameters was associated with disease category, nor outcome. crp appears useful to diagnose sirs in emergency patients, and tends to decrease during hospitalization. however, crp, neither il-6 nor tnf-a concentrations appear useful to predict the underlying disease and outcome in sirs patients. no conflicts of interest reported. calprotectin (s100a8/a9 complex) belongs to the s100/calgranulin family, and is primarily released from activated neutrophils and macrophages. serum calprotectin concentrations (cp) were shown to be increased in dogs with inflammatory diseases such as inflammatory bowel disease, pancreatitis, systemic inflammatory response syndrome, and sepsis. canine cp thus appears to be a biomarker of inflammation. considerable day-today variation of fecal canine cp was found in both healthy dogs and dogs with chronic gastrointestinal disease. however, the biological variation of canine cp in serum has not been reported. the aim of this study was to determine the biological variation of serum canine cp and its minimum critical difference (mcd). eleven healthy dogs were used for this study. biological variation of serum canine cp was evaluated over a 2.6-months period. tests for outliers were carried out at 3 levels (within-run analytical variance, intra-, and inter-individual variation). a nested analysis of variance (anova) model was used to calculate analytical (cv a ), intra-individual (cv i ), inter-individual (cv g ), and total variation (cv t ), and to determine the index of individuality (ii), index of heterogeneity (ih), and mcd. a total of 14 serial specimens were collected from 6 dogs, 13 serial samples from 3 dogs, and 12 serial samples from 2 dogs. four within-subject outliers were detected and excluded from further analysis, yielding a total of 147 serum samples and slightly right-skewed data. no outlying observations (cochrane test) or outliers among mean concentrations of subjects (reed's criterion) were detected. cv a was calculated as 3.0%, cv i as 29.9%, and cv g as 33.2%, resulting in a cv t of 66.1%. index of individuality (ii) was determined to be 0.905 and ih was 4.939, yielding a one-sided mcd of 6.4 mg/l. the analytical goal of cv a ≤ ½9cv i was satisfied. although serum canine cp remained within a relatively narrow concentration range in healthy dogs, moderate individuality was detected. moderate changes in serum canine cp (6.4 mg/l) between sequential measurements are needed to be considered clinically relevant, and using a population-based reference interval may or may not be appropriate for serum canine cp. using the mcd with the previously determined median canine cp concentration (4.9 mg/l) for the reference sample group yielded a serum canine cp concentration close to the upper limit of the previously established reference interval (11.9 mg/l), showing that the reference interval for serum ccp (0.9-11.9 mg/l) is within reasonable limits. the assay used in the study was developed at the gi laboratory, texas a&m university. most authors also work at the gi laboratory, texa a&m university. canine leishmaniasis (canl) is a multisystemic disease that is endemic in the mediterranean region. in the past, concentrations of acute phase proteins (apps), and specifically c-reactive protein (crp), haptoglobin (hp), ceruloplasmin (cp), serum amyloid a (saa) and albumin (alb), have been reported to change in dogs with leishmaniasis, and revert to normal after successful treatment, highlighting the intrinsic inflammatory reaction of the host to the parasite. since the spectrum of clinical and laboratory derangements is broad, it is possible that apps are increased specifically because of certain clinicopathological syndromes associated with canl. a total of 80 dogs with canl, diagnosed on the basis of cytological amastigote identification and ifat serology, were retrospectively included in the study. in all of them, crp, saa, hp and alb were measured at interlab-umu, murcia, spain, in aliquots of serum, which were stored in -22°c for 4 -8 years (median: 4 years). results for each of the apps were correlated to laboratory and clinical parameters (n:80), clinical and parasitological scoring (n:40), ehrlichia and leishmania serology (n:40), and clinical staging according to leishvet (n:80), using an array of linear and ordinal regression models, as well as one-way anova, t-test and fisher's lsd test. crp and alb were by far the apps most frequently correlated with clinical and laboratory abnormalities such as nutritional status, lethargy and skin ulcers (p < 0,05), as well as urinary protein to creatinine ratio (upc), total serum protein, and urine specific gravity (p < 0,02). there were limited associations between hp, cp, saa and clinicopathological parameters. a minor linear relationship was observed between crp and clinical scoring. crp and alb were also correlated with parasitological scoring in bone marrow, but not lymph node cytology (p < 0.04). dogs with ehrlichia titers had higher crp, cp and lower alb concentrations. finally, crp concentrations were higher in later compared to earlier stages of the infection, as defined by the leishvet criteria. the inflammatory component to leishmania infection doesn't seem to be exemplified by the reaction of a particular tissue, with the possible exception of glomerulonephritis. the magnitude of increase in crp and decrease in albumin is correlated with clinical staging and bone marrow parasitological scoring. no conflicts of interest reported. the consequences of abnormal platelet function in dogs and cats can be devastating and the use of anti-thrombotic therapy to prevent thrombotic events is increasingly common. the ability to measure platelet function and the efficacy of anti-thrombotic therapy is difficult due to limited availability of equipment and inability to delay platelet function analysis. the aim of this study was to adapt and validate test procedures and protocols previously developed for humans for use in dogs and cats. residual samples of citrate anticoagulated blood were used from dogs and cats presented to a specialist referral centre for various reasons unrelated to clotting abnormalities. initially the blood was stimulated using specific combinations of either arachidonic acid/epinephrine (aa/epi) or adp/u46619, designed to assess the effects of the anti-thrombotic agents aspirin and clopidogrel respectively. after 5 minutes stimulation, the blood samples were fixed using a patented platelet fixative solution developed for human platelets, which allows the delayed analysis of p-selectin an established marker of platelet activation. all analysis was performed by flow cytometry. in order to do this, specific antibodies were selected for the recognition of both canine and feline platelets. cd61 was used as a platelet identifier antibody while appropriate cd62p (p-selectin) antibodies for each species were chosen. fixed samples were repeatedly analysed at time points between 0 to 35 days following fixation to establish the stability of the fixed samples. thirteen dogs and three cats were analysed. high p-selectin expression was detected following stimulation with aa/epi and adp/u46619 in both dogs and cats following fixation. this was significantly different to unstimulated blood (p < 0.0001). there was no significant difference in detectable pselectin expression following storage of the fixed samples at any time-point up to 35 days. this confirmed the fixative was suitable as a preservative of canine and feline platelets. a limited number of dogs were evaluated whilst receiving antithrombotic medication. there was a significant difference in the activation of platelets in the dogs treated with either aspirin (p < 0.03) or clopidogrel (p < 0.004) compared with untreated dogs following stimulation with aa/epi (aspirin group) or adp/ u46619 (clopidogrel group). our results show that fixation and delayed analysis of platelet function in dogs and cats is possible for up to 35 days. this demonstrates an exciting opportunity to analyse platelet function remotely and to determine the efficacy of thromboprophylaxis in animals presenting to clinics that do not have on-site platelet analysers. no conflicts of interest reported. several authors consider thyroid hormone supplementation as a valid initial treatment option for dogs with aggression related problems. indeed, mood and behaviour modulating properties of thyroid hormones may, in part, be mediated through the interaction of thyroid hormones with neurohormones such as serotonin and prolactin. at present, prospective trials evaluating neurohormonal status or behaviour in hypothyroid dogs before and after thyroid supplementation are lacking. therefore, the aims of this study were to assess behaviour and measure serum serotonin and prolactin concentrations in dogs with spontaneous hypothyroidism before and after treatment.twenty three client-owned dogs diagnosed with spontaneous primary hypothyroidism were prospectively included in our study. after diagnosis all dogs were treated with levothyroxine (10 micrograms/kg bid). behaviour of dogs was screened at initial presentation, at 6 weeks and 6 months after initiation of therapy. owners had to fill in a hard copy of the standardized canine behavioural assessment and research questionnaire (c-barq) consisting of 101 scored questions evaluating seven behavioural categories. the average score on all questions was calculated for each dog at each of the three time periods and a paired t-test was used for comparison. serum serotonin and prolactin concentrations were evaluated at each time period using a commercially validated elisa kit and heterologous ria, respectively.results of the c-barq after six weeks of thyroid hormone supplementation when compared with the time zero demonstrated a significant increase (p < 0.05) in excitability, activity and aggression, which most likely became unmasked owing to improved overall activity of dogs. conversely, at six months period when compared with the time zero no significant changes in any of the behavioural symptoms were observed. serum serotonin was measured in 18/23 dogs colorimetrically at 450 nm. at time zero, 6 weeks and 6 months serum serotonine was 0.135 (range, 0.075-0.337), 0.141 (range, 0,071-0.392) and 0.149 (range, 0.086-0.378). no significant difference was noted between 6 week and 6 month period comparing to time zero (p = 0.84 and p = 0.37). serum prolactin concentration measured in 22/23 dogs at time zero, 6 weeks and 6 months was 3.35 ng/ml (range, 1.4-6.36), 3.57 ng/ml (range, 1.87-7.39) and 3.92 ng/ml (range, 2.01-12.92) and did not differ significantly in either time period when compared with time zero (p = 0.99 and p = 0.52).altogether, results of this study failed to demonstrate a significant role of thyroid supplementation on the majority of evaluated behavioural symptoms as well as neurohormonal status of hypothyroid dogs during 6 months of therapy. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment of hyperthyroidism in cats, but no prospective studies of the prevalence, clinical features, routine laboratory findings, or results of thyroid function tests have been reported in a series of hypothyroid cats. in this study, we describe the features of hypothyroidism in 35 cats treated with radioiodine over a 15-month period (october 2012-march 2014). during this same period, we treated %500 hyperthyroid cats with radioiodine, providing a prevalence rate of 7%. hypothyroidism was diagnosed 41-814 days (median, 120 days) after 131 i treatment, with doses ranging from 65-1000 mbq (median, 100 mbq; median pretreatment t4, 142 nmol/l). the 35 hypothyroid cats ranged in age from 9-19 years (median, 13 years). all were dsh/dlh; 28 (80%) were female and 7 were males (p = 0.0004). clinical signs in these 35 cats included overweight/obesity in 6 (17%), lethargy/dullness in 6 (17%), poor appetite in 3 (8.6%), and polyuria/polydipsia in 9 (26%). abnormalities on physical examination included dermatologic signs (dry coat, seborrhea, matting) in 5 (14%) and bradycardia (<150 bpm) in 2. twenty-two cats (63%) had no noticeable clinical features of hypothyroidism. routine laboratory abnormalities included hypercholesterolemia (>6 mmol/l) in 7 (20%) and new or worsening azotemia (>140 lmol/l) in 26 (74%) and 4 (11%) cats, respectively. median serum concentrations of total t4 (11.6 nmol/l; reference interval [ri], 10-50 nmol/l), t3 (0.6 nmol/l; ri, 0.5-0.8 nmol/l), and ft4 (15 pmol/l; ri, 10-50 pmol/l) were all in the low end of the ri. normal ri values for t4 and ft4 were maintained in 22 (63%) and 30 (86%) of the cats, respectively. serum ctsh values were high in all cats (median, 3.3 ng/dl; range, 0.47-12.0 ng/dl; ri, 0.03-0.30 ng/ml). thyroid scintigraphy showed less-than-normal amounts of residual tissue, as well as low values for thyroid-to-salivary ratio and %-uptake of pertechnetate, in 30 (86%). of those 5 cats with normal scintiscans, serum ctsh decreased into the ri without treatment when retested 1-6 months later. in conclusion, this study confirms that 131 i-induced hypothyroidism is not uncommon, with an apparent female sex predilection. serum t4 and ft4 remain normal in most cats, but high serum ctsh values and thyroid scintigraphy aid in diagnosis. unless cats have overt, long-standing hypothyroidism, most cats with subclinical disease are relatively asymptomatic, other than worsening azotemia. subclinical hypothyroidism will be transient in some cats, with normalization of ctsh values within a few months. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment for hyperthyroidism in cats. at our clinic where we use a variable 131-i dosing protocol (based on tumor volume and severity of hyperthyroidism), the prevalence of overt or subclinical hypothyroidism is at least 7%. during the 15-month period from october 2012 to march 2014, we treated 19 cats with iatrogenic hypothyroidism, which had developed 41-323 days (median, 119 days) after treatment with radioiodine (median dose, 100 mbq). these cats ranged in age from 9-19 years (median, 14 years); all were dsh/dlh; 14 (74%) were female and 5 were males. new or worsening azotemia (>140 lmol/l) was documented in 16 (84%) and 2 (10.5%) cats, respectively. diagnosis of hypothyroidism was based on the following: 1) low to low-normal serum concentrations of t4, ft4, and t3; 2) high serum tsh concentration (>0.5 ng/dl); and 3) less-than-normal amounts of residual tissue on thyroid scintigraphy. all cats were given thyroid hormone replacement as a liquid l-t4 preparation (leventa; merck animal health). cats were monitored at 1-3 month intervals by repeating serum t4 and tsh concentrations 3-4 hours after the morning l-t4 dose. ten of the 19 cats were started on a once-daily l-t4 regimen (100 lg); of these, only 2 (20%) had suppression of high serum tsh values into the reference interval (ri). of the 8 cats that had persistently high tsh values, 5 were switched to twice-daily administration (75-100 lg, bid), which successfully lowered high tsh concentrations in 4 cats. the remaining 9 cats were started on twice daily l-t4 (75 lg, bid); of these, normalization of tsh occurred in 5 cats. overall, l-t4 treatment was successful in normalizing tsh concentrations in 11 (58%) cats, 2 with once-daily and 9 with twice-daily dosing. peak serum t4 concentrations of ≥30 nmol/l were needed in most cats to normalize tsh values. higher serum t4 and lower tsh concentrations were achieved when l-t4 was administered on an empty stomach rather than given with food. a significant decrease (p < 0.003) in serum creatinine occurred after treatment with l-t4. in conclusion, our results indicate that twice-daily administration of l-t4 is needed in most cats with iatrogenic hypothyroidism to normalize high serum tsh concentrations. many cats appear to absorb l-t4 rather poorly, which can be enhanced by giving the drug on an empty stomach. the azotemia that commonly develops in cats with hypothyroidism improved or stabilized with adequate l-t4 supplementation. no conflicts of interest reported. congenital hypothyroidism (ch) has been reported in many species; the hereditary forms can be divided into thyroid dysmorphogenesis and dyshormonogenesis. while thyroid hypoplasia has been described in dogs and cats, the molecular basis remains unknown. in contrast few breeds of dogs with goiterous ch were found to have deficient thyroid peroxidase (tpo) activity. the purpose of our study was to characterize a family of domestic shorthair cats with goiterous ch and disease-causing tpo gene mutations. clinical features included dwarfism and dullness, known as cretinism and seen with ch in all species, but also constipation and megacolon which are unique to cats with ch. pedigree analysis documented an autosomal recessive mode of inheritance. affected kittens developed a goiter and had low serum thyroxine (t4) and triiodothyronine (t3) when compared to controls, but high thyroid stimulating (tsh) hormone levels indicating thyroid dyshormonogenesis. oral thyroid supplementation corrected the progression of clinical signs and prevented further constipation and reversed the megacolon. the tpo enzyme activity was extremely low in hypothyroid cats when compared to that of normal cats. genomic dna and cdna from affected, carrier, and normal cats were extracted and sequenced based upon primers developed from the feline genome database. a homozygous missense point mutation (c.1333g>a) in tpo, which results in an amino acid change (p.ala445thr), was discovered in affected cats and the mutant allele segregated within the family with goiterous ch. this is the first report of a tpo deficiency in cats. other unrelated domestic shorthair cats with goiterous ch did not have this same tpo mutation. the prevalence of this tpo mutation in the domestic cat population seems low, but ch is likely underreported in cats. supported in part by nih od 010939. some of the authors are members of diagnostic laboratories (penngen). supported in part by the nih od #010939. glucagon-like peptide-1 (glp-1)is a gastrointestinal hormone released in response to food intake that increases insulin secretion, inhibits glucagon secretion, slows gastric emptying and induces satiation. it is also assumed to stimulate beta-cell proliferation. glp-1 agonists are successfully used in humans with type 2 diabetes mellitus usually either in combination with insulin or other anti-diabetic drugs. in healthy cats twice daily (exenatide) as well as once weekly (exenatide extended-release (er)) application of glp-1 agonists induced pronounced insulin secretion. benefits of exenatide er are the regimen of once weekly injection and less side effects. the objective of the study was to assess whether administration of exenatide er in addition to standard treatment leads to improved glycemic control and higher remission rates in cats with newly diagnosed diabetes. the study was designed as a prospective, placebo-controlled clinical trial. cats were randomly assigned to two groups receiving exenatide er (group 1: bydureon â , 200 mg/kg, q7d, sc) or 0.9% saline solution (group 2: q7d, sc). both groups additionally received insulin glargine (lantus â , initial dose: ≤ 4 kg: 1.0 iu, q12 h; > 4 kg 1.5-2.0 iu, q12 h) and diet (purina dm â ). exenatide er was applied over 16 weeks or, in case of remission, for 4 additional weeks after cessation of insulin application. cats were rechecked 1, 3, 6, 10 and 16 weeks after starting therapy. remission of diabetes was defined as absence of clinical signs of diabetes and normal blood glucose and fructosamine concentrations for at least 4 weeks after discontinuing insulin injections. so far 17 cats have completed the study. mild and transient side effects in group 1 (n = 9) were reduced appetite (n = 6), nausea (n = 2), vomitus (n = 4), tiredness (n = 2) and hiding in dark spots of the house (n = 2). in group 1 remission was achieved in 4/9 (44%) cats and good metabolic control in 4/5 (80%) nonremission cats. in group 2 remission was achieved in 2/8 (25%) cats and good metabolic control in 4/6 (66%) non-remission cats. median insulin dose given during the study period was 0.47 iu/ kg/day in group 1 and 0.56 iu/kg/day in group 2. the preliminary results suggest that exenatide er can be used safely in diabetic cats. a tendency for higher remission rate, better metabolic control and lower insulin requirement was seen when exenatide er was added to the standard treatment regimen. further cases need to be evaluated to verify the potential beneficial role of exenatide er. no conflicts of interest reported. feline diabetes mellitus shares many similarities with human type 2 diabetes mellitus (t2dm), including clinical, physiological and pathological features of the disease. domestic cats spontaneously develop diabetes associated with insulin resistance in their middle age or later, with residual but declining insulin secretion. humans and cats share the same environment and risk factors for diabetes, such as obesity and physical inactivity. moreover, amyloid formation and loss of beta cells are found in the diabetic cat pancreas, as in humans. subsequently, studying the molecular mechanisms in the failing beta cells may contribute to a better understanding of the pathophysiology of t2dm in both cats and humans. the aim of the present study was to develop a method to study mrna expression of islet-specific genes in healthy and diabetic cats. previous attempts in isolating feline islets with different collagenase-based protocols have led to damaged islets or islets coated with exocrine acinar cells, which either way compromise the results obtained from gene expression studies. by using the laser microdissection technique, we were able to sample islets that were not contaminated with exocrine tissue, from both healthy and diabetic cats. high rna quality was confirmed with gel electrophoresis. by quantitative real-time pcr (qrt-pcr), mrna levels of the islet-specific genes insulin, pdx-1, iapp, chga and ia-2 were detected in both healthy and diabetic cats. we used actin b, gapdh and rps7 as internal reference genes for normalizations of our qrt-pcr data. the laser microdissection technique allows studies of islets without contamination of acinar cells, as shown in this study, and is of great advantage since it is difficult to get pure feline islets from collagenase-based isolation. differences in gene expression in healthy and diabetic cats may reveal underlying mechanisms for beta cell dysfunction and decreased beta cell mass in human and feline type 2 diabetes. conflicts of interest: the study was financially supported by the swedish juvenile diabetes foundation, the fredrik and ingrid thuring foundation, the magnus bergvall foundation, the lars hierta memorial foundation, and the foundation for research, agria insurance company. feline acromegaly is an increasingly recognised endocrinopathy among diabetic cats, caused by chronic excessive growth hormone secretion by a functional somatotrophinoma in the pars distalis of the anterior pituitary gland. the majority of human somatotrophinomas are sporadic, however up to 20% of familial isolated pituitary adenomas are caused by germline mutations of the aryl-hydrocarbon-receptor interacting protein (aip). feline acromegaly has phenotypic and biochemical similarities to human familial acromegaly with aip mutations, such as male predominance, somatotroph macroadenoma and resistance to octreotide therapy. the objective of this study was to identify the feline aip gene, identify single nucleotide polymorphisms (snps) within this gene and compare any snps with reported human aip snps. stored pituitary tissue from an acromegalic cat was used to create feline aip cdna using feline specific aip primers. stored edta blood from 10 acromegalic cats (diagnosis of insulin resistant diabetes mellitus, serum igf-1 > 1000 ng/ml and pituitary mass >4 mm identified using pituitary computed tomography or necropsy) and 10 control cats (no history of diabetes mellitus and greater than 15 years of age) were selected, dna extracted and genotyped using pcr, agarose gel electrophoresis and sanger sequencing. the feline aip gene was identified, encoding a 330 amino acid protein with 98% homology to the human aip protein. a blast search revealed this gene contained 6 exons and exon specific primers were created to enable sequencing. a single nonconservative snp was identified in exon 1 (aip:c.9g>t), encoding for an amino acid change from aspartic acid to glutamic acid in 2/10 acromegalic patients and 0/10 control cats. two additional conservative snps were also identified (aip:c.826t>c and aip:c.481t>c). exon 1 encodes for a region of the aip protein considered essential for aip-aip receptor interaction. although 70 different human aip mutations have been identified to date, a human aip:c9g>t mutation has not yet been identified. the aip n-terminal is required for the stability of the aip protein-aip-receptor complex, and essential for the regulation of translocation into the nucleus, where it binds to aryl hydrocarbon receptor nuclear translocator leading to activation of genes thought to act as tumor suppressors. loss of normal aip activity is thought to promote somatotrophinoma development. it is therefore possible that the detected aip:c.9g>t mutation predisposed to somatotrophinoma tumorigenesis in the two affected patients, and a study containing a larger number of cases is indicated. no conflicts of interest reported. hypersomatotrophism (hs) is an important cause of feline diabetes mellitus (dm). in humans surgical removal of the somatotrophinoma is generally recommended, though hypophysectomy programs have suffered from significant initial morbidity and mortality given a documented steep learning curve in newly established programs. hypophysectomy as treatment for feline hs has thus far only been described in a handful of cases, all having been treated by one single experienced hypophysectomy team. this study's aim was to evaluate the learning curve of a de novo established hypophysectomy program, through analysis of peri-and post-operative morbidity and mortality, and endocrine outcomes in the first cohort of cats with hs treated. from 2012 owners of diabetic cats with confirmed hs (igf-1 > 1000 ng/ml, pituitary mass) presented at the royal veterinary college were offered hypophysectomy. all cats undergoing surgery were operated by one neurosurgeon with previously only cadaveric experience of the procedure, through an adapted transsphenoidal approach referencing bony landmarks to computed tomographic scans reconstructed on neuronavigation software. the somatotrophinoma was extirpated using fine surgical tools. all cats received intense electrolyte and blood pressure monitoring, peri-and post-operative ddavp and intravenous insulin and hydrocortisone infusion, transitioning to subcutaneous glargine, conjunctival ddavp, oral hydrocortisone and levothyroxine. between april 2012-february 2014, 12 cats underwent hypophysectomy (median + range age: 10.2 years, 5.4-14.8; igf-1: 1846 ng/ml, 1138->2000; pituitary height 6.2 mm, 4.0-10.6). all displayed uncontrolled dm due to hs (median fructosamine: 619 umol/l); none displayed overt central neurological deficits. two cats (17%, cats 7 and 8; pituitary height (mm): 10.6 and 6.6) required mechanical ventilation post-operatively and both were euthanized. post-mortem magnetic resonance imaging revealed brain herniation and cerebral ischaemia was suspected. one cat suffered cardiac arrest post-operatively at time of jugular catheter placement, though made an uneventful recovery. four other cats developed congestive heart failure within 5 days, which was successfully treated not necessitating ongoing therapy. temporarily diminished tear production was seen in 5 cats. seven of the ten surviving cats went into diabetic remission within a median of 19.7 days (9-42); 3 others saw reduction of insulin needs by 91%. serum igf-1 normalised rapidly and significantly in all but one cat (median serum igf-1 485 ng/ml within 9 days). persistent neurological deficits or palatal wound breakdown were not encountered. starting a hypophysectomy program to treat feline hs was associated with some risk of mortality, though surviving cases benefited from the procedure with a high incidence of diabetic remission. no conflicts of interest reported. pituitary dependent hypercortisolism (pdh) in dogs is frequently associated with high serum phosphate and parathormone concentrations. the pathogenesis of such abnormalities remains unknown and the evaluation of the urinary fractional excretion of phosphate and calcium in pdh dogs might be helpful in enhancing the knowledge regarding this issue. the aim of the present study was to evaluate the serum and urinary concentrations and the urinary fractional excretion of phosphate and calcium in dogs with pdh. medical records from one referral center were retrospectively evaluated between 2003 and 2013. the diagnosis of pdh was confirmed using the cortisol to creatinine ratio, the ldds test and/or acth stimulation test, the plasma acth concentration, ultrasonography of the adrenal glands and computer tomography (ct) of the pituitary and the adrenal glands in dogs with consistent clinical signs. only newly diagnosed dogs, before treatment for pdh, were evaluated. two control groups were included: one healthy and one sick control dog (without pdh) for each dog with pdh were included. healthy control dogs (hcd) and sick control dogs (scd) were matched for age (ae6 months), breed, sex and sexual status. data were analysed using non-parametric tests and expressed as median and ranges. significance was set at p < 0.05. one-hundredsixty-seven dogs with pdh were eligible for inclusion in the study. the median age at diagnosis was 10 years (range: 3-16) and the median body weight was 14.7 kg (range: 3.0-65.5). there were 100 female (64 spayed) and 67 male (11 castrated). serum phosphate concentration (4.1 mg/dl, 1.3-10.7) was significantly (p < 0.0001) higher compared to hcd (3.4 mg/dl, 1.3-5.2) and scd (3.8 mg/dl, 1.1-32.1). serum calcium concentration (10.4 mg/dl, 7.0-14.5) was significantly higher compared to scd (10.1 mg/dl, 5.5-12.9) but not different compared to hcd (10.2 mg/dl, 8.9-12.0). urinary fractional excretion of phosphate (15.5%, 0.5-65.1) was significantly lower compared to hcd (20.4%, 0.4-67.9) and scd (16.9%, 0-83.6). urinary fractional excretion of calcium (0.37%, 0-5.70) was significantly higher compared to hcd (0.22%, 0.02-1.76) and scd (0.24%, 0-18.71). urinary calcium to creatinine ratio (4.78, 0-54.50) was significantly higher compared to hcd (2.17 0.20-16.80) and scd (2.38, 0-16.40), while urinary phosphate to creatinine ratio were not significantly different in pdh dogs, hcd and scd. in conclusion pdh dogs have lower phosphaturia and higher calciuria compared to control dogs. this findings suggest that, at least in part, the high serum phosphate concentrations are related to the renal retention of phosphate. no conflicts of interest reported. four cortisol-based methods of monitoring trilostane treatment of canine hyperadrenocorticism were compared to the results of a clinical scoring scheme based on an owner questionnaire. cases of canine hyperadrenocorticism that had received a consistent dose of trilostane for more than one month were recruited from first opinion and referral practice. each dog was used only once. owners were asked to complete a questionnaire that assessed clinical control. the dogs were then categorised as being over-controlled, well-controlled, moderately-controlled and poorly-controlled. cortisol was measured in serum samples taken pre-trilostane (peak), 3 hours post-trilostane (trough) and 1 hour post-acth injection. dogs that had an increase in cortisol after trilostane administration were excluded. a scoring system was developed for each of these 3 measurements. a fourth scoring system was developed using a novel algorithm that combined the peak and trough cortisol (peak-trough). the results of each of the 4 scoring systems categorised the dogs into those that would be expected to be over-controlled, well-controlled, moderately-controlled and poorly-controlled. weighted kappa was calculated to assess the agreement between the categorisation according to each of the 4 methods compared to the categorisation using the owners score. the pearson correlation coefficient was calculated to assess relationships between the various parameters. in total 31 tests were analysed. when compared to the results of the owner's questionnaire 16, 13, 9 and 5 dogs were correctly categorised using the peak-trough, peak alone, post-acth and trough alone respectively. amongst the miscategorised results 12, 17, 16 and 15 dogs were incorrect by 1 category and 3, 1, 6 and 6 dogs by 2 categories using the peak-trough, peak alone, post-acth and trough alone respectively. all methods correctly recognised the over-controlled dog that had been identified by the owner's score. the weighted kappas for post-acth and trough cortisol categories compared to the owner score categories were 0.09 and 0.11 respectively (defined as slight agreement). in contrast the weighted kappas for the peak and peak-trough categories were 0.36 and 0.34 respectively (defined as fair agreement). there were no significant correlations between the absolute clinical scores and cortisol concentrations. there were significant correlations between the 3 cortisol measurements. the novel methods of peak-trough and peak cortisol better reflected the level of clinical control of hyperadrenocorticism identified by the owners' questionnaire than either post-acth stimulation or trough cortisol. peak-trough and peak cortisol concentrations should be further investigated as monitoring methods for trilostane. financial support from dechra pharmaceuticals. the prognosis of canine adrenocortical insufficiency is generally regarded to be excellent. however, there is paucity of sur-vival analyses in the literature. the aim of the present study was to evaluate the survival of dogs with the diagnosis adrenocortical insufficiency based on data from a cohort of 525, 028 swedish client-owned dogs insured in one insurance company (agria pet insurance, stockholm, sweden) during the time period 1995-2006. dogs were identified by search for insurance claims with the register code for adrenocortical insufficiency. dogs were excluded from analysis if they had a previous history of hypercortisolism, and if they were born before begin of the study period. kaplan-meier survival analysis was performed. dogs were regarded as censored when the registered cause of death was other than adrenocortical insufficiency or hypercortisolism that was registered after the first claim for adrenocortical insufficiency. data from 297 dogs was included. one hundred twenty-four dogs were registered to be dead. in 81 dogs the cause of death was related to the adrenocortical insufficiency. the 1-year estimated survival-rate was 81% (95% ci, 76-86%). the 3-year estimated survival-rate was 69% (95% ci, 63-76%). the 5-year estimated survival-rate was 59% (95% ci, 51-68%). twelve dogs (4.0%) were still alive after 7 years. in conclusion, the long-term survival of dogs with adrenocortical insufficiency was reasonably good. however, the diseases-related mortality was higher than expected, and occurred mainly during the first years after diagnosis. conflicts of interest: this study was supported by grants from the swedish research council and the foundation for research, agria insurance company. the concomitant occurrence of two or more endocrine tumors and/or hyperplasias, known as multiple endocrine neoplasia (men) is a well-known entity in humans. multiple gene mutations have been identified. the two major forms are men1 and men2. in men1, the main affected organs are parathyroid, pancreas and pituitary gland. men2 occurs in 3 clinical variants: men2a, characterized by medullary thyroid carcinoma (mtc), pheochromocytoma and primary hyperparathyroidism; men 2b, characterized by mtc, pheochromocytoma and additionally abnormalities; familial medullary thyroid carcinoma. in dogs and cats only a few cases have been reported and it is unknown whether hereditary men-like syndromes exist in these species. the aim of this study was to evaluate the prevalence of multiple endocrine tumors in dogs and cats at our institution, to identify possible breed and sex predispositions and to investigate similarities with the human men syndromes. autopsy reports of dogs and cats from 2004 until 2014 were reviewed. animals with at least two endocrine tumors/hyperplasias (eth) were included. autopsy reports of 951 dogs and 1155 cats were examined. 149 dogs had eth affecting a single organ, 24 had multiple eth; 123 cats had single eth, 21 had multiple eth. in dogs with multiple eth, the most common breeds were west highland white terrier (whwt, 3/24), poodle, golden retriever, mixed-breed dogs (each 2/24). 14/24 were male (13 intact); 10/24 were female (10 neutered). median age was 12 years (range 7-18). the most common combination was multiple testicle tumors of various types (4/24). the most common affected organs were the adrenals (18/24). adrenal cortical adenomas/carcinomas/hyperplasias were mainly associated with pheochromocytomas (3/ 24), testicle tumors (3/24) and insulinomas (2/24). all 3 whwts had adrenal adenomas. both poodles had pheochromocytoma associated with pituitary adenoma or adrenal hyperplasia. 2 dogs showed tumor combinations similar to the human men1 syndrome: pituitary adenoma and insulinoma; pituitary adenoma and parathyroid hyperplasia. 19/21 cats were domestic short/long hair, 2/21 were persians. 11/21 were male (7 castrated); 10/21 were female (9 neutered). the median age was 15.5 years (range 10-19). the most common affected organs were thyroid glands (18/21), combined mostly with lesions of parathyroid (10/21) and adrenal glands (7/21). none of the cats had combinations similar to the human men syndromes. the prevalence of multiple eth in dogs and cats was 2.5% and 1.8%. men-like syndromes were extremely rare in dogs and non-existing in cats. no sex predisposition was observed. possible breed predispositions need further investigations. no conflicts of interest reported. canine angiostrongylosis is an increasingly reported disease worldwilde, including many european countries, possibly due to climatic factors, presence of foxes (acting as reservoir) or more simply, to the availability of more accurate diagnostic methods. although detection of the first-stage larvae (l1) using the baermann technique on faecal samples (preferably collected over three consecutive days) remains the gold standard, recently developed serological and molecular tests (quantitative polymerase chain reaction, qpcr) are now available. until now, the prevalence of canine angiostrongylosis among healthy and coughing dogs in belgium was unknown. the aims of the present study were (1) to describe a clinical series of recent autochtonous cases and (2) to retrospectively assess angiostrongylus vasorum qpcr in bronchoalveolar lavage fluid (balf) samples, collected over the last 7 years from a larger series of dogs, healthy or with other respiratory conditions, in order to investigate the past prevalence of the disease in belgium. seven dogs, living in southern or eastern belgium, were recently diagnosed as having angiostrongylosis (mean age= 2.3 y, mean body weight= 15.3 kg). they all presented with respiratory signs of variable severity. in 5 dogs, balf was obtained and qpcr was positive in all of them, at moderate or high level (ct from 26,7 to 29,6) while larvae were detected in the faeces of only 2 animals. in the remaining two dogs, no balf was obtained, but coproscopy was positive. all dogs responded to medical treatment, consisting in a 3-week course of fenbendazole and/or two spot-on application of moxidectin at 1-month interval. balf samples were collected between 2008 and 2014 from 10 asymptomatic client-owned dogs and 55 dogs with various respiratory conditions, including 16 dogs with confirmed bordetellosis, 18 dogs with eosinophilic bronchopneumopathy (ebp), 8 dogs with chronic bronchitis and 13 dogs with bacterial bronchopneumonia, and were retrospectively assessed with a a. vasorum qpcr assay. amongst those 65 dogs, only one balf, from a dog with ebp, yielded a positive qpcr result. in this dog, faecal analysis was negative. the present data show that, based on balf qpcr and coproscopy, presence of angiostrongylosis in healthy and coughing dogs was negligible in belgium until the last 12 months. it is now considered as an emerging condition and must be included in the differential diagnosis in coughing dogs. the present results also support that qpcr detection of a. vasorum in balf, when available, is an adequate and reliable detection technique. no conflicts of interest reported. ligneous membranitis is a rare chronic inflammatory disease associated with congenital plasminogen deficiency. it has only been described in six unrelated dogs. the objective of this study is to report the presentation, clinicopathological and post mortem findings in three related scottish terrier puppies with ligneous membranitis. ligneous membranitis is well described in humans, where it is inherited in an autosomal recessive manner. patients commonly present as infants. ocular, oral and genital lesions are most common, but other organs are occasionally involved and congenital obstructive hydrocephalus is reported in some individuals. numerous mutations and polymorphisms in the plasminogen gene have been identified in affected individuals. the affected scottish terriers (two male and one female) presented at 2 months of age with severe proliferative and ulcerative conjunctivitis and gingivitis/stomatitis; biopsy confirmed ligneous membranitis. other clinical signs included increased upper respiratory tract noise, nasal discharge and lymphadenopathy. one male was cryptorchid. clinical pathological findings included neutrophilia, proteinuria and hypoalbuinaemia. serum plasminogen activity was measured in two dogs, and was low in one. the dam and sire of the affected dogs had normal serum plasminogen activity and no history or clinical signs consistent with ligneous membranitis. no significant clinical improvement was evident following treatment with antibiotics, glucocorticoids, topical ciclosporin or heparin. one dog died of cardiopulmonary arrest in the hospital and the two other dogs were euthanized due to progressive clinical signs. post-mortem evaluation of the affected dogs revealed multiple abnormalities including severe proliferative fibrinous lesions affecting the trachea, larynx and epicardium, and multiple fibrous adhesions throughout the thoracic and abdominal cavities. the male dog had internal hydrocephalus and lacked a cerebellar vermis. this is the first report of ligneous membranitis in related dogs and the first report in scottish terriers. sequencing the plasminogen gene in the affected dogs, their parents and unrelated control dogs to identify polymorphisms or mutations that may be associated with ligneous membranitis in dogs is ongoing. the author received a travel scholarship from zoetis to attend this congress. health screening of elderly dogs is often recommended, but scientific information on clinical and laboratory abnormalities in senior and geriatric dogs is scarce. this study was undertaken to describe blood pressure measurement, physical examination (pe) abnormalities and routine laboratory test results in senior and geriatric dogs that were apparently healthy for the owner. because life expectancy in dogs is related to body size, the inclusion of 100 dogs was based on a human/pet analogy chart to determine whether a dog was senior (n = 41) or geriatric (n = 59). to verify health status, owners were asked to complete an extensive questionnaire. systolic blood pressure (sbp) was measured using the doppler technique according to the acvim guidelines. subsequently a thorough pe was performed, including body and muscle condition scoring, orthopedic examination, neurologic evaluation, indirect fundoscopy and bilateral schirmer tear test. complete blood count, serum biochemistry and urinalysis (including urinary sediment, urinary protein:creatinine ratio (upc) and bacterial culture) were evaluated. in 53 of 100 dogs sbp exceeded 160 mmhg, none of the dogs had fundoscopic lesions secondary to hypertension. body condition score was abnormal in 41 animals, 39 were overweight or obese. physical examination revealed a heart murmur in 22, submandibular lymphadenopathy in 13, moderate to severe dental plaque in 51 and one or more (sub)cutaneous masses in 56 dogs. twenty-three dogs were leukopenic, 29 had a decreased phosphorus, 32 an increased serum creatinine and one dog a decreased total thyroxine (with concurrent increased thyroid stimulating hormone). crystalluria was commonly detected (62/96) and mostly due to low numbers (<1/high power field) of amorphous crystals (82%). struvite crystals were present in 18% of the crystalluric dogs. overt and borderline proteinuria were detected in 13 and 18 of 98 dogs, respectively. four dogs had a positive urinary culture. sbp was not significantly different between the senior and geriatric group. there was no significant effect of obesity or gender on sbp. the platelet count (p = 0.014), total thyroxine concentration (p = 0.008) and the frequency of orthopedic problems (p = 0.007) and cutaneous masses (p = 0.002) were significantly higher in the geriatric compared to the senior dogs. hematocrit (p = 0.007) and body temperature (p = 0.044) were significantly lower in the geriatric group. these findings indicate that physical and laboratory abnormalities are common in apparently healthy senior and geriatric dogs. this underlines the necessity for regular health screening in elderly dogs and the urgent need for reliable and maybe age specific reference intervals in veterinary medicine. the cost of examinations reported in this study were covered by hill's pet nutrition belgium. systemic lupus erythematosus, sle, is a chronic autoimmune disorder with varying clinical manifestations and diagnosis is based on both clinical signs and laboratory findings. other systemic rheumatic diseases, referred to as sle-related diseases or immune-mediated rheumatic disease (imrd), are also described. the most common clinical signs in dogs are stiffness and pain from varying joints. one hallmark of sle and sle-related diseases in both dogs and humans is high titres of circulating antinuclear antibodies (ana), which can be demonstrated by the indirect immunofluorescence (iif) ana test. earlier studies have shown that canine iif ana positive samples may be divided into two main subgroups: homogenous (ana h ) and speckled (ana s ) iif ana fluorescence pattern. in humans, further determination of the specificity of ana positive sera is frequently employed to characterize the ana reactivity. some of these ana specificities have been demonstrated in man to strongly associate with different systemic autoimmune diseases and also with different iif ana staining patterns. presence and character of antinuclear antibodies in canine sle-related diseases are not well described. the aim of this work was to further characterize the ana specificity in dogs with sle-related disease/imrd. sera from 208 anapositive dogs, including 61 different breeds, were analyzed with elisa and line blot techniques (elisa and euroline ana profile, euroimmun, germany). the five most prevalent breeds were german shepherd dog, nova scotia duck tolling retriever, cocker spaniel, crossbreed and golden retriever. 68 sera displayed a homogenous and 140 a speckled iif ana fluorescence pattern. several specific ana-reactivities earlier characterized in human patients were identified. the majority of ana h , n = 41, 60%, showed reactivity against nucleosomal antigens and 9 (13%) against dsdna when conducted on line blot. these sera also reacted against nucleosomes and dsdna on the elisa. there were some additional positive with the elisa, so in total the elisa identified 72% with nucleosomal and 24% with dsdna reactivity. in few cases, other reactivities identified were against histones, pcna, jo-1 and rnp. in the ana s subgroup, the sm+rnp antigen evoked the most frequent reactivity, n = 30, 21% with both line blot and elisa. in few cases, reactivity against dsdna, pcna, jo-1, pmscl100kd, scl-70, ssa and ssb were identified. in several dogs no specific antigen was identified. further studies are in progress in order to in more detail characterize and identify subtypes of already known and unknown antigens with clinical importance in canine autoimmunity. one of the authors, erik lattwein, is employed by euroimmun where the analyses were performed. chronic kidney disease (ckd) has a high prevalence in cats. routine renal markers, serum creatinine (scr) and urea are not sensitive or specific enough to detect early ckd. serum cystatin c (scysc) has advantages over scr for the detection of early kidney dysfunction, both in humans and dogs. a significant higher scysc concentration in ckd cats has been demonstrated. the objective of this study was to determine the effect of age, gender and breed on feline scysc and to establish a reference interval for feline scysc. in total, 132 healthy cats between one and 16 years were included. serum cysc was determined with a validated particleenhanced nephelometric immunoassay (penia). serum cr, urea, urine specific gravity (usg), urinary protein: creatinine ratio (upc) and systolic blood pressure (sbp) were also measured. to test for difference between the groups, the f-test was used. the lower and upper value of the 95% reference interval were obtained as the 2.5% and 97.5% quantiles of the scysc observations. no significant differences in scysc concentration were observed between young, middle-aged and old cats; between female, female neutered, male and male neutered cats; and between purebred and domestic short-or longhaired cats. the 95% reference interval for feline scysc was determined as [0.58-1.95 mg/l]. there was a significant difference in scr concentration between domestic short-or longhaired cats and purebred cats. the sbp was significantly influenced by gender as well as age, while urea was influenced by both age, gender and breed. this study showed that the biological factors age, gender and breed have little or no impact on feline scysc, in contrast to scr and serum urea, making it an interesting marker. therefore, further studies are warranted to evaluate the diagnostic value of scysc as a renal marker in cats. this study recieved support from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l. ghys. assessment of renal function is often needed, however existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. in this pilot study the feasibility of a transcutaneous glomerular filtration rate measurement was investigated. the transcutaneous disappearance rate (expressed as half-life) of fluorescein-isothiocyanatelabelled sinistrin (fitc-s) was measured in three healthy research dogs and three healthy research cats. plasma clearance of sinistrin (7 data points) was performed in both species as previously described (res vet sci 1998; 64:151-6 and j fel med surg 2003; 5:175-81) and half-life was calculated using a 2-compartment model with a freely available pharmacokinetic calculator (comput meth prog bio 2010; 99:306-14) . renal elimination of fitc-s was measured transcutaneously for 4 hours (7000-8000 data points) using a miniaturized device as described previously for the same purpose in rats (kidney int 2011 79:1254-8). the procedures were performed in awake, freely moving animals using escalating doses of fitc-s (10 mg/kg, 30 mg/kg, 50 mg/kg) with a wash-out period of at least 24 h in each animal. to find the best position for the device, multiple devices were placed on each animal. the resulting fitc-s disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. in both species 30 mg/kg were adequate for kinetic calculation. the most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. transcutaneous fitc-s clearance was then repeated using the optimal dose and location and in parallel with the plasma sinistrin clearance. plasma sinistrin clearances [ml/kg/min] were 5.5, 5.0 and 3.8 in the three dogs, respectively. corresponding plasma elimination half-lives [min] were 26, 31 and 35, and corresponding transcutaneous elimination half-lives [min] were 26, 34 and 55, respectively. plasma sinistrin clearances [ml/kg/min] were 2.8, 2.2 and 1.9 in the three cats, respectively. corresponding plasma elimination half-lives [min] were 51, 60 and 61, and corresponding transcutaneous elimination half-lives [min] were 75, 96 and 83, respectively. in conclusion, transcutaneous fitc-s clearance is a feasible method for assessment of gfr in awake dogs and cats. it is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. a dose of 30 mg/kg of fitc-s seems adequate for kinetic assessment. further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions. conflicts of interest: zhp and sg are supported by the ec fp7 marie-curie programme: nephrotools. the device development was supported by the fp7 activity: place-it.ng is owner of a patent covering fitc-sinistrin and the technology for its measurement. excretion of urinary biomarkers of renal damage should occur at an early stage of chronic kidney disease (ckd), thus facilitating earlier diagnosis of renal disease. albumin and cystatin c in the renal ultrafiltrate are mostly reabsorbed by the proximal tubular cells, therefore increased urinary excretion of albumin and cystatin c (uac and ucysc) would be expected to correlate with the presence of renal tubular damage and ckd. the aim of this study was to establish biological validity of two particle enhanced turbidimetric assays (petias) for the measurement of albumin and cystatin c (previously validated for use in feline urine) by comparing the uac and ucysc between non-azotaemic cats and cats with azotaemic ckd. blood and urine samples were obtained from cats at three uk first opinion practices as part of a geriatric screening programme. haematology, serum biochemistry (including total thyroxine concentration (tt4)) and urinalysis (including urine protein:creati-nine ratio (upc)) were performed. dental disease score (calculus and gingivitis) and body condition score (bcs) were recorded. cats with tt4 > 40 nmol/l, evidence of pyuria or bacteruria, or significant systemic disease were excluded. uac and ucysc were determined in non-azotaemic cats (n = 50) and cats with azotaemic ckd (n = 10, defined as a serum creatinine concentration >153 lmol/l and concurrent urine specific gravity <1.035). comparisons between the non-azotaemic and azotaemic ckd groups were made using the mann whitney u test. correlations were assessed by spearman's correlation coefficient. data are presented as median [25 th , 75 th percentile] and statistical significance was defined as p < 0.05. uac was significantly higher in the azotaemic group than the non-azotaemic group (31.5 [9.8, 97 .6]x10 -3 vs. 11.6 [6.4, 19.0]x10 -3 ; p = 0.034), whereas upc was not significantly different between the groups (p = 0.619). unexpectedly, ucysc tended to be lower in azotaemic cats than non-azotaemic cats (3.2 [1.5, 9.1]x10 -6 vs. 11.8 [4.4, 23.2]x10 -6 ; p = 0.071). uac was weakly positively correlated with serum urea concentration (r s =0.327, p = 0.011), but was not correlated with serum creatinine concentration. ucysc was not significantly correlated with serum concentrations of urea, creatinine or tt4. uac was also weakly negatively correlated with dental calculus score (r s = -0.307; p = 0.019) and bcs (r s = -0.438; p = 0.001). uac appears to be a more sensitive test for azotaemic ckd than upc, however the apparent low specificity may limit the utility of uac as a urinary screening test for ckd. increased ucysc (determined by petia) would not appear to be a marker of azotaemic ckd in cats. no conflicts of interest reported. cystinuria is an inherited metabolic disorder that causes defective tubular reabsorption of the aminoacids cystine, ornithine, lysine and arginine (cola). the low solubility of cystine in acidic urine promotes formation of cystine crystals and uroliths in the urinary tract resulting in the clinical signs of stranguria, urinary obstruction and renal failure in affected individuals. cystinuria occurs in >70 breeds of dog and has been classified into types ia (newfoundland, landseer, labrador), iia (australian cattle dog), ib (miniature pinscher) and iii (androgen-dependent; e.g. mastiff, irish terrier). the kromfohrl€ ander is a medium-sized companion dog, bred initially as a cross between a wire fox terrier and a grand griffon vend een, first recognised internationally in1955. cystinuria has been suspected in this breed but no cases have been reported in the literature to date. we determined urinary cola concentrations in 81 adult kro-mfohrl€ ander dogs aged 1-10 years comprising 48 intact and 6 castrated males, and 26 intact and 1 spayed females. a total of 15 (31%) intact males aged 1.5 to 8.5 years had cola values >700 lmol/g creatinine and several developed cystic calculi. furthermore, 9 intact male dogs had increased cola but normal cystine levels. all castrated males had normal cola concentrations. no females had increased cola and cystine concentrations or formed any cystine calculi. we conclude that cystinuria with cystine calculi occurs frequently in adult intact male kromfohrl€ ander dogs but neither is seen in females. this appears to be an androgen dependent type iii cystinuria, as seen in mastiff-type dogs and irish terriers. thus, castration may resolve the increased urinary cola excretion and risk for cystine calculi formation and obstruction. the precise mode of inheritance is still unclear. all adult intact male kromfohrl€ ander dogs should be screened by urinary cola testing. work carried out at the author's previous place of employment (university children's hospital, frankfurt, germany). acs, rk, em, md and ug provide a diagnostic service for cystinuria and other inborn errors of metabolism in companion animals. ureteral urolithiasis is an emerging medical concern in cats. there are few reports on epidemiology, diagnosis or medical management of ureteral calculi in cats, particularly in europe. cats diagnosed with ureteral urolithiasis in the teaching hospital of the veterinary school of alfort from 2005 to 2013 were included in this study. diagnosis was confirmed with radiographs, ultrasound scan and/or laparotomy. signalment, clinical signs, clinicopathologic and diagnostic imaging findings, medical treatment and outcome were recorded. epidemiological data were compared to a reference population of 7600 cats. eighty three cats were included in the study. the occurrence of ureteral urolithiasis was significantly higher in birman to the author knowledge, it is the first time that a higher prevalence of ureteral calculi in birman cats is reported in europe. spontaneous elimination of calculus is associated with a small size (<1.5 mm). if the size of calculi tends to be bigger in cats with no improvement of renal function after medical treatment, prospective studies are still needed to determine the best medical treatment. no conflicts of interest reported. feline immunodeficiency virus (fiv) infection has been associated with kidney disease, mainly characterised by an increased prevalence of proteinuria in fiv-infected cats. however, studies evaluating renal variables in fiv-positive cats are scarce. recently, a higher systolic blood pressure (sbp) was reported in a small number of fiv-infected cats. hypertension is an important cause of proteinuria and a frequent cause of renal disease in human immunodeficiency virus (hiv) positive patients. therefore, our main objective was to describe sbp in clinically ill fivpositive cats. secondly we aimed to evaluate routine renal variables in this population. naturally infected clinically ill fiv-positive cats were prospectively included. the doppler ultrasonic technique was used to measure sbp according to acvim guidelines. serum creatinine (screat) and urea (surea) concentrations, urine specific gravity (usg) and urinary protein:creatinine ratio (upc) were determined. the study included 99 cats, with a mean age of 8.0 ae 3.7 years and a mean body condition score of 4.6 ae 1.5 on a nine-point scale. the sbp ranged from 91 to 170 mmhg, with a mean of 116 ae 18 mmhg. only two cats were hypertensive (sbp > 160 mmhg). both had isosthenuric urine, were borderline proteinuric (upc 0.2-0.4) and one of them was mildly azotemic. mean screat was 101.7 ae 64.2 lmol/l (reference interval (ri) 44.2-141.4 lmol/l) and mean surea concentration 8.6 ae 5.2 mmol/l (ri 6.2-10.8 mmol/l). thirteen cats showed increased screat levels, with decreased usg (< 1.035) in eight, proteinuria (upc > 0.4) in seven and increased surea concentrations in ten of them. five out of ten azotemic cats were proteinuric with a decreased usg. mean upc was 0.26 ae 1.06, with a wide range from 0.05 to 9.38. borderline proteinuria was present in 40/99 (40.4%) and proteinuria in 28/99 (28.3%). half of the proteinuric cats had a decreased usg. mean usg was 1.040 ae 0.013. one third of all cats had a decreased usg, with isosthenuria in seven of them. these results demonstrate that proteinuria and poorly concentrated urine are common in naturally infected clinically ill fivpositive cats, confirming previous reports in cats and humans. however, longitudinal studies of (borderline) proteinuric patients are needed to elucidate the clinical relevance. the low number of hypertensive patients and low mean sbp in our study indicate that hypertension is uncommon and unlikely to be the cause of renal damage in clinically ill fiv-infected cats. aratana therapeutics nv financially supports a clinical trial on the use of antivirals in fiv cats at our university. screeninng examinations repoorted in this trial were part of the required pre-trial investigations for that study. the presenting author is also funded by a scholarship from aratana therapeutics av. the objective of this study was to identify the prevalence of bacterial species and the susceptibility of isolates to doxycycline, trimethoprim-sulfamethoxazole (tms), ampicillin, amoxicillinclavulanic acid (amc), cephalothin, and enrofloxacin in cats with urinary tract infections (uti) with and without predisposing comorbidities. a retrospective analysis of case records between 2000 and 2009 was performed and resulted in inclusion 271 cats into the study: 128 cats with a systemic comorbidity, 36 cats with indwelling urinary catheters, 40 cats with other local comorbidities, and 67 cats with no comorbidity. the most commonly isolated bacteria were escherichia coli (e. coli), streptococcus species (spp.), staphylococcus spp., and enterococcus spp.. the proportion of gram-negative isolates was significantly higher in the cats with systemic comorbidities than in cats with indwelling urinary catheters (p < 0.001) and cats with other local abnormalities (p < 0.001), whereas gram-positive isolates were significantly more commonly isolated from cats with indwelling urinary catheters and other local comorbidities than in cats with systemic comorbidities (p < 0.001). the proportions of isolates susceptible to amc, enrofloxacin, and tms and the antimicrobial impact factors (if) were higher than the proportions of isolates susceptible to doxycycline, ampicillin, and cephalothin and the respective if. based on these findings, amc and tms would be the firstchoice antimicrobial agents for empiric treatment of bacterial the increasing rates of resistance exhibited by uropathogens represent a serious problem for the selection of an appropriate antibiotic. the aim of this study was to determine secular trends of companion animal urinary tract infection (uti) that involve extended-spectrum b-lactamase (esbl)-and carbapemenase-producing gram negative bacteria (namely, escherichia coli, klebsiella pneumoniae, proteus mirabilis, acinetobacter baumannii), methicillin-resistant-staphylococci (mrs) and ampicillin and high-level-gentamicin-resistance (hlgr) enterococci. nine hundred and twenty two uropathogenic bacteria were isolated from dogs and cats, between january 1999 and march 2014, at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. isolates were identified using standard commercial systems. susceptibility testing was performed using the disk diffusion and broth microdilution methods. clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) production was screened by double-disk synergy test. the esbl, plasmid-mediated ampc, carbapemenases, meca and aac(6')-ieaph(2'')-ia genes were detected by pcr and gene enzymes were sequenced. among enterobacteriaceae 0.7% were dhaproducers, 2.7% were esbl-producers and 3.6% were cmyproducers. all isolates were also multidrug-resistant. cefalosporinases-producer enterobacteriaceae were detected in 2000, the first being a cmy-2-producer e. coli. all the esbl-producers were e. coli or k. pneumoniae producing ctx-m-group 1 enzymes. ampicillin-resistance in enterococci was present throughout the years (15,4%, n = 8). hlgr appeared in enterococci in 2003 and was confirmed by the detection of the bifunctional enzyme that confers high level resistance to aminoglycosides (7 out of 8 isolates). in this study we showed that in the last decade the emergence of resistance to critically important antimicrobials among uropathogens from companion animals is a concerning fact. the multidrug-resistant enterobacteriaceae may compromise effective therapeutic options, namely third and fourth generation cephalosporins, fluoroquinolones, trimethoprim/sulpha combinations. the emergence of mrsa/mrsp and hlgr among uropathogens is also a therapeutic challenge. the detection of uropathogens with antimicrobial resistance is not only an animal health issue but also a matter of public health, since companion animals may act as reservoirs of antimicrobial resistant bacteria or resistance genes for humans. the author currently receives a phd grant funded by the portuguese foundation for science and technology. in the past, the author received once research support and honoraries from portuguese merial for a project on canine vector borne diseases. the aim of the present study was to use insurance data to estimate morbidity and mortality related to kidney disease in the swedish dog population. insurance company data from veterinary care-insured and lifeinsured dogs during the years 1995-2007 were studied retrospectively. incidence-and mortality rates were calculated for the whole group of dogs as well as divided by sex and breed. for the 15 breeds with the highest incidence-and mortality rates, respectively, the reasons for kidney disease were investigated by dividing the diagnoses into 10 ethiology groups. the total number of veterinary care-insured dogs was 665,245. the total incidence rate of kidney disease in this group of dogs was 15.7(15.3-16.2) cases/10,000 dog-years at risk. the number of dogs in the life insurance was 548,346 and in this group the total kidney-related mortality rate was 9.7(9.3-10.2) deaths/10,000 dogyears at risk. the 2 most commonly reported ethiologies of kidney disease were "ethiology not determined"and "infectious/ inflammatory". the 3 breeds with the highest incidence rate of kidney disease were the bernese mountain dog, miniature schnauzer and boxer. the 3 breeds with the highest mortality caused by kidney disease were the bernese mountain dog, shetland sheepdog and flatcoated retriever. in conclusion, the epidemiological information provided in this study concerning kidney disease in dogs can assist clinicians in establishing diagnoses, and can assist breeders in defining priorities for preventative measures. it can also provide valuable information for future research. the senior author has received money from the insurance company we have used data from to write our study, for another project. jens h€ aggstr€ om and ingrid ljungvall have received financial support for research from sante animale, agria insurance ltd, sveland insurance ltd, forsgren research foundation. both of these authors have also undertaken paid consulatcny work for boehringer-ingelheim, ceva sante animale. mast cell tumours represent the most common cutaneous tumour in the dog. diagnosis of a mast cell tumour can be achieved through cytological examination of fine needle aspirate. however the grade of the tumour is an important prognostic marker and requires so far histologic assessment. a 2-tier histologic grading system based on number of mitoses, multinucleated cells, bizarre nuclei and karyomegaly was recently proposed by kiupel et al. the aim of this study was to assess if the cytomorphological criteria proposed in the 2-tier histologic grading system are applicable on cytology specimens. ninety-three mast cell tumour specimens of grade i or grade iii according to patnaik with both histological specimens and fine needle aspirates were retrospectively taken from a data set and histologically and cytologically re-evaluated. according to the kiupel grading system thirty-six were diagnosed histologically as high grades and fifty-seven were considered low-grade mast cell tumours. the cytologic examination of the corresponding specimens revealed thirty-one high grade and fifty-five low-grade tumours. an agreement between histologic and cytologic diagnosis based on the kiupel grading system was achieved in eighty-six cases (accuracy 92.4%, specificity 93.9%, sensitivity 91.6%). five high-grade tumours (13.8%) were considered as low grade on cytology. cytologic grading of mast cell tumours in the dog has satisfactory accuracy, sensitivity, and specificity. histologic grading of canine mast cell tumours still remains the gold standard, but cytology already gives reliable information. no conflicts of interest reported. in canines mastocytomas are among the most frequently diagnosed neoplasms of the skin. high grade mastocytomas (grade iii, patnaik classification) are characterized by an uncontrolled growth of neoplastic mast cells (mc) and a poor prognosis. recently, the kit-targeting tyrosine kinase inhibitors masitinib and toceranib have been approved for the treatment of canine mc tumors. these drugs are able to induce responses in mastocytoma patients. however, in many patients, relapses are seen. therefore, research is focusing on new drug targets. recently, the transcription factor stat5 has been reported to play an important role in the proliferation and survival of human neoplastic mc. the aim of the present study was to evaluate the jak2-stat5 pathway in canine mastocytomas. to address this aim, the canine mastocytoma cell lines c2 and ni-1 as well as inhibitors directed against jak2 or stat5 were employed. as assessed by immunocytochemistry, c2 cells and ni-1 cells were found to express pstat5 in their cytoplasm and nuclei. intracellular expression of pstat5 was confirmed by flow cytometry. interestingly, c2 cells were found to express higher levels of pstat5 compared to ni-1 cells. next, we treated c2 cells and ni-1 cells with various concentrations of the stat5 inhibitors piceatannol and pimozide and the jak2 inhibitors azd1480 and tg101348. as assessed by 3 h-thymidine uptake, all 4 compounds were found to inhibit the proliferation of canine mc in a dose-dependent manner. drug effects were found to vary in different cell lines, with the following rank-order of potency (ic 50 values): tg101348: 0.1-0.75 lm; pimozide: 0.1-2.5 lm; azd1480: 1-2 lm; piceatannol: 5-50 lm. to further explore the mechanism of drug-induced inhibition of proliferation, we examined cell cycle progression and apoptosis in drug-exposed cells. whereas all 4 drugs tested induced only moderate cell cycle arrests in the g1 phase, these drugs were found to induce substantial apoptosis in c2 cells and ni-1 cells as evidenced by microscopy and annexin-v/pi staining. together, our data show that jak2-and stat5-targeting drugs exert anti-proliferative and apoptosis-inducing effects in canine mastocytoma cells suggesting that this signaling pathway may be a promising new therapeutic target in canine mastocytomas. the clinical relevance of this observation remains to be determined. no conflicts of interest reported. subcutaneous mast cell tumours (sqmct) in dogs are relatively uncommon compared to their cutaneous counterparts. the veterinary literature describes these tumours as a specific pathological entity with, in general, a low probability of aggressive progression. surgery is considered the main treatment modality, while medical treatment has not been described. the purpose of this study was to determine progression free survival (pfs) for a chemo na€ ıve cohort of dogs presented with non-resectable and/ or metastasized sqmct, which all underwent masitinib-based therapy. data were collected for patients with sqmct presented to participating centres in the netherlands and the uk from 01/12/ 2008 to 01/01/2014, which received masitinib-based therapy. treatment protocols employed, included masitinib alone (m), masitinib and prednisolone (mp), masitinib plus vinblastine and/ or lomustine and prednisolone (mpc). response to therapy was measured conforming to recist 1.1. adverse events were graded using vcog-ctcae 1.1. patients were grouped according to presence or absence of metastasis, treatment protocol used, previous surgery, and remission status achieved; simple comparisons were made to evaluate possible significance. twenty-five cases were identified. 18/25 were female. median age of occurrence was 9 years (2-17). diagnosis was made by histology in 11/25; proliferation indices were defined in only 4 dogs. fourteen cases exhibited metastasis at initiation of therapy. pfs for all cases ranged from 8-1275 days. median/mean pfs (days) according to treatment was m:141/317d (n = 15), mp:142/389d (n = 7), mpc:49/57d (n = 3). median/mean pfs according to metastasis status was m0:141/324d (n = 11) and m1:103/291d (n = 14). dogs who underwent previous surgery (n = 10) had a median/mean pfs of 108/344d compared to those who had no surgery 119/280d. looking at remission status, median pfs of patients who achieved a complete remission was not reached, with a mean pfs of 617d (n = 9). median/mean pfs of patients with partial remission was 86/176d (n = 11), stable disease 49/57d (n = 2), and progressive disease 12/17d (n = 3). 19/25 cases experienced suspected adverse events. three dogs, two of which ultimately died, had seven grade 3-5 adverse events (anaemia (n = 3), hepatotoxicity (n = 3), gastrointestinal toxicity (n = 1)). masitinib-based treatment is effective in the management of sqmct perceived to be aggressive. patients do not appear to benefit from prior surgery. metastatic status did not influence outcome. adding chemotherapy negatively influenced pfs. complete remission is a very favourable prognostic development. the authors have received financial support from ab science to help with the costs of statistical analyses in an unrelated project advances in distinction between morphological subtypes of canine non-hodgkin's lymphomas (nhl) have provided a better understanding of this cancer in dogs. diffuse large b-cell lymphomas (dlbcl) are the most frequent form of nhl in dogs including some distinguished morphological subtypes (mainly centroblastic polymorphic and immunoblastic) according to the who classification. few clinical studies reported dlbcl clinical outcomes under treatment while survival times of the centroblastic polymorphic subgroup were reported. the aim of this retrospective study was to evaluate the response of dlbcl to a standardized multi-agent chemotherapy protocol. medical records from dogs with a diagnosis of dlbcl between 2003 and 2013 were retrospectively reviewed. inclusion criteria were the availability of complete initial and follow-up information and the application of a standardized multi-agent l-cop chemotherapy protocol as previously described. dogs which received corticosteroids before the initiation of treatment and dogs which died for other reasons than their related disease before the end of the induction period (35 d) were excluded. response to chemotherapy was evaluated every week during the induction of treatment, then every 3 to 5 weeks. statistical analysis was performed using kaplan-meier analysis. thirty cases of dlbcl meeting all inclusion criteria were included from the initial population. seven dogs were in clinical stage iii according to the who classification, 19 in stage iv and 4 in stage v. nineteen dogs were in substage a, and 11 in substage b. on 26 dogs which have an adequate response evaluation, 20 dogs (76.9%) achieved a complete remission. the median and mean duration of first remission were 175d and 239d, respectively (range 68-725 d). the median and mean durations of survival time were respectively 311d and 319d (range 55-818 d). one year and 2-years survival rates were 32% and 3% respectively. according to the statistical analysis, neither clinical stage (p = 0.726) nor substage at presentation (p = 0.83) or morphological subtype (immunoblastic vs centroblastic polymorphic, p = 0.216) were considered as a significant prognostic factor regarding the duration of the first remission and the overall survival time. a complete response was significantly associated with longer survival times (p = 0.019). to conclude, the dlbcl displayed a good clinical response to l-cop protocol, with a median survival time of 311 days. the only significant prognostic criterion identified was a complete clinical response to the treatment. a prospective controlled study on a larger population is warranted to confirm these results. no conflicts of interest reported. canine histiocytic sarcoma (hs) is an aggressive round cell neoplasm with a poor prognosis. both lomustine and doxorubicin have been evaluated as first line chemotherapy agents with response rates of up to 46% and median survival times around 3-5 months. the aim of this study was to evaluate the response to epirubicin in a population of dogs with hs pre-treated with lomustine. medical records of dogs with a diagnosis of hs that were treated with lomustine and subsequently epirubicin were retrospectively evaluated. fifteen dogs received lomustine followed by epirubicin. there was a measureable response to lomustine in seven of 12 dogs with evident disease, 58% (3 cr & 4 pr). an additional 3 dogs achieved stable disease for an overall biological effective response of 83%. median ttp following lomustine could be assessed in 12 dogs and was 87 days (range 34-141). all fifteen dogs received epirubicin as a rescue agent: nine following progressive disease and three with stable disease on lomustine. one dog received epirubicin after completing six doses of lomustine in complete remission and two dogs in partial remission changed to epirubicin due to hepatotoxicity associated with lomustine. response rate to epirubicin was 15% and biologic effective response was 61% (1 cr, 1 pr, 6 sd, 5 pd) in 13 dogs. one dog was euthanized due to epirubicin associated gastro-intestinal toxicity and 1 dog stopped treatment with no assessment of response. median duration of response to epirubicin was 69 days (range: 40-347) and 1 dog is still alive and in remission (405 days). overall median survival time for dogs receiving epirubicin following lomustine was 218 days (range: 27-500). single agent epirubicin is a valid rescue therapy after lomustine for canine histiocytic sarcoma and results in modestly improved overall survival times in responding patients. the author received a travel scholarship from zoetis to attend this congress. the incidence of melanocytic lesions is increasing among canine population. canine malignant melanoma could have an aggressive behavior, metastasize early in the course of the disease and be resistant to most current therapeutic regimens leading to the need of finding markers with potential as therapeutic targets. the overexpression of cox-2 seems to play a key role in melanocytic tumours, having being described an association between high cox-2 immunoexpression and the malignant behavior. in order to contribute to the understanding of the role of cox-2 in melanocytic tumours, three main pathways were investigated: angiogenesis, tumour cell proliferation and inflammatory microenvironment (t-lymphocytes and macrophages). fifty one (51) melanocytic tumours [32 cutaneous (12 malignant melanomas and 20 melanocytomas) and 19 oral malignant melanomas] were included. all the samples were submitted to immunohistochemical staining carried out by the streptavidinbiotin-peroxidase method, with a commercial detection system with or without melanin blanching, for detection of the following markers (cox-2, ki-67, factor viii, vegf, cd3 and mac387). in melanocytic tumours (n = 51), both cox-2 labelling extension and intensity revealed a statistically significant association with angiogenesis by factor viii (p < 0,01), vegf (p < 0,01); ki-67 (p < 0,001), cd3 + t-lymphocytes (p < 0,001) and mac387 (p < 0,001). considering only malignant melanomas (n = 31 cases), cox-2 labelling extension revealed a statistically significant association with angiogenesis (p = 0,041) and cd3 + t-lymphocytes (p = 0,005). cox-2 intensity was also positively associated with angiogenesis (p = 0,008) and with mac387 (p = 0,015). present study demonstrated a link between high cox-2 immunoexpression and increased angiogenesis and tumoural t-lymphocyte and macrophage infiltration in malignant melanomas. these findings reinforce the usefulness of using selective cox-2 inhibitors as a valuable therapeutic tool in malignant melanocytic tumours. this study received financial support from a company (merial). neuter status and risk of malignant neoplasia is not well evaluated in the canine population, when excluding neoplasia not normally believed to be sex-hormone dependent. denmark and the scandinavian countries have a high proportion of intact dogs compared to populations from other parts of the world. in the present study it was hypothesized that there would be no difference in gender and neuter status between the population of canine patients with a non-sex-hormone dependent malignant neoplasia reported to the danish veterinary cancer registry and a general population. from august 2005 to march 2014, 3801 canine neoplasms were reported to the danish veterinary cancer registry. the total number of malignant (1788) and benign (1953) were comparable (42% and 46%). malignant neoplasms totalled 1262, when tumors from areas of distribution with known sex-hormone dependency (reproductive organs, mammary gland, perineal), and cases with unknown area of distribution were excluded. the overall distribution of malignant neoplasia was 481 (38%) intact male dogs, 157 (12.5%) neutered male dogs, 404 (32%) intact female dogs and 220 (17.5%) neutered female dogs. the distribution was even between male and female dogs (50.5% and 49.5%). compared to a known standard population of dogs, there was an overall statistically significant association of malignant neoplasia with neuter status in both sexes. for both genders this was significant for lymphoma, mast cell tumors and osteosarcomas,. for neutered females, but not males, there was increased risk of hemangiosarcoma, squamous cell carcinoma and malignant melanoma. these findings indicate that there might be an association between neuter status and development of malignant neoplasia but larger prospective studies are needed to evaluate the risk of non-sex hormone dependent cancers in neutered dogs. no conflicts of interest reported. serum acute phase proteins (apps) are considered biomarkers of the acute phase reaction, and are being increasingly used in human and veterinary medicine in diagnosis and monitoring of neoplastic diseases. in the cat, serum amyloid a (saa) is considered a positive major app, haptoglobin (hp) a moderate app, and albumin and insulin-like growth factor-1(igf-1) negative apps. the aim of the present study was to characterize the apps response in cats with mammary tumours. for that purpose, saa, hp, igf-1 and albumin serum concentrations were determined in 20 female cats with malignant mammary tumours. cats with history of previous tumours or with concomitant tumours or other diseases were excluded. information on cats age, gender, breed, tumour type, histological grade, tumour size and location, skin ulceration, vascular neoplastic infiltration, necrosis, metastasis to regional lymph nodes, thoracic or abdominal organs, and survival time from diagnosis was assessed. blood samples were collected before surgery in all cats, and whenever possible, serial samples collected on control visits. owners gave informed consent. studied population included 20 domestic short-haired cats with ages ranging from eight to 17 years (11,5 + /-2,7). all had carcinomas, including solid carcinomas (n = 10), tubulopapillary carcinomas (n = 8), one cribiform carcinoma and one carcinosarcoma. at the time of diagnosis, 70% of cats had an increase in serum concentration of hp and 20% of saa, and 25% had a decrease in concentration of albumin. mean and standard deviation values were of 2,76 + /-0,48 g/dl for albumin (reference range 2,5-3,6 g/ dl), 319,12 + /-162,89 lg/dl for igf-1, 12,95 + /-29,76 lg/ml for saa (reference value ˂5 lg/ml), and 5,12 + /-3,20 g/l for hp (reference value ˂3 g/l). a positive correlation (r = 0,60) was detected between increases in serum concentrations of saa and hp. the increase in the size of tumour was significantly associated with the concentration of saa (p˂0,05). serum hp concentrations were significantly increased in tubulopapillary carcinomas (p˂0,05), and igf-1 decreased in solid carcinomas (p˂0,05). in the cats where serial determinations were performed, development of thoracic metastasis was significantly associated with a decrease of serum concentration of albumin (p˂0,05), and with an increase of saa (p˂0,05). this study suggests that feline mammary tumours are associated with an acute phase response. according with the results obtained, saa, hp, albumin and igf-1 might be important serum biomarkers in diagnosis and monitoring of the evolution of feline malignant mammary neoplasias. no conflicts of interest reported. histiocytic sarcoma (hs) is a neoplastic proliferation of interstitial dendritic cells or tissue macrophages. dogs with hs can present with local disease or with multifocal (disseminated) involvement. disseminated hs is poorly responsive to therapy and almost always fatal. little is established regarding the aetio-pathology of histiocytic sarcoma in dogs. the purpose of this study was to establish and characterise a hs cell line from fresh tumour samples obtained from a dog with disseminated hs in order to further clarify disease pathogenesis and behaviour. with owner consent, treatment-na€ ıve tumour sections were collected from a dog with disseminated hs that was euthanased. tumour tissue was assessed with immunohistochemistry (ihc) using antibodies against canine cd18, cd3, and pax-5 to support the diagnosis of histiocytic sarcoma. primary cell cultures (hscs), established from the tumour were cultured and maintained in modified eagle's medium with 10% fetal bovine serum, l-glutamine, penicillin and streptomycin, in standard conditions. hscs were characterised by alpha naphthyl acetate esterase (anae) and lysozyme staining while pcr was used to detect cell markers cd1a, cd11c, mhc ii, cd204, ccr2, e-cadherin, and cd4. cell surface markers were compared to an established canine hs cell line (dh82). phagocytic activity of hsc cells was assessed using cellular uptake of carboxylated fluorescent beads and documented using flow cytometry and fluorescent microscopy. tumour tissue was strongly cd18 positive and negative for cd3 and pax-5. cultured cells exhibited morphological characteristics consist with dendritic cells, such as projections and pleomorphism. hsc cells stained positively for non-specific esterase (anae) and lysozyme, and pcr indicated cells were positive for cd1a, cd11c, mhc ii and cd204 and negative for cd90 and e-cadherin. hsc cells were positive for mhc ii and ccr2 while dh82 cells were negative. phagocytic activity was evident. a novel hs cell line (hsc) was established and characterized from primary tumour tissue collected from a dog with disseminated disease. hsc cells were most consistent with interstitial dendritic cell origin based on cd1a, cd11c, and mhc ii staining as well as demonstrable phagocytic activity. hsc cells also displayed expression of ccr2, unlike the established dh82 line, supporting a notion that hs consists of a variety of subtypes. ccr2 has been linked to hs growth and metastasis, suggesting it may represent a possible therapeutic target. further studies establishing and characterising canine hs cells may contribute to the elucidation of mechanisms of tumourigenesis. no conflicts of interest reported. virulent-systemic (vs)-fcv that induce cutaneous edema, ulcerations of the head and feet, and occasionally jaundice have been described in the usa and europe. here we characterize for the first time vs-fcv outbreaks in cats in switzerland and liechtenstein. the four outbreaks occurred in three geographically separated locations: schaan (liechtenstein, shelter 1), zurich (switzerland) and lausanne (switzerland, shelter 2) between november 2011 and january 2013. pcr (fcv and feline herpesvirus-1, fhv-1), virus isolation and felv/fiv testing were performed on saliva and blood samples collected from clinically affected cats. furthermore, saliva for pcr was collected from 31 additional cats in shelter 1. phylogenetic analyses were performed based on the capsid (vp1) gene sequence of fcv. vs-fcv isolates were tested for virus neutralization with sera raised against common fcv vaccine strains. outbreak 1 occurred in a cattery in liechtenstein and involved five non-vaccinated, 3-months old siblings with fever, edema, skin and tongue ulcerations. outbreak 2 occurred in a small animal clinic in zurich. a 10-year old cat presented with severe paw edema, fever, tongue and skin ulcerations, progressive hypoproteinemia and hyperbilirubinemia. outbreaks 3 and 4 happened in a cattery in lausanne five months apart and involved two litters of non-vaccinated, 2-to 3-months old kittens. the cats presented with fever, nasal discharge, edema and skin and oral ulcerations. all affected cats tested fcv-positive but negative for fhv-1, felv and fiv, except for one kitten from outbreak 4 (fiv-positive). all cats in outbreaks 1 and 3 recovered, whereas all cats in outbreaks 2 and 4 died or were euthanized because of clinical deterioration. each outbreak was caused by a phylogenetically distinct vs-fcv strain. in shelter 1, the queen and three in contact cats remained asymptomatic although infected with the same vs-fcv strain. furthermore, 7/27 other cats of shelter 1 were infected with closely related, but distinct fcv strains. the vs-fcv isolates from the two outbreaks in shelter 2 were distinct but phylogenetically related. all vs-fcv isolates from cats from the same outbreak showed a similar virus neutralization pattern, but neutralization differed between different outbreaks. in conclusion, all vs-fcv outbreaks involved multi-cat environments. the same vs-fcv strains with similar virus neutralization patterns were isolated from cats from the same outbreak. not all cats infected with a vs-fcv strain developed disease and mortality varied between the outbreaks. the sera for virus neutralization were provided by merial, france. defined herein as presence of sneezing, nasal-and/or ocular discharge, conjunctivitis and/or keratitis), but also oral cavity lesions, chronic stomatitis, limping syndrome and, rarely, virulent systemic disease. the aims of the present study were to compare cats suspected of fcv (fcv-sc) based on clinical symptoms and healthy controls (controls) and to investigate potential risk and protective factors, such as co-infection with feline herpesvirus-1 (fhv-1), mycoplasma felis, chlamydophila felis, bordetella bronchiseptica and feline retroviruses, vaccination, gender, age, breed, housing and corticosteroid and antibiotic treatment. oropharyngeal, nasal and conjunctival swabs from 200 fcv-sc and 100 controls were collected into transport medium, processed within 96 hours after collection and analyzed for fcv by virus isolation and for all tested pathogens using molecular assays. the samples were collected by randomly selected veterinary practices in 20 different areas of switzerland (10 fcv-sc and 5 controls/ area). to record clinical data, retroviral status and vaccination history of the cats, a questionnaire was filled out by the private veterinarian. the seven tested pathogens were found in the investigated population. the prevalence (fcv-sc vs. controls) was: fcv 45% vs. 8%; fhv-1 20% vs. 9%, c. felis 8% vs. 1%, b. bronchiseptica 4% vs. 2%, m. felis 48% vs. 31%, feline leukemia virus 2% vs. 1% and feline immunodeficiency virus 2% vs. 1%. fcv-sc were positive for fcv significantly more often compared with controls (or 9.2) and shed more fcv. co-infections with up to four pathogens were detected; fcv-sc were significantly more frequently co-infected (40%) compared with controls (14%). gingivostomatitis and oral ulceration but not urtd were highly associated with fcv infection. in contrast, c. felis was associated with urtd; fhv-1 was associated with nasal and ocular discharge and m. felis with conjunctivitis and ocular discharge. risk factors for fcv infection were housing in groups (especially ≥4 cats), an intact gender, maine coon breed and corticosteroid therapy. fcv-positive cats with gingivostomatitis were older and more commonly vaccinated than fcv-positive cats without gingivostomatitis. moreover they shed more fcv than cats with urtd. vaccination and primary immunization defined as two vaccinations 2-6 weeks apart with the same vaccine brand were protective factors against fcv but not fhv-1 infection. vaccination was associated with a decreased incidence of urtd in fcv-infected cats (or 0.3). further analyses will investigate cross-neutralization patterns of the prevailing fcv isolates. conflicts of interest: the study was partially funded by merial, france, and biokema, switzerland. antibody preparations are commonly used for the treatment of feline upper respiratory tract disease (furtd), although their efficacy has not been proven. the aim of this study was to evaluate efficacy of a commercial serum containing antibodies against feline herpesvirus-1 (fhv-1) and feline calicivirus (fcv) in cats with acute viral furtd. this prospective, randomized, placebo-controlled, double-blind study included 42 cats with acute (<7 days) clinical signs of fhv-1 and/or fcv infection (confirmed by quantitative pcr). all cats received symptomatic treatment and either hyperimmune serum (n = 22) (≤12 weeks 2 ml, >12 weeks 4 ml, subcutaneously q24 h, topically into eyes, nostrils, and mouth q8 h) or saline (n = 20) for three days. clinical signs, including a 'furtd score' and general health status, were recorded daily (day 0 to 7 and on day 21). fcv shedding was determined on day 0 and 21. statistical analyses included one-way analysis of variance, mann-whitney u, and student's t-test (improvement of clinical signs), fisher's exact test (fcv shedding), and spearman analysis (correlation clinical signs with virus load). clinical signs and general health status improved significantly in both groups. however, while placebo-treated cats had only improved significantly by day 7, cats receiving antibodies already significantly improved in their 'furtd score' (p = 0.046) and general health status (p = 0.032) by day 3. there was no significant difference in the number of cats shedding fcv and no correlation between viral load and clinical manifestation. administration of antibodies lead to faster improvement of clinical signs in cats with acute viral furtd, but did not influence fcv shedding. no conflicts of interest reported. different viral and bacterial pathogens can be involved in feline upper respiratory tract disease (furtd). although some clinical signs have been associated with certain pathogens, clinical signs can be variable and non-specific. aim of the study was to compare detection rates of feline herpesvirus-1 (fhv-1), feline calicivirus (fcv),and chlamydophila felis (c. felis) in cats with furtd on 4 different sampling sites, and to correlate test results and clinical signs. swabs of nose, oropharynx, tongue, and conjunctiva were taken from 104 cats with signs of furtd. on all samples, reverse transcription polymerase chain reaction (rt-pcr) was performed for detection of fcv, and polymerase chain reaction (pcr) for detection of fhv-1 and c. felis. fisher's exact test was used for all comparisons. the level of significance was p < 0.05. pathogens were detected in 89.4% of cats. of these, 55.8% were positive for fhv-1, 50.0% for fcv, and 35.6% for c. felis. fcv was isolated significantly more often from oropharynx (92.3% of fcv-positive cats) and tongue (90.4%) compared to conjunctiva (38.5%) (p < 0.001). there was no significant difference between the 4 sampling sites for detection of fhv-1 and c. felis. in addition, there was no preferred sampling site in cats with respective clinical signs, including oral ulceration, conjunctivitis, and keratitis. in cats with furtd, the oropharynx can be recommended as the preferred sampling site for detection of fcv, fhv-1, and c. felis. based upon clinical signs it cannot be determined which sampling site should be selected for detection of the pathogens. no conflicts of interest reported. pulmonary haemorrhage syndrome (lphs). s. schuller 1 , s. callanan 2 , s. worrall 2 , t. francey 1 , a. schweighauser 1 , j.e. nally 2 . 1 bern university, bern, switzerland, 2 university college dublin, dublin, ireland leptospiral pulmonary haemorrhage syndrome (lphs) is a severe form of leptospirosis, which has been increasingly recognised in humans and many animal species in the past 20 years. patients with lphs may develop rapidly progressive intra-alveolar haemorrhage, leading to high mortality. the pathogenic mechanisms of lphs are poorly understood hampering the application of effective treatment strategies. studies in humans and experimentally infected guinea pigs have demonstrated deposition of immunoglobulin and complement c3 in lphs lung tissue in the absence of significant numbers of leptospires, suggesting that lphs is, in part, caused by autoimmunity. the aim of this project was to describe the histopathologic features of lphs in dogs and to investigate whether igg and igm deposition is present in affected canine lung tissue. single-step immunohistochemistry (ihc) for dog igg, igm and leptospiral outer membrane vesicles was performed on lung tissues from 11 dogs with lphs, 4 dogs with pulmonary haemorrhage due to other causes and 4 healthy dog lungs. acute intra-alveolar haemorrhage and oedema in the absence of significant inflammatory infiltrates were present in all lphs lung tissues. three ihc staining patterns were observed in lphs lung tissue: alveolar septal wall staining with (igg n = 8/igm n = 6) and without intra-alveolar staining (igg n = 2/igm n = 0) and staining of intra-alveolar fluid only (igg n = 1/igm n = 5). intra-alveolar staining appeared to favour alveolar surfaces in some cases (igg n = 5/igm n = 5). healthy control lungs showed no staining, whereas haemorrhagic lung showed staining of intraalveolar fluid (igg/igm n = 3)) and occasional, mild and discontinuous staining of alveolar septa (n = 1). leptospiral antigens were not detected in any of the tissues. results indicate that histopathologic features of canine lphs are similar to what has been described in other species. ihc demonstrated that alveolar septal deposition of igg/igm is present in most dogs with naturally occurring lphs. while these findings support a role of the humoral immune response in the development of lphs, our findings do not indicate whether autoimmunity is a primary or secondary event in the pathogenesis of lphs. no conflicts of interest reported. leptospirosis, a zoonotic bacterial disease with a worldwide distribution, is a re-emerging disease in humans and dogs. acute renal and hepatic failure are the most frequently reported clinical manifestations of canine leptospirosis. the aim of this study was to describe clinical, laboratory and radiological features, the outcome as well as the distribution of leptospira serogroups in dogs with leptospirosis (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) . medical records of dogs diagnosed with leptospirosis were evaluated retrospectively. diagnoses were based on microscopic agglutination testing (mat), blood/urine pcr, and histopathology (levaditi staining). mat-titers ≥ 1:800 against non-vaccine and ≥ 1:3200 against vaccine serovars or a 4-fold rise of titers within 2-3 weeks were considered diagnostic. 99 dogs met the inclusion criteria. in 72 dogs diagnostic mat-titers were present (mainly against serogroups grippotyphosa (65%), australis (61%), and pomona (60%). at initial presentation, the most common clinical signs were lethargy (96%), anorexia (88%), vomitus (85%), a painful abdomen (39%), diarrhea (38%), oliguria (27%), tachypnea (26%), delayed capillary refill time (18%), pale mucous membranes (17%), fever (15%), hypothermia (15%), and icteric mucous membranes (10%). abnormal findings of the cbc included anemia (63%), thrombocytopenia (62%) and leukocytosis (57%). biochemistry abnormalities included increased creatinine concentrations (82%), increased liver enzyme activities (80%), hyperbilirubinemia (70%), hyperphosphatemia (67%), hyponatremia (63%), and hypoalbuminemia (55%). urinalysis often revealed glucosuria (77%) and an elevated urine-protein/creatinine-ratio (75%). radiological pulmonary changes were detected in 57% of the dogs initially or during the course of disease. 32 dogs died or were euthanized, 24 of them due to "leptospiral pulmonary hemorrhage syndrome". in this study, non-vaccine serogroups were the most common serogroups detected by mat. in the majority of patients renal (95%) and/or hepatic (93%) disease was detected. a pulmonary form of leptospirosis was present in 57% of the dogs. lung involvement represented a severe complication causing increased mortality depending on the severity of respiratory signs. no conflicts of interest reported. leptospirosis is a zoonotic disease that can affect multiple organs with renal and hepatic involvement being considered to be the most common. the aim of this study was to evaluate a large number of dogs with leptospirosis for cardiac and/or exocrine pancreatic involvement. a total of 59 dogs were diagnosed with leptospirosis based on clinical signs and either microscopic agglutination test, blood/ urine polymerase chain reaction, and/or histopathology. at the time of admission and, in most patients, after an average of two weeks canine pancreatic lipase immunoreactivity (cpli, as measured by spec cpl â ), ultrasensitive cardiac troponin i (ctni), and c-reactive protein (crp) were analyzed. data were analyzed with non-parametric statistics. the level of significance was set at p < 0.05. upon admission, common clinical signs reported included lethargy (n = 57), vomiting (n = 50), abdominal pain (n = 20), dyspnea (n = 16), pale mucous membranes (n = 13), oliguria (n = 11), hypothermia (n = 11), and fever (n = 10). anemia (n = 39), thrombocytopenia (n = 41), leukocytosis (n = 38), were frequently reported hematology findings. increased concentrations of creatinine (n = 48/59), phosphorus (n = 43/57), alt (n = 31/ 58), sap (n = 43/57) and bilirubin (n = 41/58) were also frequently recorded. crp (median: 48.7 mg/l; range: 0.1-60.1 mg/l, reference interval (ri): 0.1-7.6 mg/l), ctni (median: 0.137 ng/l; range: 0.005-24.063 ng/l, ri: 0-0.059 ng/l), and cpli (median: 217 lg/l; range: 29-1001 lg/l, ri: 0-200 lg/l) concentrations were above the upper limit of the reference intervals in 52/59 (88%), 42/59 (71%), and 30/59 (51%) dogs, respectively and serum cpli concentration was above the suggested cut-off value for a diagnosis of pancreatitis in 15/59 (25%) dogs. crp and ctni, but not cpli were higher upon admission compared to the re-check measurement (p = 0.0001 and 0.0056, respectively). dogs with increased serum cpli concentrations also showed a higher proportion of dogs with increased serum ctni concentrations (p = 0.001). there was no statistically significant correlation of cpli concentrations with a history of abdominal pain and/or vomiting. biochemical results were compatible with multiple organ impairment with involvement of kidneys, liver, heart, and exocrine pancreas where at least two organs were affected in 36/59 (61%) dogs. forty (68%) of 59 dogs recovered, 10 (17%) died, and 9 (15%) were euthanized. ctni and cpli were higher in non-survivors, but these differences did not reach statistical significance. however, the number of organs affected and outcome were significantly correlated (p = 0.012). our data suggest that infection with leptospira is characterized by a systemic inflammation with variable multiple organ involvement and damage, often including the heart and also the exocrine pancreas. the study was funded by texas a&m university. the primary author and two co-authors work at the gi laboratory, texa a&m university. canine bartonellosis is increasingly recognized worldwide and may be associated with diverse clinical manifestations. recent evidence suggests that bartonellosis also causes lameness and polyarthritis in dogs. however, pcr amplification of bartonella dna and isolation of bartonella species from canine synovial fluid (sf) samples have rarely been reported. canine leishmaniosis (canl) due to leishmania infantum is a multisystemic disease commonly associated with polyarthritis. based on the hypothesis that concurrent bartonella infection may be a contributing factor for the development of arthritis in dogs with canl, the main objective of this study was to investigate the microbiological and molecular prevalence of bartonella spp. in dogs with naturally-occurring canl, with or without cytologically documented arthritis. from a previous study, 38 dogs with canl were retrospectively studied for bartonella spp. infection. diagnosis of canl was based on compatible clinical and clinicopathological abnormalities, positive serology, and lymph node or bone marrow (bm) cytology. dogs with serological evidence of other vector-borne infections (anaplasmosis, borelliosis, dirofilariosis and ehrlichiosis) and dogs recently vaccinated or medicated were excluded from the study. arthritis defined as a neutrophil percentage in excess of 10% of nucleated cells in sf cytology was documented in 31/ 38 (81.6%) of dogs. a total of 74 archived specimens from 38 dogs, including 33 edta-anticoagulated blood samples, 19 bm and 22 sf aspirates were tested for bartonella spp. dna using a bartonella alpha proteobacteria growth medium (bap-gm) diagnostic platform. eight (21.1%) dogs were infected with one or two bartonella species, including candidatus bartonella merieuxii(n = 5), b. henselae sa2 (n = 3) and b. rochalimae (n = 1). bartonella spp. dna was amplified from bm in 4 dogs and from blood in 3 dogs but was not amplified from any sf sample. overall, 6 (19.4%) dogs with and 2 (28.6%) dogs without arthritis were infected with a bartonella species. the prevalence of bartonella spp. dna in the dogs with or without arthritis did not differ (v 2 test for independence, p = 0.589 the prevalence of giardia in dogs ranges between 5.4% and 55.2%, with a higher prevalence in puppies. however, the risk factors for giardia infection around weaning have been poorly described. the aim of the study was to evaluate risk factors for giardia infection in puppies during the first weeks of life and to determine an impact of this parasite on feces quality. 192 puppies from 58 litters living in a breeding kennel were followed between 4 and 9 weeks of age. each puppy was treated with fenbendazole (panacur â , msd, france, 50 mg/kg, per os, q 24 h) for 3 consecutive days at 2, 4, 6 and 8 weeks of age. for each puppy, fecal consistency was evaluated using a 13-point scale. excretion of enteropathogens was evaluated by qpcr for canine parvovirus type 2 (cpv2), qrt-pcr for canine coronavirus (ccv), coproantigens quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for any eggs and oocysts. a generalized linear mixed model (proc glimmix) with giardia infection as a binary outcome was used to assess the following effects: breed size, age, and cpv2, ccv and isospora ohioensis infections. a linear mixed model (proc mixed) with fecal score as outcome was used to determine the following effects: breed size, age, and giardia, cpv2, ccv and i. ohioensis infections. a total of 277 fecal samples were collected; cpv2, giardia, i. ohioensis and ccvwere detected in respectively 13.4%, 17%, 30.3% and 67.9% of the samples. the risk of giardia infection increased with age (odd ratio= 1.5; 95%ci=1.1-2.1; p = 0.015). neither breed, nor cpv2, ccv and i. ohioensis infections influenced risk of giardia infection (p = 0.299; p = 0.213; p = 0.892; p = 0.282 respectively). giardia infection did not impact feces quality (p = 0.11), whereas a significant influence of cpv2 (p < 0.001), ccv infection (p = 0.030) and breed size (p < 0.001) was evidenced. this study underlines that even with an adapted deworming program the eradication of giardia is difficult to obtain in large dog packs. the higher prevalence of giardia in puppies of 7 weeks and older could be linked with the immunity gap during this period. giardia was not associated in our study with an increased risk of diarrhea. the lack of pathogenicity of the parasite per se could be hypothesized, but also an efficacy of the treatment for the prevention of the clinical signs or a local and systemic immunity limiting clinical signs. financial support from royal canin. hepatic encephalopathy (he) can occur in dogs as a complication of primary liver disease or as consequence of congenital portosystemic shunts (pss). the aim of this study was to assess magnetic resonace spectroscopy (mrs) usefulness in he diagnosis. twenty dogs with a presumptive diagnosis of he were enrolled. inclusion criteria were: a clinical examination, a blood cell count and biochemical panel, at least one plasmatic ammonia determination higher than 100 lmol/l, optional hystopatology of the liver, requirement of magnetic resonance imaging (mri) investigation by the clinician. even though mrs represents a non-invasive procedure, informed consent was obtained from dogs'owners. he diagnosis was confirmed in 20/20: 16/20 had a pss, 3/20 showed hepatic microvascular dysplasia and 1/20 had hepatic cirrhosis histologically confirmed. the control-group was made of 23 patients retrieved from our database that underwent mri without showing any abnormalities on brain scans. the mr protocol included t2-weighted fast spin-echo, t1-weighted sequences, proton density, and radio-frequency pulses gradient-echo. on mri examination cerebral atrophy was evident in all the patients (20/20), ranged from mild to severe. in 17/20 patients symmetrical bilateral hyperintensities in the globus pallidus on t1-weighted images were evident. mrs was performed using a short te (30 ms) and it was defined a volume of interest in the parieto-occipital region by 10-mm side cube (1 cc of volume). glutamine-glutamate (gln), n-acetylaspartate and n-acetyl aspartyl glutamate (naa), choline derivatives (cho), and myo-inositol (ins) were analyzed. comparing spectra from the he affected dogs with those from the control-group decreased values of ins, cho, naa as well as high field mri combined with brain mrs provide accurate and non-invasive diagnosis of canine he. in accordance with human medicine publications, it could be state that mrs has a role in he diagnosis and follow-up with particular mention monitoring. no conflicts of interest reported. diagnosis of hepatobiliary diseases often requires hepatic tissue sampling for histologic evaluation. the laparoscopic technique is a safe method and allows acquisition of tissue by wedge and needle biopsies from different liver lobes in a minimally invasive way.specimens obtained with an 18-gauge biopsy needle must be interpreted with caution due to considerable variability in tissue involvement with certain disease processes. we hypothesized that needle biopsy specimens would produce findings divergent from those produced by wedge biopsy specimens. the goal of the study was to compare histological findings from two laparoscopic biopsy methods (wedge and needle) and assess which sampling technic can represent the overall disease process. procedure: all dogs included in this prospective study study were suspected diffuse hepatic disease and underwent laparo-scopic hepatic biopsy (wedge and needle 14-16g) between 2012 and 2014. all biopsy specimens were examined on the basis of morphologic criteria and a comparison was made between the two types of biopsies procedures according to wsava liver standardization group morphologic criteria. results: twenty-two dogs were included. no complications were reported during the laparoscopic procedure. the median number of portal triads per needle biopsy specimen was 6 (range, 4 to 8) compared to 20 (range 14;25) with wedge biopsy specimen. the median length of needle biopsy specimens was 10 mm; (range, 6 to 16 mm) and 6 mm for all wedge biopsies. on the basis of biopsy interpretation, the diagnosis was overall similar with the two methods: 8 dogs had vacuolar hepatopathy, 4 acute cholangitis, non-specific acute form in 3 dogs. chronic hepatitis with cirrhosis was found in 2 cases, 3 dogs had diffuse neoplasia and 2 miscellaneous hepatic disorders. the fibrosis was considered to be severe in 3 dogs, moderate in 3 dogs and mild in 11 dogs. no quantitative and qualitative difference was observed between the two types of biopsies specimen. this study demonstrates that the biopsies with a needle length of at least 10 mm brings satisfactory information for the evaluation of most of the inflammatory, vacuolar hepatopathies, fibrosis and diffuse tumoral infiltrations. wedge biopsies allow to examine the largest number of portal triad, more contributory for certain forms of cholangitis affecting larger canals and for a single case, images of peri-hepatitis were counted at the level of the capsule. fibrosis does not seem to be more important in the sub-capsular zone contrary to what is observed in human pathology. no conflicts of interest reported. indocyanine green (icg), a fluorescence dye, is excreted solely by the liver without enterohepatic re-circulation. hence it has been used for decades as an ideal albeit invasive marker of hepatic function and blood flow in cats and dogs. here we evaluated the feasibility of a minimally invasive transcutaneous icg clearance to assess hepatic function instantaneously. transcutaneous icg clearance was performed in 3 healthy research cats and 3 healthy research dogs with normal liver function (bile acid stimulation test, ammonia tolerance test) using a modified device (kidney int 2011 79:1254 with an excitation wave length of 760 nm and an emission wave length of 820 nm. the devices were placed on different locations (lateral thoracic wall, ventrolateral abdomen, metatarsus and antebrachium) and fixed with a light bandage. to find a suitable dose to reach adequate transcutaneous peak concentrations escalating doses of icg (0.1, 0.2 and 0.3 mg/kg) were injected intravenously with a wash out period of at least 24 h in-between. measurement was continued for 1 hour after injection in awake animals moving freely. the resulting icg disappearance curves were visually inspected to find best location (minimal artifacts, acceptable background noise) and dose. in all animals a dose of 0.2 mg/kg was deemed ideal and the thoracic and abdominal wall gave consistent results. half-life of icg clearance was calculated using a one-compartment model. half-lives in cats were 4.99, 5.23 and 7.19 minutes, respectively; in dogs: 9.66, 12.51 and 15.58 minutes, respectively. in conclusion, the transcutaneous assessment of icg clearance is feasible in a clinical setting. results are obtained within one hour and can be assessed instantaneously. the procedures are minimally invasive and well tolerated by the animals. given that most patients with a presumed liver problem undergo abdominal ultrasound no further clipping of hair is necessary as the device might be placed in this area. further studies are necessary to obtain reference values in healthy pets and those with various conditions leading to impaired hepatic function. no conflicts of interest reported. gallbladder diseases like gallbladder mucocele and cholecystitis can reduce gallbladder motility and may lead to cholestasis. since impaired gallbladder emptying contributes to sludge and gallstone formation, the evaluation of gallbladder motility requires accurate and appropriate methodology. three-dimensional (3d) ultrasonography has been shown to be accurate and appropriate tool for measurement of gallbladder volume in humans. therefore, we applied this novel technique for the first time to study preprandial and postprandial gallbladder volume in 10 healthy mixed-breed dogs and compared the results to twodimensional (2d) ultrasonography. the dogs were placed in dorsal recumbency to obtain ultrasonographic measurements of the gallbladder. measurements by both 2d and 3d ultrasonography were recorded in preprandial state and after ingestion of full-fat milk. the preprandial and postprandial gallbladder volumes determined by 3d ultrasonography were significantly higher than corresponding volumes by 2d ultrasonography (1.11 ae 0.07 vs 0.77 ae 0.06 and 0.81 vs 0.61 ml/kg, respectively, p < 0.05). in 2d ultrasonography, most dogs (8/10 [80%]) had a preprandial gallbladder volume ≤ 1.00 ml/kg. however, in 3d ultrasonography, 6/10 (60%) of dogs had a preprandial gallbladder volume ≥ 1.00 ml/kg. gallbladder contraction index was higher in 3d ultrasonography than 2d ultrasonography, however, it did not reach statistical significance (p = 0.25). in conclusion, 3d ultrasonography showed larger gallbladder volumes than 2d ultrasonography in healthy dogs. it seems that 3d ultrasonography is appropriate adjunct device to 2d ultrasonography to estimate gallbladder volume when 2d ultrasonography could not detect whole gallbladder volume. more research is needed to determine clinical value of 3d ultrasonography in canine gallbladder imaging. no conflicts of interest reported. the aim of our study was to compare high-definition oscillometry (hdo) and doppler ultrasonographic measurements with direct blood pressure measurements in conscious dogs. the doppler study was performed by three investigators and by using 3 different sphygmomanometers with 3 different sized cuffs. devices and measurement sites were changed randomly among the investigators. cuffs were wrapped around the antebrachium in the forelimb or around the mid-metatarsus in the hind limb. in addition to the limb sites, cuffs were also placed around the base of the tail in case of the hdo method. cuff sizes were 45-78% of the measured limb circumferences during the doppler measurement. for the hdo method all measurements were performed by the same investigator and the cuffs provided by the manufacturer were used: the smallest cuff was used for the hind limb and tail, and the medium cuff was used for the forelimb measurements. dogs were gently held in lateral recumbent position, measurement were performed on the nondependent limbs. radio-telemetry transducers were implanted to the right femoral artery some months to a year preceding the blood pressure measurements for reasons unrelated to our study. direct blood pressures varied 110-214 mmhg and 139-192 mmhg during the doppler and hdo measurements, respectively. two-hundred paired simultaneous doppler and direct measurements from 11 dogs and 100 paired simultaneous hdo and direct telemetric measurements from 7 dogs were obtained. at least 3 successful consecutive measurements could be obtained by the same investigator at the same site during the doppler or hdo measurement in 53 and 22 cases, respectively. thus, the mean of these measurements could be calculated similarly to the established everyday clinical practice. bias (mean difference), precision (standard deviation) and limits of agreements were calculated both from the individual paired measurements and from the means of the consecutive measurements using bland-altman spot analysis. systolic measurement performed on the tail with the hdomethod yielded the smallest bias and deviation and the best limits of agreement during this study. both doppler and hdo-measurements performed on the forelimb overestimated, while hind limb measurement underestimated the direct telemetric pressures. results of all three measurement sites by hdo performed better than forelimb or hind limb doppler-measurements, however hdo-measurements were more difficult to obtain and more often resulted with measurement failure compared to the doppler technique. cuff size above 55% of the measured limb circumference showed better results than smaller cuff sizes during the doppler measurements. no conflicts of interest reported. the doppler technique is considered the most repeatable indirect method to measure systolic arterial pressure (sap) in dogs. however, recent studies emphasized the effect of body position and used limb on sap measurement. the aim of this study was to determine whether a difference existed in sap measured simultaneously in dogs using different limbs, with two doppler units by two different operators. sixty clientowned dogs, admitted to the veterinary hospital for different reason, were enrolled. they were divided in 3 groups based on body size: 20 small breed dogs (<15 kg); 20 medium breed (15-30 kg); 20 large breed (> 30 kg). for each dog the anxiety status was recorded. sap was measured via doppler technique when dogs were in right lateral recumbency in a quite environment. right and left forelimb sap and left forelimb and left hindlimb sap were recorded simultaneously, with two identical doppler units equipped with headphones, by two operators. measurement was performed based on the acvim guidelines. five measurements were recorded, the higher and lower values were discarded from the analysis. the relationship of mean sap for each limb with body weight, sex, anxiety status and sap value was evaluated. mean ae sd sap was significantly higher for the right forelimb (175.81 ae 37.15) compare to the left forelimb (165.14 ae 33.35) on overall population. the difference was significant for large breed dogs, males and dogs with sap ³ 180 mmhg. sap was higher for the left forelimb (163.67 ae 32.11) compare to the left hindlimb (151.34 ae 34.31) on overall population. the difference was significant for medium and large breed dogs, females, calm animals and dogs with sap ³ 180 mmhg. the mean sap from the left forelimb recorded by two different operators at two different moments, were compared and no difference was evident. in conclusion, sap measurement from different limbs, in dogs in right lateral recumbency, is poorly correlated. measurement of sap from the left forelimb is more repeatable during time and between different operators. sap trend monitoring should be done using the same measurement site for any animal. no conflicts of interest reported. amlodipine has been considered the treatmenf of choice for hypertension in cats for more than a decade. there is, however, an unmet need for a cat-specific formulation. the aim of the study was to assess the efficacy of chewable amlodipine tablets in reducing systolic blood pressure (sbp) in cats diagnosed with hypertension. seventy-seven client-owned cats were included in the study (mean age 14 years). the study was randomised, double-blind, placebo controlled, and consisted of two phases. in the blinded phase, 42 cats received 0.125 mg/kg amlodipine once daily for 14 days. if they responded the dose remained the same to day 28. for non-responders, the dose was increased to 0.25 mg/kg. thirty-five cats received placebo following the same protocol. arterial blood pressure was measured using a high definition oscillometry method. at day 28 a responder was defined as a cat showing a decrease of sbp to ≤ 150 mmhg or a decrease from baseline of at least 15%. after 28 days all cats continued with amlodipine for 2-3 months in an open phase with the placebo cats repeating the same dose escalation protocol as in the blinded phase. the responder rate was 63% in the amlodipine group and 18% in the placebo group following the dose escalation from day 14 being applied to 54% and 80% of cats receiving amlodipine and placebo respectively. cats receiving amlodipine were 7.9 (95% ci 2.6 to 24.1) times more likely to be classified as responders when compared to those receiving placebo (logistic regression model, p = 0.0003). from a baseline value of 181.6 ae 12.5 and 179.3 ae 10.8 mmhg the mean sbp decreased to 153.6 ae 16.9 mmhg with amlodipine and to 167.7 ae 20.5 mmhg with placebo (repeated measures analysis of covariance model, p < 0.001) by day 28. the responder rate was not influenced by factors other than amlodipine treatment (e.g. baseline blood pressure, concomitant ace inhibitor therapy, renal disease). there were no differences between the amlodipine and placebo groups in the frequency of adverse events reported during the 28-day blinded phase. likewise, there were very few changes in the laboratory values over time in either group. the present study is the first large clinical trial to show that amlodipine is clearly superior to placebo in the treatment of cats with hypertension. the chewable amlodipine formulation effectively reduced sbp, had a good palatability and was well tolerated. it can be used concomitantly with ace inhibitors and in cats with renal disease. conflicts of interest: m. huhtinen and j. aspegr en are employees of the sponsor j. elliott has the following information to disclose: amlodipine is the treatment of choice for feline hypertension. limited published data exist on serum concentrations achieved in hypertensive cats. the aim of the study was to assess serum amlodipine concentrations in cats treated with a new formulation of amlodipine and relate these to the blood pressure reduction achieved. seventy-seven client-owned hypertensive cats were enrolled into a randomized, double-blind and placebo controlled study consisting of two phases. in phase one, 42 cats (group a) received 0.125 mg/kg amlodipine once daily for 14 days. if they were deemed to have responded (see below) the dose remained the same to day 28. for non-responders, the dose was increased to 0.25 mg/kg. thirty-five cats (group b) received placebo following the same protocol. blood pressure was measured using high definition oscillometry. a responder was defined as a cat showing a decrease of systolic blood pressure (sbp) to ≤150 mmhg or a decrease from baseline of ≥15%. following day 28 (phase 2), group a continued on amlodipine and group b switched to amlodipine and the dose was adjusted as per phase 1. both groups were followed for 90 days on amlodipine. blood was collected at days 28 (group a) and 90 (both groups) and serum [amlodipine] measured by liquid chromatography mass spectrometry. the sbp measured on treatment was calculated as percentage of the baseline sbp and plotted against serum [amlodipine] using a sigmoidal emax model (winnonlin software). data are expressed as mean ae se. the serum concentrations of group a cats that remained on 0.125 mg/kg were 29.8 ae 2.5 ng/ml whereas those switched to 0.25 mg/kg were 51.2 ae 7.8 ng/ml. when data from groups a and b were pooled, a sigmoidal relationship between percentage baseline sbp and serum [amlodipine] was found. estimated values of lowest percentage baseline blood pressure on treatment (emax) was 83.1 ae 1.7%, with an ec 50 value of 10.4 ae 2.6 ng/ ml and a slope function of 2.5 ae 1.2. the serum concentration required to reduce blood pressure by 15% was estimated to be 20 ng/ml. the present study related blood pressure reduction to serum [amlodipine] in feline clinical hypertension. the limitations of this study were the limited number of blood samples collected and lack of information relating to the exact timing of blood sampling relative to dosing in some cats. however, these data could be used to define appropriate therapeutic serum [amlodipine] in hypertensive cats. m huhtinen is an employee of the sponsor; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd.; l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health. commercial assays for the measurement of canine and feline pancreatic lipase immunoreactivity (spec cpl â and spec fpl â , respectively; idexx laboratories, westbrook, me, usa) have been available for a few years and have previously been analytically and clinically validated. recently, new commercial assays for the measurement of these parameters have become available, though neither one of these assays have been analytically or clinically validated in the literature. thus, the goal of this study was to compare these newly available assays to the established assays. leftover serum samples from diagnostic submissions to the gi laboratory were collected based on certain parameters (e.g., results throughout the working range of the assay, good quality sample or hemolytic, lipemic, or icteric sample) and were assigned random sample id numbers. the samples were evaluated by spec cpl â or spec fpl â , sent on dry ice to the klinik am hochberg, and one aliquot of each sample was blindly submitted to laboklin for measurement of cpli and fpli by their newly released in-house assay and also to the gi lab at texas a&m university for repeated analysis by spec cpl â and spec fpl â to exclude any effect of shipping. there was no significant difference between serum cpli or fpli concentrations before or after shipping at the gi lab (pvalues for wilcoxon matched-pairs signed rank tests: 0.593 and 0.672, respectively). in contrast, there was a significant difference between serum cpli or fpli concentrations between the newly released assays and the previously established assays (pvalues < 0.0001 and 0.0241, respectively). while there was a significant correlation between the newly released and the previously released assays (spearman r: 0.775 and 0.7386, respectively), this correlation was very poor for assays that supposedly measure the same analyte. also, the interpretation for serum cpli and fpli results between the previously developed assays and the new assays did not agree for many of the samples. finally, both newly developed assays showed some erratic results. in conclusion, the newly released assays for the measurement of cpli and fpli do not agree with previously established and validated assays, provide different interpretations, and show erratic results. thus, further research is needed before these newly released assays could be recommended for clinical use. dr. steiner serves as director and dr. suchodolski serves as associate director of the gastrointestinal laboratory at texas a&m university. dr. steiner also serves as a paid consultant to idexx laboratories, westbrook, me, usa. both the gastrointestinal laboratory and idexx laboratories offer cpli and fpli testing on a fee-for-service basis. digestive health is a main concern for growth, morbidity and mortality in weaning puppies. fecal immunoglobulin a (iga) has been suggested as a useful noninvasive biomarker for mucosal immunity. the purpose of this study was to evaluate the effect of infection with enteropathogens on fecal iga concentrations in puppies and that of physiological factors such as age and breed size. 282 puppies from 33 breeding kennels were included in the study. puppies were between 5 and 14 weeks of age (meanaestandard deviation (sd): 7.8 ae 1.5 weeks). depending on the mean adult body weight of their respective breed, the puppies were divided into small (if mean adult body weight < 25 kg) or large (>25 kg) breed puppies. for each puppy, fecal consistency was evaluated using a 13-point scale and feces were collected for the evaluation of presence of fecal enteropathogens and fecal iga concentrations. the presence of enteropathogens in fecal samples was evaluated by qpcr for canine parvovirus type 2 (cpv2), qrt-pcr for canine coronavirus (ccv), coproantigen quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for other parasite eggs and oocysts. fecal iga concentrations were measured by an elisa test. statistical analyses were performed using sas software. a linear mixed model (proc mixed) with fecal iga concentration as outcome was used to determine the following effects: enteropathogen infection, breed size, age, and fecal score. the respective influence of litter and breeding kennel as random effects was also determined. data is presented as mean ae sd. small breed dogs represented 27.3% (77/282) of the total number of dogs included. at least one enteropathogen was identified in 76.2% of puppies (214/281). fecal iga concentration was significantly influenced by fecal enteropathogens (p = 0.037). puppies infected with at least one enteropathogen had significantly lower fecal iga concentrations than puppies without any enteropathogens (5.0 ae 4.4 lg/g vs. 6.9 ae 5.5 lg/g). breed (p = 0.029), but not age (p = 0.082), influenced iga concentration. small breed puppies had significantly higher fecal iga concentrations than large breed puppies (6.8 ae 4.8 lg/g vs. 5.0 ae 4.7 lg/g). no significant relationship between fecal iga concentration and feces quality was evidenced (p = 0.165). this study suggests that fecal iga concentration is a promising marker for subclinical infection by at least one enteropathogen and confirms that digestive physiology varies with the breed size. a link between lower digestive immunity and higher susceptibility to enteropathogen infection needs further investigation. conflicts of interest: financial support of royal canin. canine chronic enteropathies (cce) include diet-responsive, antibiotic-responsive, and immunosuppressive-responsive enteropathies (ire). this prospective study was designed to evaluate a commercial hypoallergenic dry diet a containing oligopeptides as the only protein source for the management of dogs with ire and as an alternative to immunosuppressive therapy over a 10 week period. nineteen dogs across france and quebec entered the study. dogs with food or antibiotic-responsive chronic enteropathy, hypoproteinemia, or treated with immunomodulating drugs were excluded from the study. dogs were included in the study after complete clinical, ultrasonographic, endoscopic evaluation and histopathological evaluation of intestinal biopsies showing signs of intestinal inflammation. the owners were instructed to feed exclusively the study diet a . canine inflammatory bowel disease activity index (cibdai) scores, fecal scores as observed by the dog-owners, and body weight were evaluated at baseline, 2, 5 and 10 weeks after inclusion. dietary treatment was regarded successful if the cibdai score was reduced by at least 75%. the protocol has been reviewed and accepted by royal canin ethics committee and owners completed an informed consent. results are presented as meanaesd (range). statistical comparisons were performed with a wilcoxon test. thirteen dogs (7 intact males, 4 neutered and 2 intact females) completed the trial. seven dogs were excluded (2 diagnosed with giardia, 4 s with no histological evidence of inflammation, 1 with hypoadrenocorticism). mean age was 4.4 years ae 3.01 (1.1 -11.2), mean body weight 16.2 kg ae 13.36 (2.5 -43.0). cibdai score was 9.4 ae 3.43 (4-16) at inclusion, was 2.7 ae 1.84 (0-6) after 5 weeks and was 1.5 ae 1.31(0-3) after 10 weeks (p = 0.0015 and p = 0.0022 vs inclusion, respectively). fecal scores after 5 [4.0 ae 0.58 (3-5)] and 10 weeks [4.0 ae 0.60 (3-5) ] were improved compared to inclusion scores 1.9 ae 1.12 (1-4) (p = 0.0033 and p = 0.0051, respectively). the low molecular weight poultry feather hydrolyzed proteinbased dry extruded diet a appears to be effective in the management of idiopathic ibd without any concurrent immunosuppressive drug over the 10 week period of this pilot study. these preliminary findings should be confirmed by a prospective, randomized double blind study. feline pancreatitis is the most common exocrine pancreatic disorder with varied mortality. however, there is no available and reliable method to evaluate the severity and prognosis of the disease. ninety-two cats diagnosed as pancreatitis with acute onset of compatible clinical signs and a positive snap â fpl tm test between october 2011 and september 2013 were enrolled in this study. all cats were divided into survival (n = 48) and nonsurvival (n = 44) groups. fifty-two parameters including signalments, clinical signs, physical examinations, clinicopathological examinations, diagnostic images, complications and concurrent diseases were analyzed and compared between the two groups. parameters with p ≤ 0.05 were considered for further analyses. the mortality in this study was 47.8%. hematocrit, albumin, bun, creatinine, total bilirubin, calcium, phosphorous, body temperature, systolic blood pressure, the body cavity fluids, complications, e.g. systemic inflammatory response syndrome (sirs) and acute renal failure (arf) were found to be significantly associated with disease severity and prognosis, and were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each cat by summing all weighting factors. the total scores of each cat ranged from 12 to 83. the severity scores in this study achieved an area under receiver operating characteristic (auroc) of 0.88. the optimal cut-off point for discriminating outcome was 32.5 with the sensitivity of 89.6% and specificity of 77.3%, respectively. the mortality was 87.2% with a score ≥ 33, whereas 18.9% with a score ≤ 32. there was significant difference (p < 0.001) between the two groups of the cut-off point. furthermore, the mortality reached to 100% when the score more than 56. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in cats with pancreatitis. no conflicts of interest reported. convincing evidence for the role of clostridium (c.) perfringens as a primary pathogen in acute haemorrhagic diarrhoea syndrome (ahds) in dogs was recently found. it is suspected that clostridial toxins, especially c. perfringens enterotoxin, play a relevant role in the disease process. however, to date enterotoxigenic c. perfringens strains have only been described in single case reports. thus, the aim of this study was to indentify the specific c. perfringens genotype involved in adhs. small intestinal biopsies were collected with a sterile single-use biopsy forceps from ten dogs with ahds and immediately cultured. in 8/10 dogs, clostridial strains were isolated and identified as c. perfringens by mass spectrometry using maldi-tof ms. c. perfringens colonies from each dog were submitted for specific detection of the four major toxin genes (alpha, beta, epsilon, and iota), the enterotoxin gene, and the beta2 toxin by multiplex pcr. every clostridial isolate was typed as c. perfringens type a based on the detection of the alpha toxin encoding gene. in 5/8 isolates, additionally the beta2 toxin gene was identified, however, none of clostridial strains encoded for the c. perfringens enterotoxin gene. the results of this study suggest that c. perfringens type a is the most important c. perfingens genotype involved in the disease process of dogs with ahds. although c. perfringens enterotoxinhas been associated with intestinal diseases in humans, dogs, horses, pigs, and other animal species, this enterotoxin is most likely not responsible for the intestinal lesions in dogs with ahds. no conflicts of interest reported. p < 0.000). lack of treatment response was significantly associated with il (il: 3/4, sre: 6/20, fre: 0/63, are: 0/11; p < 0.000). anemia, thrombocytopenia, and increased plasma urea were significantly associated with il (anemia: il: 4/5, sre: 13/22, fre: 11/64, are: 5/11, p < 0.000; thrombocytopenia: il: 3/5, sre: 0/22, fre: 4/64, are: 0/11, p = 0.004; increased urea: il: 3/5, sre: 1/22, fre: 2/62, are: 1/11, p = 0.003). hypoalbuminemia (<20 g/l) and hypocobalaminemia (<200 pg/ml) occurred significantly more frequently in dogs with sre (hypoalbuminemia: il: 0/5, sre: 10/22, fre: 1/62, are: 1/11, p < 0.000; hypocobalaminemia: il: 0/3, sre: 9/20, fre: 8/63, are: 1/11, p = 0.015). results of this study show that elderly and large breed dogs were more frequently affected with il and sre compared to other etiologies and both il and sre were associated with greater disease severity and/or a negative outcome. in comparison, anemia, thrombocytopenia, and increased plasma urea were most frequently detected in il whereas severe hypoalbuminemia and hypocobalaminemia were significantly associated with sre. no conflicts of interest reported. alpha 1 -proteinase inhibitor (a 1 -pi) is a proteinase-resistent protein that can be quantified in fecal, urine, and serum samples from dogs. recently, increased fecal and urinary canine a 1 -pi (ca 1 -pi) concentrations have been described in dogs with gastrointestinal diseases (e.g., inflammatory bowel disease [ibd] , but also in dogs with exocrine pancreatic insufficiency) and in dogs with chronic hepatitis or chronic kidney disease, respectively. decreased serum ca 1 -pi concentrations have been reported in dogs with ibd, protein-losing enteropathy (ple), and hypocobalaminemia. treatment protocols for dogs with ibd and/or ple commonly include corticosteroids, but the effect of corticosteroid therapy on serum ca 1 -pi concentrations have not yet been reported. the aim of this study was to evaluate the effect of hydro-cortisone on serum ca 1 -pi concentrations in healthy dogs. twelve healthy beagle dogs were randomly allocated to a placebo-group (n = 6) and to a treatment group (n = 6; hydrocortisone-group). the placebo-group received an empty gelatin capsule po q12 h, whereas the hydrocortisone-group was treated with hydrocortisone at a dose of 8.5 mg/kg po q12 h. serum samples were obtained at baseline and on day 1, 5, 28, 56, and 84 during treatment as well as day 1, 5, 28, 56, and 84 post-treatment for all dogs. serum ca 1 -pi concentrations were measured at all time points using an in-house radioimmunoassay. a mann-whitney u test was used to compare the baseline measurements of both groups. the effect of hydrocortisone-treatment on serum ca 1 -pi concentrations was evaluated by comparing ca 1 -pi at baseline and during treatment and between baseline and posttreatment period using a manova. baseline serum ca 1 -pi concentrations did not differ between the hydrocortisone-and the placebo-group (p > 0.05). serum ca 1 -pi concentrations increased significantly (p = 0.0004) during the treatment period in the hydrocortisone-group (baseline [median in mg/l: 1, 796] ). in contrast, no difference was observed between both groups when comparing serum ca 1 -pi concentrations at baseline and during the post-treatment period (p > 0.05). this study showed that hydrocortisone-treatment over 12 weeks did affect serum ca 1 -pi concentrations in healthy dogs. whether corticosteroid therapy has any effects on fecal or urine ca 1 -pi concentrations in healthy dogs remains to be determined. the author works at texas a&m university, whose gi lab currently offer a commercial assay for faecal alpha1-proteinase inhibitor. canine chronic enteropathy (ce) is a common, but poorly understood syndrome, with variable response to therapy and prognosis. there is a need for novel biomarkers that are specific for intestinal disease and that provide objective measures of disease severity, progression, and prognosis. serum citrulline is a useful biomarker in human intestinal disease as it is specific to the small intestine and indicates globally reduced enterocyte mass and absorptive function in various disease states. it is used to determine, quantitatively, intestinal integrity at the enterocyte level and is not influenced by nutritional or inflammatory status. the aim of this study was to determine whether serum citrulline can be used as a biomarker for ce in dogs. in this retrospective study, computer records from the university of liverpool small animal teaching hospital were used to identify dogs with ce. disease severity was quantified by cibdai. controls were age-and breed-matched dogs without gastrointestinal disease. serum citrulline was measured by ultra-high performance liquid chromatography with tandem mass spectrometry. in dogs with ce, serum citrulline concentration was measured at presentation and at various time points after starting treatment. serum citrulline was measured in 49 dogs with ce and 69 controls. 17 dogs responded to dietary manipulation (food-responsive enteropathy, fre) and 3 responded to antibacterials (antibiotic-responsive diarrhoea, ard), with a further 2 having invasive mucosal bacteria, of which one responded to antibacterials and one was refractory. 27 dogs were diagnosed with idiopathic ibd (on the basis of exclusion of known causes and failure to respond to therapeutic dietary and antibiotic trials), of which 12 responded to immunosuppressive therapy, 13 were refractory, and 2 were lost to follow-up. serum citrulline concentration did not differ between dogs with ce (median 8.5 lg/ml, range 1.4-20.6) and controls (median 8.2 lg/ml, range, 1.2-34.9, p = 0.96). there was also no difference in serum citrulline concentration amongst dogs with fre, ard, ibd, and controls (p = 0.48). serum citrulline did not differ between dogs that responded well, or were refractory to treatment (p = 0.39), between 23 dogs with and 26 without protein-losing enteropathy (p = 0.67), or between 35 dogs that survived and 8 that were euthanased because of ce (p = 0.65). serum citrulline did not correlate with cibdai (r 2 = 0.10). these findings do not support the use of serum citrulline as a biomarker in determining diagnosis, prognosis, or quantifying severity in dogs with ce. one of the co-authors (marco caldin) has a diagnostic laboratory offering citrulline assays. chronic enteropathy (ce) is a multi-factorial disease, which involves aberrant immune responses to commensal bacteria or dietary antigens. macrophages have an important role in human disease but little information is available in canine intestine. data to date have relied solely on macrophage identification using mac387, an antibody directed against calprotectin, which recognizes both macrophages and neutrophils. in this study an alternative antibody for macrophages, am-3k, directed against a scavenger receptor (cd163) was used and distribution of both markers was compared. this antigen is of interest as positive cells accumulate in intestine of humans with ce. endoscopic duodenal biopsies were obtained from seven crossbreed dogs. serial histologic sections were stained with mac387 or am-3k. positively-stained cells were counted from 5 random areas from both villous and crypt regions. stained cell localisation was subjectively evaluated and the percentage of positively stained cells from the total nucleated cells per 10,000 lm 2 in the villus or crypt was compared between both antibodies using a wilcoxon signed-rank test. mac387 and am-3k did not co-localize on serial sections. there were significantly more am-3k positive cells than mac387 in the crypts (3.6% [0-7.0] versus 0.8% [0-7.5], p = 0.005). in contrast there was no difference in expression of either markers in the villi (3.2% [0-8.16] versus 1.8% [0-11.2], p = 0.27). this study reports for the first time the existence of two populations of macrophages in canine intestine. these results in normal dogs will be used to explore further the distribution and function of macrophages in dogs with ce. no conflicts of interest reported. chronic diarrhea and vomiting are common clinical signs in dogs. primary (e.g., inflammatory, infectious, neoplastic, mechanical, or other) and secondary gastrointestinal diseases (e.g., exocrine pancreatic, hepatic, renal, or endocrine disease) are possible underlying causes. the aim of this study was to evaluate the final diagnoses in dogs with chronic diarrhea and/or vomiting and to determine the prevalence of various primary and secondary gastrointestinal diseases in dogs with these gastrointestinal signs. medical records of 209 dogs presented between july 2011 and august 2013 with chronic diarrhea (d), vomiting (v) or both (diarrhea and vomiting [vd]) were retrospectively reviewed. dogs were included if a minimum work-up (hematology, plasma biochemistry profile, and fecal parasitology) had been performed and if a final diagnosis was recorded (155/209). a primary gastrointestinal disease was recorded in 83% of the cases (129/155) and included inflammatory diseases (90/ , exocrine pancreatic insufficiency, hypoadrenocorticism, polyendocrinopathy, dilated cardiomyopathy, and leukemia in one dog each). in total, 44% of the dogs were presented with d (69/155) followed by 33% with vd (51/155), and 23% with v (35/155). d and vd were significantly more frequent in dogs with primary gastrointestinal disease (d: 61/129, vd: 46/129), compared to dogs with secondary gastrointestinal disease (d: 8/26; vd: 5/26; p = 0.001, chi square test). v was significantly more common in dogs with secondary gastrointestinal disease (13/26) as compared to dogs with primary gastrointestinal disease (22/129; p = 0.001). in this study, food responsive enteropathy (36%) was the most commonly diagnosed cause of chronic gastrointestinal signs. chronic pancreatitis was the most frequent cause of secondary gastrointestinal disease (46%). diarrhea was significantly associated with primary and vomiting with secondary gastrointestinal disease. no conflicts of interest reported. matrix metalloproteinases (mmps) 2 and 9 are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. intestinal mucosal levels of mmp-2 and -9 have been shown to be increased in animal models and human ibd. to our knowledge, the presense of mmp-2 and -9 has not been studied in the intestinal mucosal samples of healthy dogs as well as in canine spontaneous ibd. thus, the main aim of this study was to identify the presence of mmp-2 and -9 in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from 12 healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava histology standards, recorded findings of all samples were considered insignificant. pro-mmp-2 and -9 activities were found in 17/48 (35%) and 25/48 (52%) of the samples, respectively. among four different parts of the intestine of 12 dogs, the ileum had the highest positivity rates of 7/12 (58.3%) and 8/12 (66.7%) for pro-mmp-2 and -9 activities, respectively. however, statistical analysis showed no significant difference of pro-mmp-2 and -9 activities between the separate parts of the intestine (p > 0.05). the enzyme activities ranged for pro-mmp-2 between 0.015 and 6.449 arbitrary units (au) and for pro-mmp-9 between 0.018 and 5.680 au. none of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active mmp-2 and mmp-9. this study showed that pro-mmp-2 and -9 could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of mmp-2 and -9 in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases. no conflicts of interest reported. digestive perforation is called spontaneous when it arises in the absence of foreign body ingestion, gastric dilatation and volvulus, external trauma, or previous digestive surgery. in dogs many predisposing factors have been identified, including antiinflammatory administration, severe hepatic or renal disease, stress, shock, gastric hyperacidity, neoplasia and idiopathic inflammatory bowel disease. spontaneous digestive perforation has been uncommonly reported in cats. the objectives of this study were to describe the clinical characteristics of spontaneous digestive perforation in cats, and to compare the frequency of malignant versus non-malignant causes for these perforations. to be included in this study, the perforation had to be spontaneous and confirmed by exploratory surgery. the medical records of 13 cats diagnosed as having spontaneous digestive perforation between 2010 and 2013 were reviewed. the mean age of cats was 7.6 years (9 months to 17 years). five cats had concurrent illnesses including viral upper respiratory tract disease, pancreatitis and chronic kidney disease. the most frequently reported signs included anorexia (85%), vomiting (61%), and lethargy (54%). histological examination was performed in 11 cats and diagnosed alimentary lymphoma in 54% and inflammatory lesions in the other 46% of them. six cats had received anti-inflammatory within the previous 3 months. half of them were finally diagnosed with lymphoma. five cats with lymphoma received chemotherapy.three cats died early in the postoperative recovery period, 5 cats were euthanized 12 to 146 days after surgery, and 2 cats were still alive at the end of this study. in the absence of pneumoperitoneum, clinical signs and clinicopathological abnormalities are not specific enough to allow differentiation between cats with gastrointestinal ulceration and those with perforation. in most cases, there is no diagnostic test that individually determine whether perforation has occurred or is impending, and clinicians should use of multimodality diagnostic procedures such as radiography, ultrasonography, endoscopy, and abdominal fluid cytopathology to avoid delay in diagnosis of digestive perforation. histological examination of ulceration is essential as lymphoma should be suspected in all cats presented with spontaneous perforation. the link between anti-inflammatory administration and spontaneous perforation in cats is not established. no conflicts of interest reported. campylobacter species are commonly isolated from faeces of dogs and cats with c. upsaliensis (cu) and c. helveticus (ch) being the most frequently isolated. these two species are usually not considered pathogenic in dogs and cats and are closely related to each other and to c. jejuni, the most common cause of bacterial gastroenteritis in humans in the developed world. interestingly, despite their close genetic relationship, in humans cu is considered a pathogen while ch is not. this study aimed to describe whole genomes of cu and ch isolated from dogs and cats and to in silico investigate their pathogenic potential with comparison to several published genomes of c. jejuni and c. coli. genomic dna was extracted from three isolates of each of cu and ch recovered from the faeces of healthy dogs and cats. sequencing was performed using an illumina miseq to generate 250 base paired reads. reads were trimmed for both length and quality. contigs were assembled using the velvet assembler. the concatenated contigs generated for each assembly were quality ranked (by number, size, maximum length and n50) and the three top ranked assemblies were annotated using the prokka annotation tool. ribosomal mlst nucleotide sequences were used as a proxy for the core genome to compare the phylogeny of cu and ch with other species in the campylobacter genus and visualised as a neighbornet using splitstree. annotated draft genomes were clustered using orthomcl and pathogenic traits were investigated in silico using pathogenfinder and viru-lentpred software. the cu and ch draft genomes were~1.732 mb and 1.844 mb in size, and comprised on average 110 and 151 contigs, and on average 1782 and 1942 predicted genes, respectively. of these cu had on average 497 and ch 622 hypothetical proteins. using orthomcl, a core genome of 1459 and 1751 genes resulted for cu and ch, respectively. neighbornet trees based on ribosomal mlst nucleotide sequences and the core genome confirmed the close phylogenetic relationship of ch and cu within the campylobacter genus. pathogenfinder predicted all isolates as human pathogens with probabilities of 88.3-91.5%. both pathogenfinder and virulentpred identified many pathogenic proteins in cu and ch of different functions (e.g. chemotaxis, transporter and motility systems) but considerably fewer than in c. jejuni and c. coli. this study provides many insights into the pathogenic potential of pet-associated emerging campylobacter pathogens and is to our knowledge, the first to report a draft genome of ch. no conflicts of interest reported. there are only few laboratory markers being evaluated for diagnosing and/or monitoring canine chronic enteropathies, including inflammatory bowel disease (ibd). s100a12 belongs to the s100/calgranulin-protein family and has been proposed to play a central role in both innate and acquired immune responses. it has been reported to be increased in stool samples, serum and/or intestinal mucosa in human patients with ibd. myeloperoxidase (mpo) is an enzyme found mostly in granulocytes. intestinal mucosal levels of mpo have been shown to be increased in animal models and human ibd. to date, s100a12 and mpo levels in intestinal mucosal samples have been reported neither from healthy dogs nor from dogs suffering from ibd. to start investigating this aspect in dogs, the objective of this study was to evaluate mucosal s100a12 and mpo levels in the small and large intestines by using enzyme-linked immunoassay (elisa) and spectrophotometric methods, respectively. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from 12 healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava standards the histologic findings of all samples were considered insignificant. s100a12 concentrations were from the highest to the lowest: ileum, 71.5 (38.9-141.9) lg/l; colon, 23.2 (6.7-75.6) lg/l; duodenum, 11.4 (6.9-28.5) lg/l; and jejunum, 8.5 (5.1-19.3 ) lg/l. the concentration in the ileum was significantly higher than in all other segments (p < 0.05), and the colonic mucosal concentration was higher than the jejunal (p < 0.05). the highest mpo activity was found in the ileum (0.49 [0.19-1.05] da/min), followed by jejunum (0.36 [0.28-1.70] da/min), duodenum (0.26 [0.09-0.59] da/min), and colon (0.09 [0.04-0.14] da/min). mpo activity was significantly higher in ileal and duodenal than in colonic mucosal samples (p < 0.05). the jejunal mpo activity was higher than the colonic and duodenal activity (p < 0.05). this study showed that using elisa and spectrophotometry allow the detection of canine intestinal mucosal s100a12 and mpo, respectively. the levels on s100a12 and mpo seem to differ between certain parts of the intestinal mucosa of healthy dogs. both assays appear to be useful to further evaluate the role of s100a12 and mpo in the pathogenesis of canine chronic enteropathies, including ibd. dr. heilmann, dr. suchodolski, and dr. steiner have a patent pending that includes the canine s100a12 assay used in this study. the authors declare that they have no further conflicts of interest. the aim of the present study was to establish the incidence of innocent cardiac murmurs in a fairly large number of clinically healthy puppies. a second aim was to evaluate a possible correlation between the presence of an innocent cardiac murmur and a lower hematocrit value. puppies of certain breeds are routinely screened for the presence of congenital porto-systemic shunts in the netherlands. breeders bring their nests to our clinic for individual measurement of blood ammonia concentration. in one year time (from february 2013 until january 2014) 389 dogs of 11 different breeds were examined, with 295 of them being cairn terriers. the age of the dogs varied from 20 to 108 days (mean 53 days). while the breeders were waiting for the blood results, the cardiac auscultation was performed by a single board-certified cardiologist (vsz). hematocrit was measured with an automatized hematology analyzer system from the surplus blood sample. cardiac murmur was found in 59 dogs (15%). in all cases this was a soft (1-2 out of 6) systolic murmur, most of the time with a musical character and with the point of maximal intensity on the left hemithorax, compatible with the description of an innocent cardiac murmur. no murmurs were found that could be compatible with a congenital cardiac anomaly. the hematocrit was significantly (p = 0.003) lower in the group of dogs with a murmur (mean 30.9%, standard deviation 2.8%) compared to the group without a murmur (mean 32.2%, standard deviation 3.1%). multivariate analysis shows that the presence of murmur is correlated with the hematocrit, but not with the age of the dogs. physiologic anemia has been long suspected to be one of the possible causes of innocent cardiac murmurs in young animals and children. however, according to the authors knowledge, no published reports exist that looked for a possible correlation. which other factors contribute to the presence of an innocent murmur is largely unknown. because of a large overlap between the hematocrit values of dogs with and without a cardiac murmur, measuring hematocrit in a particular pup would not help a less experienced first line veterinary practitioner to decide whether a murmur is innocent or the result of a congenital cardiac anomaly. limitation of this study is that no echocardiography was performed to rule out congenital cardiac anomalies as the cause of the murmurs. neither were the dogs re-checked for spontaneously disappearance of the murmur. no conflicts of interest reported. low intensity systolic murmurs, with point of maximal intensity over the outflow tract on the left side of thorax, are not uncommonly heard during cardiac auscultation of apparently healthy siberian husky dogs, often with excellent exercise tolerance. the origin of these murmurs in athletic dogs such as huskies is unlikely to be due to heart disease but more likely due to turbulent blood flow in the outflow tract caused by a large stroke volume and forceful cardiac contractility in early systole. the differentiation of these murmurs from "pathological"significant murmurs can however be problematic in general practice. the aim of the present study was to investigate the prevalence of murmurs in a sample of successfully racing siberian husky dogs and furthermore to study the phonocardiographic characteristics of these murmurs. phonocardiograms and ecgs were recorded in 37 actively racing siberian husky dogs, with normal or excellent exercise tolerance. normal stamina was confirmed by successful racing. phonocardiograms were easy and rapid to record on a pc laptop connected to the meditron stethoscope in ambulant "field"practice. systole was measured as the duration measured from the onset of the first heart sound to the onset of the second heart sound and the murmur duration from the onset the first heart sound to the end of the murmur. the duration of the first heart sound plus the murmur was measured and calculated as a percentage of the duration of systole. cardiac murmurs of grade 1-2 were heard in 22% of dogs examined. phonocardiogram from these dogs revealed early systolic crescendo-decrescendo or decrescendo murmurs with a duration of maximally 75% into systole. all dogs with murmurs had a silent pause at the end of systole. ecg was normal in all dogs. murmurs in adult athletic dogs should not be regarded as a definite sign of heart disease. physiological flow murmurs of up to 75% of systole is a common finding in active siberian husky dogs (prevalence 22% in the examined sample). phonocardiography is a rapid and practical method for differential diagnoses between pathological murmurs and physiological flow murmurs. no conflicts of interest reported. nt-probnp has a degree of overlap with clinically normal animals, particularly those with mild or subclinical heart disease. prior studies have evaluated the sensitivity and specificity of a point-of-care second generation elisa that utilizes snap technology. the snap feline probnp test uses the same biological reagents as the cardiopet probnp test but provides results in 10 minutes. we sought to prospectively validate the assay in a population of clinically normal cats. cats were recruited based upon the absence of a heart murmur, gallop, and/or arrhythmia. all cats received physical examination, non-invasive blood pressure measurement, complete biochemical analysis including a t4, urinalysis and echocardiogram. only cats considered free of underlying cardiac or systemic disease were enrolled. sixteen adult cats were enrolled and blood samples were obtained for nt-probnp concentrations at 0, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr. samples were placed in edta tubes and centrifuged within one hour and split into two tubes for duplicate samples at each time point and stored at -80°c. once all samples were collected, they were shipped on dry ice overnight and run in one batch (idexx laboratories) for measurement of nt-probnp concentrations. snap tests were visually evaluated by one blinded reader. comparison of snap assay vs. quantitative elisa revealed a 1.0 (auc) degree of correlation between assays, and that a positive snap test result was associated with a nt-probnp concentration of 126.4pmol/l or greater. the average bnp concentration of abnormal cats (191.1 ae 5.8) determined by the snap assay was significantly greater than the normal (26.4 ae 1.2). this study was funded through idexx and the university of florida college of veterinary medicine resident grant competition. we acquired 2d echocardiographic cineloops from the left apical 4-chamber view optimized for the la, and analyzed atrial longitudinal strain (st) and strain rate (sr) in 27 dogs (10 healthy dogs and 17 dogs with mmvd -5 acvim stage b1, 5 stage b2 and 7 stage c). endocardial la ste curves were obtained and peak atrial longitudinal strain (pals), peak atrial contraction strain (pacs), conduit atrial longitudinal strain (cals); pals-pacs) and contraction strain index (csi -pacs/pals*100) were calculated. la sr curves were similarly obtained to determine the peak positive strain rate (srs) during left ventricle systole, the first negative peak strain rate (sre) during early diastole and the second negative peak strain rate (sra) during atrial contraction. for all variables, a mean of 3 measures was used for the statistical analysis. we compared each of these variables between each acvim stage by kruskal-wallis tests and post-hoc pairwise comparisons, with comparison-wise a=0.05. normal dogs had higher pals and cals than dogs with mmvd (p < 0.0001 and p = 0.0005); stage c dogs had lower pals, pacs and cals than all other dogs (p < 0.0001, p = 0.0022 and p = 0.0005), but csi did not differ between groups (p = 0.1). stage c dogs had lower srs (p = 0.0005), higher sre (p = 0.0029) and sra (p = 0.0004) than other dogs. normal dogs had lower sre and sra than dogs with mmvd (p < 0.001). our data suggest that ste might be useful in assessing la function in dogs with mmvd, and might potentially differentiate dogs with severe subclinical disease from dogs with congestive heart failure. no conflicts of interest reported. sighthounds are athletic dogs and they have been claimed to have larger hearts compared to similar sized breeds. the left ventricle (lv) may enlarge in response to cardiac disease, but also in response to training, so called athlete's heart syndrome, which is a benign condition. to distinguish abnormal echocardiographic measurements from normal, breed-specific reference values are needed. the aim of this study is to establish normal reference ranges for echocardiographic measurements in the saluki breed. the study comprised 78 clinically healthy salukis (41 males and 37 females), mean age 72 months (ae sd 28 months), bodyweight (bw) 24,7 kg (ae 3,7 kg). case history was ascertained and dogs underwent physical examination, complete blood count, serum biochemistry profile, thyroid profile, blood pressure measurement and 3-min ecg. standard m-mode and 2d echocardiographic measurements were obtained. dogs with systolic murmur 1/6, and dogs with mitral valve regurgitation (mr) <15% (mr color flow jet area/left atrium areax100% in apical view) were considered normal. linear regression models were used to establish reference ranges. heart rate (hr) varied from 44 to 120 bpm (81 ae 17 bpm). bw was a significant predictor for lv dimensions, i.e. m-mode lv diameter and 2d volume in diastole (lvidd and lvedv) and systole (lvids and lvesv), and mitral valve end point septal separation (epss). hr was a significant predictor for fs % (fractional shortening). predicted values (95% prediction intervals) were calculated from regression models where mean bw (24,7 kg) and age (72 months), and median hr (80 bpm) were used. normal reference ranges were: lvidd 46,0 mm (40,0-52,0), lvids 33, 4 mm (27, 5) , lvedv 86,3 ml (64,6-108,0), lvesv 44,2 ml (29,2-59,2), fs%: 27,5% (20,3-34,6), ejection fraction ef%: 48,9% (38,6-59,1), epss 7,3 mm (4,4-10,2), sphericity index 1,6 (1,4-1,9), interventricular septum in diastole 10,9 mm (8,6-13,3) and systole 13,8 mm (10,5-17,1), lv free wall in diastole 10,4 mm (8,3-12,4) and systole 13,1 mm (10,0-16,1), left atrial (la) diameter 28,5 mm (23, 3) , aortic (ao) diameter 23,9 mm (20,1-27,8) , la/ao 1,2 (1,0-1,4), and aortic and pulmonic flow velocity 1,4 m/s (0, 9) and 1,2 m/s (0,8-1,6), respectively. this study provides echocardiographic values for normal salukis which can be used as a reference values. no conflicts of interest reported. in mitral valve disease, atrial remodeling is an indicator of evolution and prognosis, the duration of the p wave being considered suggestive of the dilatation of the left atrium. in humans' studies, neurological conditions have a significant impact on cardiac electrophysiology by altering the electrical impulse conductibility. the aim of this study is to examine the duration of the p wave in dogs suffering from mitral valve disease in comparison to dogs diagnosed with different neuropathies without cardiac abnormalities. we analyzed standard electrocardiograms (5 min of ecg, on 6 peripheral leads) performed on three polymorphic groups of dogs (different age, weight and breed): group 1 (n = 14) healthy dogs, group 2 (n = 30) dogs diagnosed with mitral valve disease and group 3 (n = 27) dogs suffering from different neuropathies (without any associated or previously diagnosed cardiovascular disease). the duration of the p wave was measured for all dogs (five consecutive p waves without anomalies or artifacts) and reported to the degree of atrial remodeling, assessed by left atrium/ aorta ratio on echocardiography. the interpretation of the ecg and echocardiography was made by the same examiner (md). the results were statistically evaluated in a specialized program (ibm spss vs. 21). the p wave recorded average values was 0.061 ae 0.028 seconds for the mvd group with significant differences between the stages of heart failure (p = 0.008). no correlations were found between its increase and the dilation of the left atrium (r 2 = 0.012). there was no statistically significant difference regarding p wave duration when compared dogs of the neuropathy group and those of the mitral valve disease group (the p wave recorded average values = 0.068 ae 0.018 sec.). both, atrial tissue lesions (as in mitral valve disease) and autonomic nervous system anomalies (secondary to a neurological condition), may change the conductibility of the electrical impulse in the left atrium. the conductibility of the electrical impulse at this level does not seem to be influenced by its actual dilation, but by the impairment of the intra-atrial and inter-atrial conduction pathways. caution must be given when p wave is analyzed in dogs with concurrent cardiologic and neurologic condition. no conflicts of interest reported. newfoundland dogs were prospectively recruited among those undergoing screening for congenital and acquired heart disease. screening includes patient history, physical examination, and systemic arterial pressure measurement by doppler flow meter and transthoracic echocardiography (m-mode, 2d and echo-doppler). screening is performed on conscious dogs of at least 1 year of age. dogs without historical, clinical, electrocardiographic and echocardiographic signs of cardiovascular disease were included in the study. unpaired, two-tailed student's t-test and linear regression were performed to evaluate the influence of gender, age and body weight (bw) on echocardiographic parameters. echocardiographic measurements were compared to previously reported reference values. the reference limits of echocardiographic parameters in the newfoundland dogs were calculated. forty-six healthy adult newfoundland dogs of both genders (20 males and 26 females), 1 to 6 years of age (mean 2.6 ae 1.6 years), 40 to 72 kg (mean 54.7 ae 8.84 kg) fulfilled the inclusion criteria. significant but weak correlations were detected between aortic diameter (ao) and age (p = 0.012, r 2 = 0.186), left atrial to aortic ratio (la/ao) and age (p = 0.047, r 2 = 0.192), e-point to septum separation (epss) and bw (p = 0.038, r 2 = 0.117), m-mode left ventricular internal diameter (lvid) in diastole (d) and systole (s) and bw (respectively p = 0.002, r 2 = 0.201 and p = 0.006, r 2 = 0.158), and between ao and bw (p = 0.008, r 2 = 0.203). none of the echocardiographic measurements was statistically different between males and females. left ventricular internal diameter in diastole, lvids, ao, epss increased with bw, as expected. the aorta appears to become wider with advancing age. a proportion of the studied population had m-mode parameters below the allometric scaling reference range, suggesting that this method can over-estimates m-mode parameters in this breed. these findings stress the importance to report newfoundland breed specific normal ranges for echocardiographic parameters. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease usually diagnosed by thoracic ct-scan that mainly affects west highland white terriers (whwt). pulmonary hypertension (ph), a severe co-morbid condition with a challenging diagnosis, may develop in cipf dogs. the ratio between the right pulmonary vein and pulmonary artery (pv/ pa) has been described as an echocardiographic indicator of ph in cipfdogs. this study was intended to investigate whether ct-scan angiography cardiac findings are 1) altered in dogs with cipf compared to healthy control dogs and 2) correlated with pv/pa measured by echocardiography (pv/pa us ). thoracic cta images from 6 whwt with cipf (group a) and 9 healthy controls from various breeds (group b) were retrospectively reviewed by one observer. all measurements were obtained in transverse post-contrast images displayed in a soft tissue window. pv and pa were measured dorsal to the right atrium, perpendicular to the long axis of these vessels. in addition, pulmonary trunk (pt) was assessed just ventral to the division of pulmonary arteries, perpendicular to its long axis. ascending aorta (ao) was also measured perpendicular to its long axis. transverse reformatted images were obtained to have a view equivalent to the standard 4 chambers-echocardiographic view where right ventricle (rv) and left ventricle (lv) were measured. three ratios were calculated pv/pa ct , pt/ao and rv/lv, compared between groups and correlated with pv/pa us in both bi-dimensional (bd) and m-modes (mm). statistical analyses were performed with xlstat â software. values are given as mean ae sd. statistical significance was set at a p ≤ 0.05. pv/pa ct was lower in group a (0.64 ae 0.18) in comparison to group b (1.00 ae 0.23, p = 0.008) and correlated with pv/pa us (bd: r = 0.796, p = 0.001; mm: r = 0.730, p = 0.003). pt/ao was higher in group a (1.18 ae 0.08) compared to group b (0.92 ae 0.14, p = 0.001) and correlated only with pv/pa us measured in bd mode (r = -0.623, p = 0.02). the rv/lv ratio was increased in group a (0.91 ae 0.11) in comparison to group b (0.70 ae 0.08, p = 0.001) and a correlation between rv/lv and pv/pa us was found (bd: r = -0.726, p = 0.005; mm: r = -0.657, p = 0.01). in conclusion, in whwt with cipf, pv/pa ct , pt/ao and rv/lv ratios measured on thoracic cta images are correlated with pv/pa us and may serve in the assessment of ph. no conflicts of interest reported. companion animals presenting to the emergency room in distress need to be assessed rapidly and accurately to implement life-saving therapies. focused cardiac ultrasound (focus) can be a useful adjunct to the physical examination in assessing dyspneic animals in the emergency room. rapid bedside ultrasound evaluations performed by ec are commonly used in human medicine, however feasibility and utility of focus by ec in veterinary medicine has not been fully evaluated. the purpose of this study is to determine the baseline accuracy of focus performed by ec and whether or not a basic training session could improve accuracy compared to evaluation by a cardiology specialist. fifteen ec including 6 boarded emergency-critical care specialists and 9 emergency res-idents performed focus on four animals; a normal cat and dog, and a cat and dog with severe valvular and myocardial heart disease, respectively. ec semi-quantitatively assessed 6 thoracic and echocardiographic parameters including left atrial dimension, left ventricular systolic function and wall thickness, right heart dimension, and presence or absence of pleural or pericardial effusion before and after a structured didactic lecture and hands-on practical session. primary outcome was the level of agreement with examination performed by a cardiologist. level of agreement regarding ec assessment of all parameters improved from 0.70 to 0.78 after training (p < 0.01). level of agreement concerning left atrial diameter improved from 0.52 to 0.75 (p < 0.01). ec confidence in their overall focus evaluation and findings improved from 51% to 70% (p < 0.0001). in summary, ec accuracy and confidence in semi-quantitatively assessing basic cardiac parameters using focus were improved following a simple structured training session. focus might be a valuable tool to rapidly assess simple thoracic and cardiac parameters in the emergency setting. no conflicts of interest reported. obesity is an increasing health problem in dogs. success of weight-loss programs is often limited by compliance issues. the purpose of this study was to determine the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned dogs, under typical household conditions. the objectives were 1) to evaluate weight loss parameters in dogs fed a ndwmf* and 2) to assess the owner's perception of the dog's quality of life. overweight/obese, otherwise healthy, client-owned dogs (>3/5 body condition score -bcs) were enrolled in the study (n = 162). initial veterinary evaluation included physical examination, nutritional assessment, determination of ideal body weight (ibw), and development of weightloss feeding guidelines. daily energy requirement (der) for weight loss was calculated as der = 70 x ibw kg 0.75 . initial and follow-up evaluations (monthly for 6 months) encompassed determination of body weight, bcs, body fat index (bfi), muscle condition score (mcs), and feeding practices. quality of life assessment by owners included dog's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis comprised scatterplots, regression analysis, summary statistics as appropriate for the type of analyses performed (continuous or categorical variables, distribution), and a mixed model anova to assess changes over time (with statistical significance at p < 0.05). ninety four percent of the dogs lost weight (n = 153) with an average weight loss of 14.5% (sem, 1.1%) over 6 months and an average weekly weight-loss rate of 0.73% (sem, 0.04%). the mean duration of weight loss was 127 days (sem, 4.3 days) with an average of 33 days (sem, 0.7 days) between rechecks. thirty nine percent of dogs achieved ibw (0.39, ci: 0.31-0.48). fifty five percent of dogs ate more calories from ndwmf* than the recommended der for weight loss (median fed above der=8%) and 94% of these dogs (0.94, ci: 0.87-0.98) still lost weight. thirty six percent of dogs received treats. bcs and bfi decreased significantly over time compared to baseline. owners perceived a significant increase in energy and happiness in the dogs that lost weight without changes in appetite or begging behavior. in conclusion, this clinical study confirmed the effectiveness of the ndwmf* in achieving weight loss in overweight/obese client-owned dogs in spite of higher than recommended caloric intake. owners reported significant improvements in dog's quality of life without negative side effects. * haptoglobin is a moderate acute phase protein in cats. as a part of the innate immune system its concentration rises within 24-48 hours after tissue damage. aim of the study was to validate an elisa which was recently developed for the measurement of feline haptoglobin and to compare it with a commonly used spectrophotometric assay. the concentration of haptoglobin was measured in 38 healthy and sick cats using a sandwich-elisa (tecomedical group, rheinbach, germany). the validation included the detection of intra-assay and inter-assay variation, dilution linearity, spike recovery and lower detection limit. a spectrophotometric assay (tridelta development ltd, maynooth, ireland) was used as a reference method. all samples were measured in duplicate. statistical analysis was performed using ibm â spss â statistics 20 (ibm corporation â ) and included descriptive statistics, spearman correlation (rs) and coefficients of variation (cv). the coefficients of variation were 2.3%, 2.9% and 4.6% for intra-assay variability and 5.1%, 8.8% and 11.2% for inter-assay variability. the ratio of observed to expected dilutional parallelism of 4 serum samples diluted 3 times ranged from 108 to 118%. the ratio of observed to expected spike recovery of 4 serum samples ranged from 91% to 94%. the lower detection limit was 0.19 mg/ml. the correlation between the 2 assays was significantly strong (rs = 0.94, p < 0.001). the recently available sandwich-elisa provides a high accuracy and precision and can therefore be used for the measurement of feline haptoglobin. the 3rd and 4th author (m. hennies and c. wienen) work for the company tecomedical group that developed the elisa which was evaluated in the study. they provided the kits and they helped with performing the tests, but they did not have any influence on the results and the interpretation of the data. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease that mainly occurs in the west highland white terrier (whwt) breed. the cipf diagnosis commonly relies on thoracic high-resolution computed tomography (hrct) findings and ultimately on histopathology. as those tests are not easily performed in practice, identification of measurable markers of fibrosis, that might help to diagnose and/or monitor the course of cipf, is helpful. vegf is an angiogenic regulator involved in a variety of physiological and pathological processes. in human ipf, serum vegf concentration has been shown to be higher in ipf patients compared to healthy volunteers and may reflect the severity of the lung disease. the aims of the present study were (1) to investigate the potential role of vegf as a peripheral blood biomarker in cipf; and (2) to investigate possible breed-related differences in basal vegf concentration, that might explain the high predisposition of the whwt breed for cipf.therefore, vegf was determined by elisa (canine vegf quantikine elisa kit, r&d systems) in the serum of 14 whwt with cipf confirmed by hrct and/or histopathology (median age 11 years, range 5-14), 18 healthy whwt (9, 3-17), and 85 healthy dogs of other breeds, including: 14 scottish terrier (st) (5, 1-10), 16 jack russell terrier (jrt) (7, 1-12), 15 maltese (6, 1-13), 14 king charles spaniel (kcs) (6, 1-10), 12 labrador retriever (lr) (6, 2-12) and 14 malinois belgian shepherd (6, 2-8). health status was based on clinical examination, serum biochemistry and haematology in all healthy dogs and a thoracic hrct was performed in 9/18 healthy whwt. the khi² test with the threshold 5% was used for the statistical analysis (xlstat â software). eight cipf whwt (57%) have serum vegf concentrations above the kit detection limit (39.1 pg/ml) compared to 1 whwt (0.05%) in the group of healthy dogs (p = 0.001). concerning inter-breed differences in healthy dogs, most values obtained were below the kit detection limit with only 3 kcs (21%), 3 jrt (19%), 3 lr (25%) and 1 st (7%) having vegf serum levels above 39.1 pg/ml (p = 0.147). results of the present study show that (1) vegf might be an interesting blood biomarker for cipf; (2) canine vegf quantikine elisa kit is not appropriate for measurement of serum vegf levels in healthy canine populations. no conflicts of interest reported. the total protein (tp) concentration and cell count of pleural and abdominal fluid is used to differentiate a transudate from an exudate. tp can be measured by automated wet chemistry analyser or more easily using a refractometer. the aim of this study was to assess if refractometer values of tp are useful for this purpose. retrospectively samples from canine pleural and abdominal effusions in which tp concentration was measured both with a refractometer as well using pentra 400 (abx horiba, montpellier) were included. samples were collected into heparinized tubes and analysed within 12 hours. bland-altman diagrams were created and correlation between both measurements was calculated by spearman 0 s nonparametric correlation. over a 48-months period, 93 pleural and 147 abdominal effusion samples were analysed with both techniques. median (range) tp concentrations in pleural effusion measured by refractormeter or by pentra was 33 (2-57) g/l and 29 (7-68) g/l, respectively. median (range) tp concentrations in abdominal effusions measured by refractometer or pentra was 42 (2-70) g/l and 34 (2-57) g/l, respectively. tp measurement between refractometer and pentra values were significantly correlated in pleural (r = 0.919, p < 0.0001) and abdominal (r = 0.907, p < 0.0001) effusion. the bland-altman graph showed a bias in the thorax and abdomen of 2.8 and 5.7. the refractometer is an acceptable, rapid and efficient method for determination of total protein concentration in pleural and abdominal effusions in dogs to differentiate transudates from exudates. no conflicts of interest reported. coagulation factor vii (fvii) deficiency has been reported in beagles since the 1960's. deficient dogs show a mild hemorrhagic tendency, but often remain asymptomatic and are incidentally discovered by an isolated prolonged prothrombin time due to <10% plasma fvii activity. factor vii deficiency occurs commonly in beagles, alaskan klee kais and scottish deerhounds. in these 3 breeds it is caused by a single missense mutation (c.407g>a, p.gly136glu) in the second epidermal growth factorlike domain of fvii, which drastically reduces the secretion and activation of fvii. research beagles were also commonly affected which may have pharmaco-toxicologically affected studies but specific dna screening programs have been established. we report here on the discovery of fvii deficiency in welsh springer spaniels (wss) in finland based upon a novel screening panel for~100 known mutations underlying inherited disorders in different canine breeds (www.mydogdna.com). among 31 wss initially tested, 12 were heterozygously (39%), and 1 homozygously affected for the same fvii mutation, which was confirmed by sequencing in all dogs. in order to determine whether the mutation causes fvii deficiency also in this breed, we recruited 6 littermates and their mother. none of these wss had shown an increased hemorrhagic tendency, but affecteds bled excessively following blood collection. we found that the 3 homozygous affected dogs of the litter exhibited markedly prolonged prothrombin time but normal partial thromboplastin time. they also had drastically reduced fvii activities but normal to high fviii and fix activities compared to their littermate controls. the 3 heterozygous carriers tested did not show any prolongations in their prothrombin time, but had half normal fvii activity. in conclusion, we document here the presence of fvii deficiency in wss based upon dna and coagulation activity testing. the common gly136glu mutation must have arisen prior to the separation of the very different fvii deficient breeds. there is no knowledge of an advantage of the heterozygote state. while there is only a mild hemorrhagic tendency, bleeding dogs could be treated with fresh frozen or cryo-poor plasma or human recombinant fviia. this preliminary study indicates a high carrier frequency in wss. screening by new platform dna methods for this and other ancestral defects is helpful to detect additional hereditary diseases and genetic predispositions in different breeds, while other mutations are new and restricted to one or related breeds. authors are affiliated with genetic disease screening test laboratory. remarkably little has been published on haematological and serum biochemical phenotypes of the domestic dog. information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database; similar information was collected from all dogs with normal serum biochemical profiles, considering all parameters other than glucose as inclusion criteria. normal haematological profiles were available for 6046 dogs, 5447 of which also had machine platelet concentrations within the reference interval; normal serum biochemical profiles were available from 3045 dogs, 1495 of which also had accompanying normal serum glucose concentrations. for the haematological data, 75 pure breeds plus a mixed breed control group were represented by 10 or more dogs, while for the serum biochemical data, 60 pure breeds plus a mixed breed control group were represented by 10 or more individuals. all measured haematological parameters except mean corpuscular haemoglobin concentration (mchc), and all serum biochemical analytes except sodium, chloride and glucose, varied with age. concentrations of white blood cells (wbcs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex, as did total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, and activities of alanine aminotransferase (alt), creatine kinase (ck), amylase and lipase. neutering status had an impact on haemoglobin concentration, mean corpuscular haemoglobin (mch), mchc, and concentrations of wbcs, neutrophils, monocytes, lymphocytes and platelets, as well as all serum biochemical analytes except albumin, sodium, calcium, urea and glucose. principal component analysis (pca) of haematological data revealed 37 pure breeds with distinctive phenotypes, while pca of serum biochemical data revealed over 50 pure breeds with distinctive phenotypes. furthermore, all haematological parameters except mchc and all serum biochemical analytes except urea and glucose showed significant differences between specific individual breeds and the mixed breed group. twenty-nine breeds had distinctive haematological phenotypes and 21 breeds had distinctive serum biochemical phenotypes when assessed in this way. tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. this study represents the first large-scale analysis of haematological and serum biochemical phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of haematological and biochemical traits, triangulating phenotype, breed and genetic predisposition, as well as the urgent need for breed-specific reference intervals in clinical practice. the author has received funding from bbsrc, petplan charitable trust, and cruk), but none of thesegrants were for this study. for each cat aged 10 years or older, irrespective of their suspected thyroid status, presented to eight veterinary practices in portugal, the veterinarian and the pet owner had to complete a questionnaire and the veterinarian had to take a venous blood sample (into a plain tube) from the cat, after obtaining signed owner consent. the veterinary questionnaire included history, attitude, activity, heart rate and thyroid palpation. cats aged <10 years and those diagnosed previously with hyperthyroidism were excluded. blood samples were centrifuged and the serum harvested and stored frozen until collection by the laboratory within 2 days of sampling. total t4 was measured using a chemiluminescent method (immulite 1000, siemens). cats were classified as hyperthyroid, equivocal or euthyroid based on a total t4 concentration of >51 nmol/l, 30-51 nmol/l or <30 nmol/l, respectively. repeat measurement of total t4 after 4-6 weeks was recommended for all equivocal cases. the individual cat was the statistical unit. descriptive statistics was used to summarise the data and associations between different clinical signs analysed using chi-square, fisher's exact test or the mann-whitney u test. the level of significance was set at 0.05. thirty cats were excluded from the prevalence analysis because they were aged <10 years (between 4 and 9 years, n = 11) or their age was not stated (n = 19, four of these cats were hyperthyroid). by the end of february 2014, samples had been submitted from 197 cats that met the inclusion criteria. based on the thyroid hormone analysis, there were 14/197 (9%) hyperthyroid, 28 (14%) equivocal and 152 (77%) euthyroid cats. very few follow-up blood samples were taken. hyperthyroidism appears to be not uncommon in portuguese cats. getting owners to return for follow-up blood sampling appears to be problematic. under-reporting of hyperthyroidism appears to be a significant problem in portugal, as has been reported for some other countries. thyroid palpation should form part of routine physical examinations, especially of middle aged and older cats. older cats in portugal should be screened for hyperthyroidism even in the absence of a detectable thyroid nodule. both authors are employees of msd animal health. msd animal health funded the study. msd animal health has an approved veterinary medicinal product for the treatment of feline hyperthyroidism. this product is available commercially in some eu markets but not in portugal. diabetes mellitus is one of the most commonly encountered endocrinopathies in cats and its prevalence has increased in the past. similar to human type 2 diabetes, feline diabetes is associated with comparable lesions occurring in the pancreatic islets, namely islet amyloidosis and beta-cell loss. studying the pathophysiology of feline diabetes and the molecular mechanisms through which glucose metabolism is disturbed is largely hampered by the lack of a method for the isolation of pure pancreatic islets. the aim of this project was to improve a previously established method for the isolation of pancreatic islets; in particular enhancing the purity of isolated islets in this species. cats that died or were euthanized due to severe illness other than pancreatitis or other pancreatic disease were enrolled. pancreata were perfused post-mortem with 50 ml collagenase type iv (0.5 mg/ml) through the pancreatic duct. the perfused organ was then digested for 30', 40' and 60' at 37°c in a water-bath and purified using a filtration method. islet cell viability and purity were determined by thiazolyl blue tetrazolium bromide (mtt assay) and dithizone staining, respectively. perfusing the pancreas through the pancreatic duct allowed collagenase to access the islets using anatomical structures and to improve islet yield compared to previously established protocols in this species. the digestion time of 60' provided the best islet yield. after digestion, feline pancreatic islets remained satisfactorily viable for 5 days in the culture system following regular media changes. the current study has successfully optimized the isolation, purification and culture maintenance of feline islets. the successful yield and viability of islets isolated through the suggested protocol may provide promising potential as a source of islets for diabetes research in cats. no conflicts of interest reported. nesidioblastosis describes a syndrome of acquired hyperinsulinaemia and associated hypoglycaemia secondary to focal or diffuse (non-neoplastic) beta cell hyperplasia within the pancreas. beta cell dysregulation is thought to occur secondary to pancreatic injury. this syndrome has been reported in humans with increasing frequency, but it has not previously been described in domestic pets. a six year old, de-sexed female british shorthair cat presented with acute onset weakness and mental dullness. upon initial presentation the cat was mildly hyperglycaemic (9.9 mmol/l; 3.3-6.7 mmol/l). over the following 12 hours the cat developed central blindness, tremors, intermittent seizures and opisthotonus. repeat blood sampling revealed a marked hypoglycaemia (0.8 mmol/l). an insulin level (performed on serum obtained while the cat was hypoglycaemic) was inappropriately elevated (10938 pmol/l; reference range 72 -583 pmol/l). an intravenous bolus of 5% glucose resulted in rapid resolution of all clinical signs and mild transient hyperglycaemia (12.5 mmol/l). despite frequent feeding, the hypoglycaemia (2.0 mmol/l) recurred, so an intravenous glucose continuous rate infusion was commenced. an abdominal ultrasound was unremarkable, although three cranial mesenteric lymph nodes were noted to be prominent (3 mm in width). an exploratory laparotomy revealed a firm and erythematous left limb of the pancreas. the body and right limb of the pancreas appeared grossly normal. following surgical resection of the left limb of the pancreas, the cat returned to a euglycaemic state after a brief rebound hyperglycaemia. histopathology revealed pancreatic fibrosis with marked multifocal micronodular hyperplasia of exocrine and endocrine cells, mild lymphoplasmacytic inflammation and ductular ectasia. synaptophysin immunohistochemistry confirmed nodular beta cell hyperplasia. mild granulomatous lymphadenitis and hydropic change within hepatocytes was also noted. the cat recovered uneventfully without any further intervention. it gained weight and remained euglycaemic over the following six months. while beta cell hyperplasia has been reported as an incidental histopathological finding in euglycaemic young beagles, this is the first reported case of clinically significant hypoglycaemia secondary to nesidioblastosis in a domestic pet. while this condition is rare, nesidioblastosis is being increasingly recognised in the human field and it is an important differential to consider when investigating hypoglycaemia as it cannot be differentiated from insulinoma without histopathological evaluation. age of onset may provide a clue to this non-neoplastic disease, as this cat was much younger than all previously reported cases of feline insulinoma (all > 12 years of age at diagnosis). while recurrence has been reported in humans, a favourable outcome is anticipated following partial pancreatectomy. no conflicts of interest reported. hyperthyroidism is the most common feline endocrinopathy of geriatric cats worldwide. nonetheless, data concerning the accurate prevalence of feline hyperthyroidism (fh) is scarce; and apparently exhibit geographical variation, which can be an important instrument in investigating risk factors through the analysis of exposure to different factors in areas of high and low prevalence. in europe, fh is considered more frequent in the northern than in the southern countries. the aims of this study were to determine the occurrence of fh in a region of portugal, to characterize clinical presentation and potential risk factors. during an 18-month period, 120 geriatric cats ≥ eight years from aveiro (central region of portugal) were selected. cats were excluded if presented in shock or moribund, or in treatment with drugs that might affect total t4 (tt4) serum concentration. the tt4 concentration was determined through chemiluminescence (immulite â , siemens), and diagnosis of fh established if tt4 serum concentration ≥ 4.0 lg/dl (reference values 0.8-3.9 lg/dl) associated with compatible clinical signs. information on age, gender, breed, weight, housing conditions (indoor vs outdoor), use of external parasiticides, food (dry vs canned food and flavor), use of litter box, environment, clinical signs and laboratory data was collected. all owners gave informed consent. population studied included 49 males (40.8%) and 71 females (59.2%), mainly domestic short-haired cats (88.3%). ages ranged from eight to 21 years old (11.8 + /-3.0). eight (6.7%) cats were diagnosed with hyperthyroidism. if only cats ≥ 10 years of age were considered (n = 89), prevalence raised to 9.0%. hyperthyroid population comprised four males and four females, ranging from 13 to 21 years old (15.6 + /-2.7). increasing age (p = 0.001), polyphagia (p˂0.001), weight loss associated with increased (p˂0.001) or normal appetite (p˂0.001), presence of thyroid uni or bilateral nodules (p˂0.001), vomiting (p = 0.04) and hyperactivity (p = 0.006) were significantly associated with hyperthyroidism in the geriatric population studied. environment was also significantly associated with development of fh (p˂0.001), with cats from urban or semi-rural areas at higher risk of developing the disease than cats living in a rural environment. no other significant associations were found between hyperthyroidism and other factors analyzed. in our knowledge, no epidemiologic studies on fh have been performed in portugal, a country where the occurrence is believed to be low, but in which the population of pet cats, the feline geriatric population and the clinical cases diagnosed have been increasing. conflicts of interest: the study was partially supported by laborat orio segalab s.a. and dechra veterinary products. canine diabetes mellitus (cdm) has been proposed to be a spontaneous animal model of human autoimmune diabetes, and comparative research can be undertaken to investigate the interaction between genetic and environmental factors. most epidemiological studies of cdm have been performed in northern european and north american populations. our aim was to evaluate the epidemiology and clinical features of the diabetic dog population from the canary islands, with special focus on immune-mediated disease. dogs attending our veterinary teaching hospital were included from january 2009 to january 2012. previously diagnosed and new cases were considered. prevalence was calculated as number of cdm/total number of dogs attending the hospital and incidence as newly diagnosed cases divided by the same value per year. anti-insulin antibodies were assessed by elisa. genotyping for dog leukocyte antigen (dla) and measurement of canine anti-gad65 and anti-ia2 antibodies by radio-immunoprecipitation assay were performed in dogs with suspected immune-mediated diabetes. twenty-nine dogs with cdm were identified from a mean population of 4302 (3741-4581) dogs per year (mean prevalence 0.23% and mean incidence cases per year 16 per 10,000). age at diagnosis was 9.45 years (range: 3.5-14y). most dogs were not neutered (87% females; 83% males). nine breeds were represented, including poodle (21%) and andalusian wine-cellar rathunting dog (7%). seasonality was observed in the diagnosis with peaks in december and march-april. diabetes was classified as dioestrus diabetes (55%), idiopathic/immune-mediated (21%), iatrogenic (7%) and secondary to pancreatitis (14%) or other endocrine disorders (3%). insulin-treated dogs were negative for anti-insulin antibodies (n = 19). from the suspected immune-mediated cases (n = 5), autoantibody reactivity was shown in two cases (anti-gad65, n = 1; anti-ia2, n = 1). no previously described, diabetes-risk dla-types were identified. although age, prevalence and incidence did not differ from previous studies, the high proportion of entire females likely explained the high frequency of dioestrus diabetes. the andalusian wine-cellar rat-hunting dog was identified as a high-risk breed for cdm. most of the dla-types seen have not been previously described, but at least two have been associated with increased risk of autoimmunity in dogs. further population-based studies are needed in different regions, to assess the heterogeneous nature of this disease. no conflicts of interest reported. the cortisol-dehydroepiandrosterone (dhea)-ratio is widely used in human medicine as a marker for stress however it is not clear whether it could also help in distinguishing hyperadrenocorticism (hac) from other diseases which might have a negative impact on the outcome of a dexamethasone low dose test. therefore the aim of the study was to evaluate the cortisol-dhea-ratio as an additional diagnostic marker for hac in dogs. to achieve this aim, a reference range of this ratio depending on the sex should be evaluated in healthy dogs and compared with dogs having a hac. in 55 healthy dogs (age: 1 -11.4 years) and in 20 dogs with hac (age: 7.1 -14.6 years) of different breeds the plasma concentration of cortisol (immulite system, siemens healthcare diagnostics) and dhea (beckman coulter) was measured and the ratio was calculated. all dogs were patients of the small animal clinic except five of the healthy dogs which were recruited from the institute of pharmacology, toxicology and pharmacy of the university. with these data the cortisol-dhea-ratio was calculated for male dogs (healthy dogs n = 18; dogs with hac n = 3), neutered males (healthy dogs n = 9; dogs with hac n = 5), female dogs (healthy dogs n = 21; dogs with hac n = 5) and spayed females (healthy dogs n = 7; dogs with hac n = 7). the statistical analysis was performed with sigma stat. the plasma cortisol-dhea-ratio of healthy male dogs was the lowest ratio of all sexual categories (mean average 84.8 ae 128) and it differed significantly to all other sexes (neutered males = 231 ae 138, p = 0.002; females = 244 ae 124, p < 0.001 and spayed females (183 ae 60.0, p = 0.006). the cortisol-dhea-ratio showed no significant difference between male and female dogs with hac. spayed females with hac had significantly higher cortisol-dhea-ratios (501 ae 310) than healthy spayed females (p = 0.035) but no significant differences were found in other sexual categories. this preliminary data indicates that the cortisol-dhea-ratio might not be a very promising tool for the diagnosis of hac. in addition, the significant gender-dependency of this parameter has to be considered and may generally limit its clinical usefulness. this study is financially supported by the bruns-stiftung. no conflicts of interest reported. hyperthyroidism is common in older cats. the aim of this study was to assess the prevalence of feline hyperthyroidism and potential intrinsic risk factors in a hospital population in southern germany. total thyroxine (t4) was prospectively measured by enzyme immunoassay (eia) in sera of 425 cats older than 8 years that were presented to the clinic of small animal medicine. a standardized physical examination was performed, and body condition score (bcs) and thyroid palpation score (tps) were assessed. association between signalement, bcs and tps was analyzed by student 0 s unpaired t-test, chi-square, and mann-whitney test. level of significance was set at 0.05. fifty nine cats were diagnosed with hyperthyroidism leading to a prevalence of 13.9% (ci 10.9-17.5). hyperthyroid cats were older than non-hyperthyroid cats (p < 0.0001) and more often female (p = 0.024, odds ratio 1.920). domestic short or long hair cats were more often affected than pedigrees (p = 0.015). hyperthyroid cats had higher tps (p < 0.0001) and lower weight than non-hyperthyroid cats (p < 0.0001) although bcs was not different (p = 0.290). in 11 (2.5%) cats, the elevated t4 was an incidental finding. in 3 of those, the disease was confirmed later (the others were dead due to unrelated diseases). in 152 patients, hyperthyroidism was considered a differential diagnosis and was confirmed in 32 (21.1%) cats although in 5 cats additional diagnostic means were necessary. older female domestic cats are predisposed to hyperthyroidism which is frequently diagnosed after the initial clinical suspect. in a few affected cats an elevated t4 is not present or can precede clinical signs. conflicts of interest: the study was partially funded by msd intervet. the main endocrinopathy affecting both humans and pet felines is diabetes mellitus. accurate diagnosis is the most important aspect in the future outcome of the disease. a computer based decision support system (dss) is targeted on assisting clinicians with one or more steps of the diagnostic process. the novelty of our dss emerges from the possibility of assisting both clinical and paraclinical diagnosis stages of diabetes mellitus and all common combination of disorders associated with this endocrinopathy. the motivation behind the development of such system is the desire to maximize the reliability of clinical decisions. the design of our feline diabetes mellitus dss emerges from the syndrome of polyuria-polydipsia, with the possibility of spotting the accompanying pathologies. fuzzy logic is used for dealing with knowledge representation and uncertainty. the fuzzy rules proposed to represent this knowledge emerge from anamnesis, clinician's input, clinical and paraclinical description, and confirmation diagnostic tests. clinical signs such as polyuriapolydipsia, persistent hyperglycemia, polyphagia, weight fluctuations, administration of drugs with a diabetogenic potential, were considered decisive in the pattern of diagnosis establishment. registered medical records of 29 cats, 16 males and 13 females, whit ages from 7 to 18 years old, were analyzed in order to validate the dss. using matlab software, the dss was implemented and tested. for any case with polyuria-polydipsia the system provides, via a friendly graphical user interface, the diagnosis with the highest probability. the set of diagnoses which can be generated by the dss consists in: a) diabetes mellitus; b) diabetes mellitus induced by (b.1) hypersomathotropism, (b.2) hyperthyroidism, (b.3) hyperadrenocorticism and (b.4) diabetogenic medication; c) diabetes mellitus in association with (c.1) chronic kidney failure and (c.2) heart failure; d) ketoacidodic diabetes mellitus; e) pancreatitis. the dss was applied with success on all 29 cases, revealing the following diagnoses / no of cases: (a) -8, (b.1) -2, (b.2) -1, (b.4) -8, (c.1) -3, (c.2) -3, (d) -4. an adequate treatment protocol requires an accurate and complete diagnosis. advanced computational systems accompany clinicians in their decision making, leaving a reduced space for medical errors and superfluous, expensive and time consuming tests. future work will be targeted on exploring the possibilities of combining the dss with an artificial neural network model for diabetes mellitus. this can be the foundation of a complete case oriented management system for feline diabetes mellitus and associated disorders. no conflicts of interest reported. activins are cytokines belonging to the transforming growth factor (tgf)-b superfamily. it is thought that activins may be the key intermediary in tgf-b1 mediated fibrotic response. activin a has been suggested to participate in the pathogenesis of human idiopathic pulmonary fibrosis (ipf), but studies regarding the role of activin b are still spares. canine ipf (cipf) is a chronic, incurable interstitial lung disease occurring particularly in west highland white terriers (whwts). during the disease course, acute exacerbations (aes), with poor prognosis, can occur. histopathologically aes of cipf are featured by diffuse alveolar damage, which is also a key feature in acute respiratory distress syndrome (ards). our objective was to study the expression of activin a and b by immunohistochemistry in the lung tissue of cipf whwts (n = 5), cipf whwts with concurrent ae (n = 4), and dogs of various breeds with ards (n = 4), and to compare these findings to healthy whwts (n = 3). in addition, western blot analysis of activin b from bronchoalveolar lavage fluid (balf) of cipf whwts (n = 6) and healthy whwts (n = 6) was conducted. we demonstrated that activin b, but not activin a, is strongly expressed in the altered alveolar epithelium in lungs of diseased whwts as well as in ards lungs. furthermore, activin b was detected in balf of cipf whwts, most notably in samples from dogs with ae, but not in balf of healthy whwts. this novel finding suggests that activin b participates in the pathophysiology of cipf and might act as a potential marker of alveolar epithelial damage. no conflicts of interest reported. dogs of the breed nova scotia duck tolling retriever (nsdtr) are affected by several immune-mediated diseases, in particular steroid-responsive meningitis-arteritis (srma) and an immune-mediated rheumatic disease (imrd). imrd is a systemic lupus erythematosus-related disease characterized by chronic stiffness and pain in several joints. the aim of this study was to investigate the morbidity in nsdtrs and to test the hypothesis that nsdtrs are predisposed to srma and imrd. insurance data from a swedish insurance company (agria insurance company, stockholm, sweden) from 1995-2006 was used for the study. approximately one third of swedish dogs are insured by agria and the insurance database is a validated tool for epidemiological studies. assessment of morbidity was based on veterinary care events. disease diagnoses were grouped in both general and specific disease categories. individual diagnoses that were likely to represent imrd were combined. morbidity was defined as incidence rates and presented as number of cases per 10 000 dog years at risk (dyar). relative risk (rr) for nsdtrs compared to other breeds combined was calculated. the study included 445 336 dogs, 2890 were nsdtrs. the most common general causes of veterinary care for nsdtrs were injuries followed by gastrointestinal and musculoskeletal disorders with significant increased risk (rrs between 1.2 and 1.3) for nsdtrs compared to other breeds. the highest relative risk for nsdtrs was for systemic lupus erythematosus (rr 19.0). compared to other breeds, nsdtrs had an increased risk for srma (rr 11.5) and imrd (rr 11.8) with an incidence rate of 19.6 cases per 10 000 dyar for srma and 8.8 cases per 10 000 dyar for imrd. the incidence rate for srma and imrd in nsdtrs were also compared to dogs of other retriever breeds. the comparison revealed that nsdtrs also had a significant increased risk for both srma (rr 20.8) and imrd (rr 10.1) when compared to other retrievers only. this study is the first to investigate the morbidity for imrd in nsdtrs, which is important for further research and breeding practice. for several reasons the incidence rates might be underestimated and exact numbers should be interpreted with caution. however underestimation of incidence rates should not differ between dogs of different breeds, therefore not affecting the risk calculations. it can be concluded that nsdtrs are predisposed to the diseases srma and imrd with an increased risk compared to other breeds and to other retrievers. brenda n. bonnett consults with agria insurance company on various projects. agria insurance company has also funded work leading to the development of the insurance data base that my study was based on. canine infectious respiratory disease (cird) is a multifactorial contagious disease caused by respiratory viruses and selected bacterial pathogens. cird has been shown to be a predisposing factor in the development of bacterial pneumonia (bp) in dogs housed in dense populations such as kennels and rehoming centers. the aim of this study was to determine the prevalence of viral co-infection and to assess its effects on disease severity in household dogs diagnosed with bp. a prospective cross-sectional observational study was conducted and 20 dogs diagnosed with bp caused by opportunistic bacteria were included. 13 dogs with chronic (> 30 days) tracheobronchitis caused by bordetella bronchiseptica were included as controls for virus analysis. diagnosis was confirmed by thorough clinical examinations as well as with cytological and bacterial analysis of bronchoalveolar lavage (bal) or transtracheal wash (ttw) samples. canine parainfluenssavirus (cpiv), canine adenovirus, canine herpesvirus, canine distempervirus, canine respiratory coronavirus (crcov) and canine pneumovirus were analysed in bal or ttw samples using rt-pcr assay. cpiv was detected in 7/20 (35%) and crcov in 1/20 (5%) respiratory samples in dogs with bp. respiratory viruses were not detected in dogs with chronic tracheobronchitis. there were no significant differences in the duration of hospitalization (p = 0.427) or arterial pao 2 at presentation (p = 0.343) between bp dogs with and without a viral co-infection. these results indicate that co-infections with respiratory viruses are common also in household dogs with bp. additionally, viral co-infections did not cause a more severe course of bp. the author's researcjh is financially supported by the finnish foundation of veterinary r and the finnish veterinary foundation. esvim-p-4 causes of canine anemia in taiwan: a five-year retrospective survey. e.c.y. lin 1 , p.c. liu 1 , l.l. chueh 1 , b.l. su 2 . 1 graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, 2 institute of veterinary clinical sciences, national taiwan university, taipei, taiwan anemia is a common hematologic disorder in dogs, however, few data are available regarding epidemiology and causes in taiwan. to investigate the causes of anemia, 3174 anemic cases (pcv< 37%) collected between january 2008 and december 2012 at national taiwan university veterinary hospital (ntuvh) were analyzed. most dogs (72.1%, n = 2889) presented with a mild form (25%≦pcv<37%), which was followed by a moderate form (15%≦pcv<25%; 20.8%, n = 660) and a severe form (pcv<15%; 7.1%, n = 225). among the 2037 dogs with identifiable causes, 70.4% (1435 dogs) were induced by single cause, whereas 29.6% (602 dogs) by multiple causes. neoplasia-related anemia (n = 460), infectious pathogens-related anemia (n = 287), renal disease-related anemia (n = 251) and post-surgery/ traumarelated anemia (n = 182) account for 32.1, 20.0, 17.6 and 12.7% of single-cause cases, respectively. furthermore, 123 of them (8.6%) presented with severe anemia. severe anemia primarily resulted from infectious disease-related anemia (61.8%), followed by imha (11.4%), and tumor-related anemia (11.4%). of the 76 infectious disease-related severe anemic dogs, the most common diagnosed pathogen was babesia gibsoni (88.2%, n = 67), followed by ehrlichia canis (6.6%, n = 5)and babesia canis (3.9%, n = 3). taken together, tumors, infectious diseases, and renal failure are the most frequently causes of canine anemia in taiwan, furthermore, b. gibsoni appeared to be the most important infectious pathogen causing severe anemia which may be associated with the climate in this geographical area. no conflicts of interest reported. bordetella bronchiseptica (bb) is one of the primary causative agents of canine infectious respiratory disease (cird). this contagious disease, commonly seen in young dogs, is often self-limiting, although a wide range of respiratory signs can be found, from mild illness to severe pneumonia leading to death. although mycoplasma cynos (m. cynos) was recently identified as an emerging and possibly lethal pathogen in cird 1 , the role of m. canis and m. cynos as primary respiratory pathogens still remains unclear. detection of these bacteria is now improved by quantitative polymerase chain reaction (qpcr). in dogs with cird due to bb, the frequency of co-infection with mycoplasma spp, in par-ticularm. cynos, and their possible role in the severity of the clinical signs are unknown. the aim of the present study was to investigate the presence of m. canis and m. cynos in a population of dog infected with bb, compared with 2 other populations: healthy dogs and dogs with bacterial bronchopneumonia where bb was not involved (bbp). therefore, bb, m. canis and m. cynos were detected by qpcr in the bronchoalveolar lavage fluid (balf) sample in 16 dogs with bb (mean age = 0.9 y, mean bw = 8.8 kg), 10 dogs with bbp (3.9 y, 15.0 kg), and 10 healthy dogs (6.1y, 20.2 kg). bordetellosis was diagnosed based on clinical findings together with demonstration of pleiomorphic cocci/coccobacilli adhering to the cilia of the epithelial cells on cytospin balf preparations, and positive qpcr on balf. a clinical severity index (csi 0 to 12) was assigned based on clinical signs (cough 0-2, dyspnea 0-2, lethargy 0-1, fever 0-1), thoracic radiographic pattern (0-3), and balf score (0-3). bbp was diagnosed based on clinical findings, balf cytology and culture. m. canis was indifferently detected in healthy (5/10, 50%), bbp (4/10, 40%) and bb dogs (3/16, 19%) while m. cynos tended to be more frequently detected in bb group (8/16, 50%) than in healthy (2/10, 20%) and bbp dogs (1/10, 10%) (khi² test, p = 0,068). in bb dogs, no correlation could be detected between csi and presence of m. cynos (khi² test p = 0,26). in conclusion, the present data suggest that, in cird, coinfection with bb and m. cynos is frequent, but is not correlated with clinical disease severity. further studies are required to investigate whether coinfection of bb and m. cynos deserves specific therapeutic considerations. no conflicts of interest reported. canine idiopathic eosinophilic bronchopneumopathy (ebp) is a disease characterized by eosinophilic infiltration of the lung and bronchial mucosa in young adults. aetiology remains unclear although immunologic hypersensitivity is clearly suspected, while inciting antigens are generally unidentified. in humans as in cats, infections with mycoplasma spp. have been discussed as potential triggers in inflammatory bronchial disease 1,2 . bordetella bronchiseptica (bb) is a recognized pathogen agent of canine infectious tracheobronchitis. detection of bb and mycoplasma spp, especially mycoplasma cynos (m. cynos), and their potential role of in canine inflammatory bronchitis, have not been investigated. the aim of the present study was to investigate the frequency of bb, mycoplasma canis (m. canis) and m. cynos in canine ebp. therefore, presence of bb, m. canis and m. cynos were retrospectively assessed by quantitative polymerase chain reaction (qpcr) in bronchoalveolar lavage fluid (balf) samples from 18 dogs with ebp (mean age = 4.5 y, mean body weight = 21.1 kg) as well as in 8 dogs with aspecific chronic bronchitis (7.3 y, 22.3 kg). based on clinical signs, a clinical severity score (css, 0-5) was assigned each ebp dog. although all balf culture and cytology were negative for this bacteria, bb was more frequently detected by qpcr in ebp dogs (5/18, 28%) than in cb dogs (0%) (khi² test, p = 0,009). presence of bb in ebp dogs was independant of age but significantly associated with css (khi² test, p = 0,041). results of qpcrwere positive for m. canis and m. cynos in 6 (33%) and 2 (11%) ebp dogs and in 1 (13%) and 1 (13%) cb dogs, respectively. there was no difference between the 2 groups for any of the organisms. any relation between age or css and presence of m. spp in ebp dogs was observed. in conclusion, m. canis and m. cynos do not seem to be predominantly involved in the pathogenesis of canine ebp. however, bb is more frequently detected in balf from ebp dogs than from dogs with aspecific cb and its presence is associated with clinical severity. whether bb is able to trigger eosinophilic inflammation or is only more easily collected in an inflamed environment is unclear. but ebp dogs could potentially act as bb carriers and source of infection. therefore, bb should be systematically searched for in canine ebp cases and treated accordingly. no conflicts of interest reported. distal renal distal renal tubular acidosis (drta) was recently reported in three dogs with imha. the purpose of this study was to explore the hypothesis that drta is an underdiagnosed concurrent disorder in dogs with imha. we report the clinical presentation and outcome of three dogs where the combination of imha and drta was strongly suspected. the medical records of dogs diagnosed with imha at the university of edinburgh hospital for small animals between january 2008 and may 2013 were reviewed to identify cases where venous blood gas analysis and urinalysis had also been carried out. for the purpose of this retrospective study imha was defined by the presence of anaemia with pcv < 30%, and one or more of the following criteria; a positive slide agglutination test, positive coombs' test or moderate to marked spherocytosis. the criteria for diagnosis of drta included moderate to marked hyperchloremic metabolic acidosis with a normal anion gap; urine ph (> 6.0) in the face of metabolic acidosis; hypokalaemia (< 3.5 mmol/l). fifty-seven records were evaluated, with 39 cases being excluded due to insufficient clinical information, including inability to determine urinary ph due to the severity of pigmenturia in four cases. of the 18 cases where there was sufficient clinical data to assess the likelihood of drta only one case fulfilled all the criteria; two cases fulfilled all but one of the criteria and drta was strongly suspected based on clinical progression and persistence of urine ph > 6 in the face of severe metabolic acidosis. of the three cases where concurrent imha and drta was suspected, two survived to discharge; one was still alive at the time of writing (13 months after discharge) and the other was euthanased 2 months after discharge following the development of multiple joint effusions and skin lesions suggestive of sle. venous blood gas analysis and assessment of urine ph should be considered in all cases of imha to exclude the possibility of concurrent drta, particularly where persistent hypokalaemia is detected. prospective evaluation of a larger cohort of imha cases is required to determine the actual incidence of concurrent drta. no conflicts of interest reported. persistent renal proteinuria is considered an early marker of chronic kidney disease (ckd) and it is listed among the initiation factors and progression factors according to kdoqi guidelines. nevertheless, few data are available about the prevalence of proteinuria in cats affected with ckd, in which it is assumed that nephropathy is mainly characterized by tubulointerstitial damage. the aim of this study is to determine the prevalence of proteinuria in cats affected with ckd and to valuate the relations between urine protein to creatinine ratio (upc) or iris substaging by proteinuria, towards purebred, sex, age, haematology, biochemistry and urinalysis. wilcoxon test, linear regression and chi-square test were used for the statistical analysis. data from 251 cats were considered. non-renal proteinuria was an exclusion criterion. proteinuric cats (upc>0.4) were 15.4% in ckd cats, while 15.9% could be substaged as bordeline proteinuric (0.2) in cats, proteinuria tends to increase with aging (p < 0.0001) and with worsening of the nephropathy (p = 0.002). proteinuria was related to the anaemic state in ckd cats: upc significantly increases with rbc count, hb, ht and mch decreasing (p < 0.0001 and p = 0.049 respectively). proteinuria tends to increase with wbc count (p = 0.0001) and neutrophils increasing (p = 0.0001), while tends to decrease with lymphocytes increasing (p = 0.008). furthermore, upc significantly increases in presence of an inflammatory serum protein electrophoretic pattern. upc tends to increase with phosphorus and alp increasing (p < 0.0001 and p = 0.0005 respectively); while the role of phosphorus in ckd is well known, the increase of alp is questionable: it has been hypothesized that higher alp levels in ckd could be related to b-alp increase due to bone remodelling in secondary renal hyperparathyroidism. considering urine parameters, upc increases when urinary specific gravity and ph decrease (probably related to worsening of ckd and development of a metabolic acidosis) and when glicosuria is present, regardless of the cause. furthermore, proteinuria increases in presence of rbc in urinary sediment and in samples where casts were observed, in particularly when rbc casts (considered always pathological and indicative of glomerular damage) were present. upc values assessed in proteinuric cats and data analysis suggest the need of deepen the analytical variability of upc and the opportunity to reconsider the intervals of substaging by proteinuria in cats. no conflicts of interest reported. the aim of this retrospective study was to evaluate: a) the relations between urine culture results and urinalysis parameters; b) the results of the antimicrobial susceptibility tests. urine samples were collected by cystocentesis from 252 dogs and 52 cats, whose diagnostic workup included a differential diagnosis of uti: all samples underwent a complete urinalysis, upc ratio assessment and urine culture. infected vs sterile results were related to urine physical, chemical parameters and observations from urinary sediment analysis. statistical analysis was performed using jmp 7.0 (sas institute inc.). a p value <0,05 was considered significant. urine culture resulted positive in 104 dogs (41%) and 18 cats (33%). the presence of uti was significantly related to urine physical properties (color and turbidity), usg and leukocyturia: infections tended to be more frequent in urine samples characterized by a light yellow color, cloudy or sub-limpid aspect and low usg. nevertheless, urine was limpid in 30% of infected samples, and a normal usg was found in 28.6% of dog's uti but only in 4.8% of cats. although leukocyturia tends to become higher in infected samples both in dogs and cats (p < 0.0001), in 23.1% of infected sediments wbc count was normal. haematuria detected by dipstick was significantly related to uti in dogs but not in cats, nevertheless the rbc count in sediment was not related to infection in both species: rbc count was normal in 30.4% of infected feline samples and in 36.4% of canine samples. no significant relation between presence or absence of uti and albuminuria, bilirubinuria, glycosuria was detected, while upc tends to become significantly higher in dogs. although the chi-square test showed a significant relation between infection and the detection of bacteria in urinary sediment, a pseudobacteriuria was found in 20.5% of samples; furthermore bacteria weren't observed in 25.1% of infected samples (usg<1.013). e. coli was isolated in the majority of samples (45,9%), compared to other species: staphylococcus(13.6%), proteus (10.2%) and streptococcus. (8.5%). the urinalysis pitfalls and the high antibiotic resistance verified towards the most widely used molecules (penicillins, cephalosporins, quinolones) strongly indicates the importance to perform antimicrobials susceptibility tests to avoid the risk of failure associated with the use or abuse of empiric therapies in utis. no conflicts of interest reported. azotemia in dogs with chronic heart failure may reflect impaired renal function not only because of inadequate renal perfusion, but also due to organic renal injury. impaired renal function is observed in 50% of dogs with heart failure. altered renal hemodynamics due to decreased cardiac output results in renal hypoperfusion, and resultant elevation of blood urea nitrogen and creatinine, defined as azotemia. azotemia is a prognostic factor in dogs with mitral regurgitation, therefore, preservation and/or restoration of renal function is thought to improve prognosis. medical treatment for heart failure, however, includes angiotensin converting enzyme inhibitors and loop diuretics, which has been shown to increase the risk of developing azotemia. we hypothesized that mitral valve repair surgery ameliorates renal function by improvement of systemic hemodynamics. the change in renal function in dogs with mitral regurgitation was assessed by evaluating time-dependent changes in glomelular filtration rate by inulin clearance before and after cardiac surgery. eighteen dogs with severe mitral regurgitation with azotemia (plasma urea nitrogen level > 28 mg/dl, plasma creatinine level >1.9 mg/dl) were included in this study. the glomerular filtration rate in all dogs were evaluated by determining inulin clearance before and 3 months after surgery. serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, and plasma creatinine concentration were measured at each time point as well as during the initial staging of heart failure based on the international small animal cardiac health council (isachc). left atrial/aorta ratio by echocardiography and vertebral heart size by thoracic radiographs were also measured. glomerular filtration rate significantly increased 3 months after surgery (40.0 ml/min/m 2 [25.6 -123.0], 2.7 ml/min/kg [1.0 -5.3]) compared to before surgery (38.4 ml/min/m 2 [12.6 -50.3], 2.2 ml/min/kg [0.7 -3.8]) (p < 0.05). the isachc stage of heart failure was improved at 3 months after surgery compared to before surgery. in addition, serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, la/ao and vhs significantly decreased after surgery (p < 0.05). the use of diuretics decreased after mitral valve repair surgery and consequently, a decrease in plasma urea nitrogen and creatinine levels were observed. therefore, this suggests that the main cause of azotemia in dogs with mitral regurgitation may be due to inadequate renal blood flow and exacerbation by the use of diuretics. no conflicts of interest reported. glomerular filtration rate (gfr) is generally considered to be the gold standard measurement of kidney function. gfr can be calculated by measuring serum iohexol clearance using concentrations at 2,3 and 4 hours following a bolus injection. for validation, serum samples were spiked at low (0.017 mg/ ml), medium (0.27 mg/ml) and high (2.16 mg/ml) iohexol concentrations. they were analysed, along with standard calibration curves (8 concentrations ranging from 0.017 to 2.16 mg/ml), using deltadot's label-free high performance capillary electrophoresis (hpce) system. data were analysed using deltadot's general separation transform (gst). clinical and spiked serum samples were also sent for analysis by mass spectrometry (ms) at a reference laboratory (14 samples for comparison). concentrations obtained by hpce and ms were compared in a bland altman plot. gfr for clinical samples was calculated from the measured iohexol concentrations using the method reported by bexfield (2008) . a validated method was produced, with a lower limit of detection of 0.009 mg/ml and an lower limit of quantification of 0.017 mg/ml. the upper limit of quantification was 2.16 mg/ml. the standard curve had excellent linearity (r 2 = 0.993). maximum inaccuracy was less than 11.5% of the true value, except at lloq, where it was within 24.2%. average within day variability was less than 13.1% at all levels, while between day variability was less than 5.6%, except at lloq, where it was less than 15.4%. agreement between the results obtained by measurement with hpce and ms was good (bias 1.3%, lower and upper limits of agreement of -15.4 and 18.0%, respectively). method specificity was confirmed by the absence of matrix effect in six serum specimen obtained from clinical dogs. clinical samples were analysed and gfr reported with a 3 day turnaround time. in conclusion, hpce provides an accurate and precise method for measuring iohexol in canine serum. conflicts of interest: l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health; s williams is an employee of the rvc and works in collaboration with deltadot ltd; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd. esvonc-p-1 cystic pancreatic neoplasia in cats. c.m. borschensky 1 , k. steiger 2 , a. staudacher 3 , m. schlitter 2 , i. esposito 2 , h. aupperle 1 . 1 laboklin gmbh&co. kg, bad kissingen, germany, 2 tu m€ unchen, institute of pathology, m€ unchen, germany, 3 veterinary clinic dr. staudacher, aachen, germany pancreatic neoplasms in the cat mostly exhibit a solid growth pattern and are diagnosed as carcinomas. in contrast, only few reports about cystic pancreatic lesions exist. until now, only benign cystic pancreatic lesions are described in the literature. according to the histological pattern, they have been termed as cysts, (acinar) cystadenoma or pseudocysts. in man, cystic pancreatic neoplasms are classified according to the localisation (intra-/extraductal), growth pattern and differentiation (mucinous, (tubulo)papillary, serous, acinar). the aim of this study was to characterise feline pancreatic neoplasms in more detail, based on the human classification system with a special view on cystic lesions. pancreatic masses sent to laboklin from 19 domestic cats (7-14 years) were investigated routinely macroscopically and by histological methods (h.&e. stain). the neoplasms showed a cystic (n = 8) or solid (n = 11) pattern. cystic pancreatic tumors were up to 7 cm in diameter and were classified as benign variants in five and malignant variants in three cases. based on the human classification system, they were classified as tubulopapillary (n = 2), acinar (n = 2) and mixed (n = 1) adenomas and mixed carcinomas (n = 3), respectively. solid pancreatic nodules were diagnosed as carcinomas with a tubular (n = 5) or acinar (n = 6) differentiation pattern. in summary, the gross structure (solid versus cystic) seems to be of prognostic relevance. in contrast to solid tumors, cystic pancreatic lesions in the cat behave benign in a higher percentage of cases, resulting in a better prognosis. therefore, surgical excision of these cystic masses can be recommended. with respect to the human classification system, three different subtypes of cystic pancreatic neoplasms were detected in the cat that have not been described before in veterinary medicine: tubulopapillary, acinar and mixed. to best of our knowledge, this is the first report of cystic adenocarcinomas in feline pancreas. further corresponding clinical and histological investigations are needed for a better diagnostic (ultrasound, mri) and prognostic characterisation of cystic lesions in feline pancreas. no conflicts of interest reported. the immunohistochemical detection of cyclooxygenase-2 (cox-2) expression in canine mast cell tumours was recently described by our team (prada et al., 2012) . however its prognostic value needs to be established. the aim of the present work was study the prognostic value of cox-2 expression by investigating the relationship with several clinical and pathological variables including the overall survival (os) time. we included 57 dogs with mast cell tumours (18 grade i; 21 grade ii and 18 grade iii). cox-2 immunohistochemical expression was carried out by a streptavidin-biotin method. for the cox-2 immunoreactivity evaluation were considered the number of positive cells (cox-2 extension), the intensity and the score of cox-2. the following clinical and pathological features were considered: animal age, sex, tumour anatomical location, tumour size, skin ulceration, histological grade, histological safety margins and number of mitosis. cox-2 expression was correlated with the clinical and pathological data and with the overall survival. cox-2 intensity was statistical significantly associated with skin ulceration (p = 0,009); histological grade (p = 0,006) and absence of histological safety margins (p = 0,043), high mitotic number (p = 0,024) and with overall survival (p = 0,02). both cox-2 extension and cox-2 immunohistochemical score present no statistical relationship with the variables considered neither with the overall survival. our results suggest that cox-2 have an important role in dog mast cell tumours progression and could constitute a promising therapeutic target in this neoplasia. however, our study also demonstrated that in mcts, is the cox-2 intensity that has the prognostic value, not the number of cox-2 positive cells (cox-2 extension) and not the cox-2 immunohistochemical score. consequently, cox-2 intensity should be elected for evaluating the cox-2 positivity in mcts immunohistochemical studies. merial provided financial support for immunohistochemical analayis. the research centres has also received financial suppoprt from cecav, ceca and citab. dogs which were radiated for a subcutaneous sarcoma between 2006 and 2011 were included. medical records were reviewed and patient characteristics, treatment protocols, adjuvant therapies and outcome were analysed. follow-up information was obtained from medical records and by phone conversations with veterinarians or pet owners. thirty-two dogs were included into this study. mean age was 9 years and mean body-weight was 32 kg. male dogs were slightly overrepresented (59.4%). curative intent radiotherapy was applied in 22 dogs and palliative intent in 10 dogs with a mean total dose of 51 and 28.4 gray, respectively. in 17 dogs microscopic disease was radiated. five dogs received liposomal doxorubicin concomitantly with radiotherapy, two received adjuvant doxorubicin and one intralesional cisplatin. overall median survival time was 914 days with curative and 513 days with palliative treatment. overall median survival time in dogs with macroscopic disease was 369 days and in patients with microscopic disease it was not reached. radiotherapy was generally accepted as new treatment modality by pet owners and referring veterinarians. comparable to the literature, best outcome was achieved for dogs radiated with microscopic disease conflicts of interest: no conflicts of interest reported. oncept â , is indicated for the treatment of stage ii or iii oral melanoma after local control with survival times significantly increased following vaccination. a similar improvement in survival times has also been reported with digit melanoma. medical records of dogs diagnosed with melanoma between march 2009 and december 2013 were retrospectively evaluated. inclusion criteria were a histopathological diagnosis of melanoma, surgical excision of the tumour, and vaccination using the oncept â vaccine. 34 dogs met the inclusion criteria. nificant renal involvement in dogs with cvl. this result indicates staffordshire terrier (2), bouvier, giant schnauzer, maltese, irish setter, kerry blue terrier, golden retriever, scottish terrier, and great dane (1 of each). 6 dogs had stage ii digit melanoma with an equal sex distribution and a median age of 7.8 years (range 3-10). currently 5 still alive and one dead, the latter following surgery for resection of a rib osteosarcoma the median survival time of the dogs still alive is 25.5 months (range 5-40) versus 13.4 months (range 2-25) for the dogs that have died. none of the dogs showed any adverse effect to the vaccine infected macrophages can cause injury in different organs, including the kidney. cvl is known as a common cause of glomerulonephritis. thus, this study aimed to investigate and characterize the renal lesions in dogs seropositive for leishmania sp. in brazil. this project was approved by the animal ethics committee of uece, brazil. twenty adult dogs seropositive for cvl from center for zoonosis control were randomly selected for this experiment. cvl was diagnosed by immunofluorescence and elisa. urine and blood sampling and kidney harvesting were performed immediately after euthanasia that the glomerulonephritis is a common sequelae related to leishmaniasis infection. even dogs in stage 1 of ckd showed significant renal histopathological changes. animals infected with leishmania sp. may have severe renal damage and risk of progressive chronic kidney disease even when no increase of creatinine levels or proteinuria is detected.conflicts of interest:the authors received funding to pay the phd scholarship of one student (conselho nacional de pesquisa e desenvolvimento -cnpq-brazil) and received a research grant from fundac ßão cearense de apoio ao desenvolvimento cient ıfico e tecnol ogico -funcap. anaplasma phagocytophilum, the causative agent of canine granulocytic anaplasmosis, is an obligatory intracellular bacterium transmitted by ixodes ticks. transmission via blood transfusion has rarely been described in human medicine and once in a dog. in the berlin/brandenburg area the seroprevalence rate in dogs was 43% regardless of health status.the aim of this study was to evaluate pcr screening results for a. phagocytophilum in canine blood donors between 2006 -2012 in order to estimate the risk of transfusion-transmitted infection.917 edta blood samples from 517 dogs were submitted for a. phagocytophilum real-time pcr testing (targeting the msp2gene). altogether 158 dogs were tested up to 11 times. clinical and laboratory data were examined before each donation. statistical analysis was performed using spss 17.0.the pcr test was positive for 21 of the 917 samples. none of the dogs tested pcr positive more than once. positive results were most often detected in june (8), may (5), and july (3), but also in five other months. in three dogs a mild increased in rectal temperature (≥ 39,0°c) was documented. mild laboratory abnormalities were noted in 11 dogs: thrombocytopenia (3), leukocytosis (2), leukopenia (2), anemia (1) and hyperproteinemia (6/18); four dogs had more than one abnormality. there was no significant difference between the pcr negative and positive blood samples in regard to laboratory abnormalities.altogether, 2.3% of blood samples from healthy canine blood donors were pcr positive for a. phagocytophilum. therefore, blood donors should be screened by pcr in endemic areas all year round. no conflicts of interest reported. the study population consisted of 358 cats, including 98 control cats recruited from 29 veterinary practices across the country. among the 260 disease cats, 127 cats presented urtd, 149 cats had conjunctivitis and 153 cats suffered chronic gingivostomatitis, many of them presenting more than one clinical sign. pcr for the above-mentioned pathogens was performed from pooled conjunctival and oropharyngeal swabs for each cat. a questionnaire regarding signalment (age, breed, sex, neuter status), environment (indoor, number of cats in household) and vaccination history was obtained. data was analysed by multivariable logistic regression with alpha equal to < 0.05.the prevalence for the four pathogens has been previously reported in detail. briefly, the prevalence among the four groups (including controls) ranged from 6 to 28% for fhv-1, 15-58% for fcv, 2-20% for chlamydophila felis and 20-46% for mycoplasma felis.in the univariate analysis, age, neutering status, being purebred, indoor keeping, number of cats in the household and body weight were variably associated with the different groups of disease and the presence of the pathogens. in the multivariable analysis, only the following factors remained significant. in the multivariable analysis, only the following factors remained significant: urtd was significantly associated with positive results for fhv-1, chlamydophila felis and mycoplasma felis (in addition to being male and not castrated); conjunctivitis was significantly associated to positive results for fhv-1 and chlamydophila felis (in addition to being young, not castrated and purebred) and cgs was significantly associated to positive results for fcv (in addition to being young, male and purebred). not being properly vaccinated was a significant risk factor only when all three groups were analyzed together. the number of cats in the household was an independent risk factor for detecting each of the pathogens studied. the age was also a significant factor in cats with fcv and chlamydophila felis, being older cats predisposed to fcv and younger cats predisposed to chlamydophila felis.the present study describes important epidemiological data for cats presenting urtd, conjunctivitis and/or cgs, and emphasizes the complex interrelationships occurring among the different pathogens. our results also support the role of fcv in cats with chronic gingivostomatitis.conflicts of interest:the study was funded and designed by merial laboratories. reports of methicillin-resistant staphylococci (mrs) in animals have become more frequent in last years. various studies have demonstrated the transmission of mrsa between animals and humans in daily contact with animals, however there is only limited data so far available on the transmission of methicillinresistant coagulase-negative staphylococci between animals and humans. the objective of this study was to investigate the frequency of methicillin-resistant staphylococci (mrs) carriage in healthy veterinarians, veterinary nurses, veterinary assistants, veterinary students and farm workers from several veterinary hospitals, clinics and farms.nasal swabs were collected from 72 veterinarians (60 small animal veterinarians and 12 pig veterinarians), 8 veterinary nurses, 53 veterinary students, 4 veterinary assistants and 11 farm workers. mrs were screened on brilliance tm mrsa2 agar (oxoid) or chromid tm mrsa (biom erieux). after 24-48 h of incubation at 37°c, suspected colonies on both media were subcultured onto blood agar plates. species identification was obtained by species-specific pcr. methicillin-resistance was confirmed by pcr amplification of the meca gene. mrsa isolates were characterized by mlst.thirty-nine mrs were identified in 38 humans (23 veterinarians, 2 veterinary nurses, 7 veterinary students, 2 veterinary assistants and 5 farm workers). the mrs isolates were identified as staphylococcus aureus (mrsa, n = 23), s. epidermidis (mrse, n = 9), s. pseudintermedius (mrsp, n = 1), s. haemolyticus (mrsh, n = 1) and 5 mrs coagulase-negative staphylococci. the frequency of colonization by mrs was similar in both small animals and pigs veterinarians (ae 30%). one veterinary student was colonized simultaneously with an mrse and an mrsh. the predominant st in humans in contact with small animals was st22 and in humans in contact with pigs was st398.in our study the frequency of colonization by mrsa was high, but the frequency of mrse should not be underestimated. mrsa isolates in this work belonged mainly to the st22 lineage which is the most frequent in small animals and humans in europe. humans in daily contact with animals can become colonized by mrs of animal origin and thus are important keys for infection control programs in veterinary hospitals and farms.conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party (awp) and member of the antimicrobial advice ad hoc expert group (ameg) of the european medicines agency (ema). , species not determined [2]) did not. all 17 cats underwent antibiotic treatment (doxycycline or a fluoroquinolone); 12 cats received blood transfusions and/or oxyglobin â . three cats were euthanatized within 11 days due to concurrent disease (fiv, pancreatitis/cholangitis) or financial constraints, one cat due to persistent anemia after 11 weeks. four cats were lost to follow-up. the remaining 9 cats underwent follow-up for a period of 11-199 weeks (median 24). hemoplasma pcr analysis was conducted 2-7 times on blood samples at variable time points from 6 of the 9 follow-up cats. the first negative pcr in 4 cases occurred after 3 (cmhm, during antibiotic treatment), 7 (cmhm, during antibiotic treatment), 10 (cmhm, during antibiotic treatment) and 23 (mhf, after completion of antibiotics) weeks. one cat remained pcr positive (cmhm) at 4, 13, and 18 (all during antibiotic treatment) weeks, and another cat (cmhm) was pcr positive at 199 weeks. reactivation of the hemoplasma species (documented by hemolysis and positive pcr) occurred in 2 cats (both cmhm) 1 and 3 times, respectively, up to 177 weeks after initial presentation. reactivation was suspected (no pcr testing available) in 2 additional cases (cmhm [1], mhf [1]). four of the 9 follow-up cats were euthanatized after 14-180 weeks (median 24) due to concurrent disease (cardiomyopathy, immune-mediated thrombocytopenia, postoperative complications, diabetes mellitus). infection with hemoplasmas is often chronic, can reactivate months later and is rarely the reason for euthanasia. no conflicts of interest reported. canine distemper (cd) is a worldwide occurring infectious disease caused by a morbillivirus of the family paramyxoviridae. cdv infection can result in a systemic infection. dogs presented with neurologic signs revealed the terminal stage of the disease and usually failed to therapy. additional passive immunotherapy is hypothesized to be beneficial in the early stage of cdv infection. porcine anti-cdv antibody subunit f (ab') 2 [f(ab') 2 ] was produced by animal technology laboratories, agriculture technology research institute. eighteen cdvnaturally infected dogs showing respiratory signs but no neurological signs were treated with the combination of f(ab') 2 and supportive therapy (group 1). group 2 included 33 dogs in a similar clinical signs (without neurological signs) that received only supportive therapy. the survival rate was 72.2% (13/18) in group 1 and 33.3% (11/33) in group 2, respectively, with a significant difference between the two groups (p < 0.05). the progressive rates of developing neurological signs during therapy of group1 and group2 were 44.4 and 63.6%, respectively. there was no significant difference between the two groups. the survival rates of dogs developing neurological signs during therapy were 50% (4/8) in group 1 and 4.8% (1/22) in group 2, respectively, with a significant difference between the two groups. in conclusion, additional administration of porcine anti-cdv antibody subunit f(ab') 2 before developing of neurological signs could decrease the mortality and furthermore reduce the rate of developing neurological signs. no conflicts of interest reported. key: cord-022597-9b1a8cri authors: nan title: hematopoietic tumors date: 2009-05-15 journal: withrow & macewen's small animal clinical oncology doi: 10.1016/b978-072160558-6.50034-4 sha: doc_id: 22597 cord_uid: 9b1a8cri nan the etiology of canine lymphoma is largely unknown and likely multifactorial; however, current investigations are shedding significant light on the subject. recent advances in molecular cytogenetics (see chapter 1, section a), including gene microarray techniques, have been and currently are being applied to investigations of chromosomal aberrations in dogs with lymphoma. [10] [11] [12] breen's group has documented gain of canine chromosomes 13 and 31 and loss of chromosome 14 as the most common aberrations in a group of 25 cases analyzed. 11 the recent publication of the canine genome and the commercial availability of canine gene microarrays (e.g., genechip canine genome 2.0 array; affymetrix santa clara, california) certainly will lead to advances in our understanding of the genetic events in lymphoma in the very near future. 13 several genetic predispositions have been reported for a pedigree of bull mastiffs, 14 a group of related otter hounds, a family of rottweilers, and a breeding pair of unrelated scottish terriers. 15 germ line and somatic genetic mutation in the p53 tumor suppressor gene (see chapter 2) and the n-ras gene have been documented in bull mastiffs and in a dog with lymphoma, respectively. [16] [17] [18] in addition, differences in the prevalence of immunophenotypic subtypes of lymphoma among different breeds have been shown to indicate heritable risks. 19,19b epigenetic modifications also have been investigated in dogs with lymphoma; deoxyribonucleic acid (dna) hypomethylation (see chapter 1, section a) was a feature of neoplastic cells in most lymphoma cases and in one third of the leukemia cases investigated and likely is involved in malignant transformation of lymphoid cells. 20 in humans, characteristic chromosomal abnormalities are being described with increasing frequency as more precise banding and other high-resolution techniques are applied. chromosomal aberrations are nonrandom in human lymphoma, and several aberrations serve as markers for various subtypes of lymphoma. in addition, several oncogenes that may play a role in the pathogenesis of lymphoma have been detected based on the identification of cytogenetic abnormalities. [21] [22] [23] chromosomal aberrations also have been reported in canine lymphoma. [10] [11] [12] 24 a study of 61 dogs with lymphoma demonstrated a treatment advantage in dogs with trisomy of chromosome 13 (25% of the dogs studied), as evidenced by an increase in duration of the first remission and overall survival time. 24 as our knowledge of molecular events and tumorigenesis has expanded, several molecular aberrations have been implicated in various canine tumor types, and some associated with lymphoma have been identified. altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and death-pathway alterations are common in human lymphomas and likely are also involved in the dog. as mentioned earlier, n-ras and p53 aberrations, although rare in dogs, have been implicated in some dogs with lymphoma. [16] [17] [18] [25] [26] [27] telomerase activity (see chapter 14, section d) also has been documented in canine lymphoma tissues. [28] [29] [30] alterations in cellular death-pathways, particularly the bcl-2 family of proapoptotic and antiapoptotic governing molecules, have been implicated in human non-hodgkin's lymphoma 31 and currently are under investigation in canine lymphoma. the hypothesis that a retrovirus may be involved in the pathogenesis of canine lymphoma has not been confirmed. 32 however, viral particles with properties similar to those of retroviruses have been identified in short-term cultures of canine lymphoma tissue. [33] [34] [35] [36] in physician-based oncology, a direct association has been made between helicobacter infections and the development of gastric lymphoma. 37 although this has not been shown definitively in dogs, evidence indicates that helicobacter infection in laboratory beagles results in gastric lymphoid follicle formation, which is considered a precursor of mucosa-associated lymphoid tissue (malt) lymphoma in humans. 38 some evidence has accumulated that implicates phenoxyacetic acid herbicides, particularly 2,4-dichlorophenoxyacetic acid (2,4-d) , in the development of human non-hodgkin's lymphoma. 39, 40 a population case control study of non-hodgkin's lymphoma in kansas farmers reported a twofold to sixfold higher risk in individuals who frequently mixed or applied herbicides (specifically 2,4-d). 41 a published, hospital-based case control study of dogs indicated that owners in households with dogs that developed malignant lymphoma applied 2,4-d herbicides to their lawn or employed commercial lawn care companies to treat their yard more frequently than owners of dogs without lymphoma. 42 the risk of canine lymphoma was reported to rise twofold (odds ratio, 1.3) with four or more yearly applications of 2,4-d. the results of this study since have drawn criticism, and three follow-up investigations have not validated the assertion of increased risk. [43] [44] [45] in another study, dogs exposed to lawn treatment within 7 days of application were more than 50 times more likely to have urine levels of 2,4-d of 50 µg/l or higher. 46 the highest concentration was noted 2 days after application. in a study of 28 dogs with lymphoma, the tumors of 20 dogs with known exposure to 2,4-d were analyzed using polymerase chain reaction (pcr) technology for cellular n-ras oncogene mutations. 47 the ras genes influence cell proliferation and may induce differentiation through signal transduction pathways. mutation in the ras genes results in ras proteins that promote cell growth. one dog in the series showed a mutation of n-ras, indicating that such mutations are uncommon in canine lymphoma, a finding similar to that for humans with lymphoma. 47 in an environmental case control study performed in europe, two variables, residency in industrial areas and use of chemicals (defined as paints or solvents) by owners, modestly increased the risk of lymphoma; however, no link was found between the use of pesticides and risk. 48 a weak association between lymphoma in dogs and exposure to strong magnetic fields was observed in a preliminary epidemiologic study. 49 in this hospital-based, case control study, dogs categorized as having high or very high exposure had an increased risk of lymphoma (odds ratio, 1.8). more thorough studies are necessary to evaluate this association further. impaired immune function has been identified in dogs with lymphoma. 50, 51 immune system alterations in the dog (e.g., immune-mediated thrombocytopenia), independent of age and gender, have been associated with a higher risk of subsequent development of lymphoma compared to the normal population. 52, 53 additional evidence for the role of the immune system in the development of lymphoma comes from observation in human transplantation patients. individuals with immunosuppression have a higher risk of lymphoreticular cancer, 54, 55 and organ transplant patients have a higher incidence of non-hodgkin's lymphoma. 56 a case of lymphoma that developed in a dog after treatment with cyclosporine, although only one case, supports a link to immunosuppressive therapy in the species. 57 the classification of malignant lymphoma in dogs can be distinguished on the basis of anatomic location, histologic criteria, and immunophenotypic characteristics. the most common anatomic forms of lymphoma, in order of decreasing prevalence, are the multicentric, craniomediastinal, gastrointestinal, and cutaneous forms. primary extranodal forms, which can occur in any location outside the lymphatic system, include the eyes, central nervous system (cns), bone, testes, bladder, heart, and nasal cavity. eighty percent of dogs with lymphoma develop the multicentric form, which is distinguished by the presence of superficial lymphadenopathy (figure 31-1) . 58 lymph node enlargement usually is painless, rubbery, and discrete and may be localized initially to the mandibular and prescapular nodes. most animals are asymptomatic at the time of presentation, but approximately 20% to 40% have a history of weight loss, lethargy, anorexia, and febrile episodes. 59, 60 diffuse pulmonary infiltration also may be seen in 27% to 34% of affected dogs, as detected by radiographic changes (figure 31 -2). [61] [62] [63] [64] based on bronchoalveolar lavage, the actual incidence of lung involvement may be higher. 62, 65 hepatosplenomegaly is the most common manifestation of abdominal involvement and usually is associated with an advanced stage of multicentric disease. the alimentary form of lymphoma is much less common, usually accounting for 5% to 7% of all canine lymphomas. 58 this form is reported to be more common in male dogs than female dogs, 8 similar to observations in humans. 66 dogs with infiltrative disease of the intestinal tract show weight loss, anorexia, panhypoproteinemia, and evidence of malabsorption. 67, 68 primary gastrointestinal (gi) lymphoma in dogs usually occurs multifocally and diffusely throughout the submucosa and lamina propria of the small intestine, with frequent superficial ulceration and occasional transmural infiltration of the serosa. lymphocytic-plasmacytic inflammation can be seen adjacent to or distant from the primary tumor. 67 pathologically, some of these neoplasms may resemble plasma cell tumors, and aberrant production of immunoglobulins may occur. histopathologically, distinguishing between gastrointestinal lymphoma and lymphocytic-plasmacytic enteritis (lpe) can be difficult. 67 some have suggested that lpe may be a prelymphomatous change in the gi tract. a syndrome of immunoproliferative intestinal disease characterized by lymphocytic-plasmacytic enteritis has been described in basenjis, which subsequently develop gastrointestinal lymphoma. 69 in addition, plasma cell-rich areas with heterogeneous lymphomatous infiltration may resemble lesions of lpe. only a few reports specifically identify the immunophenotype of the lymphocyte subpopulations in alimentary lymphoma. historically, it was presumed that they most likely originate from b cells; however, recent evidence suggests that most gastrointestinal lymphomas in dogs originate from t cells. 70 the boxer and shar-pei breeds appear to be overrepresented in alimentary lymphoma and are reported to have morphologic features (epitheliotropism) consistent with t-cell disease. 71 lateral thoracic radiograph of a dog with diffuse interstitial infiltration with lymphoma secondary to multicentric lymphoma. the mediastinal form of lymphoma occurs in approximately 5% of cases. 58 this form is characterized by enlargement of the craniomediastinal lymph nodes or the thymus, or both ( figure 31-3) . however, as previously noted, 20% of dogs with multicentric lymphoma have radiographic evidence of craniomediastinal lymphadenopathy. 63 hypercalcemia is reported to occur in 10% to 40% of dogs with lymphoma and is most common with the mediastinal form. 72, 73 in a study of 37 dogs with lymphoma and hypercalcemia, 16 (43%) had mediastinal lymphoma. 74 the mediastinal form in dogs is most commonly associated with a t-cell phenotype. 60, 75 cutaneous lymphoma can be solitary or more generalized and usually is classified as epitheliotropic (mycosis fungoides) or nonepitheliotropic. [76] [77] [78] [79] [80] [81] [82] [83] [84] cutaneous lymphoma may also involve the oral mucosa, 76 and extracutaneous involvement can occur, most often in the lymph nodes, spleen, liver, and bone marrow. canine epitheliotropic cutaneous lymphoma is the most common form of cutaneous lymphoma and usually originates from t cells, 85 similar to its development in humans. in dogs the t cells are cd8+ cells, whereas in humans they are mostly cd4+ cells. 86 a rare form of cutaneous t-cell lymphoma is characterized by generalized skin involvement with evidence of circulating malignant t cells in the peripheral blood. these lymphocytes usually are large (15 to 20 mm in diameter) and have folded, grooved nuclei. in humans this is called sézary syndrome, 87 which also has been reported in dogs and cats. 79, 80, 88, 89 b-cell cutaneous lymphomas usually spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis. hepatosplenic lymphoma is a relatively uncommon, distinct presentation in the dog marked by a lack of peripheral lymphadenopathy in the face of hepatic, splenic, and bone marrow infiltration with malignant lymphocytes, usually of t-cell origin. biologically, this form of lymphoma is extremely aggressive and poorly responsive to therapy. in humans the tumor usually is composed of γδt cells (i.e., t cells that express the γδt-cell receptor), and this immunophenotype has been confirmed in at least one dog in the veterinary literature. [90] [91] [92] intravascular (angiotrophic, angioendotheliomatosis) lymphoma is a distinct form of lymphoma defined as proliferations of neoplastic lymphocytes within the lumen and wall of blood vessels in the absence of a primary extravascular mass or leukemia. it has been reported several times in the veterinary literature, and in most cases it involves the central and peripheral nervous system (including the eye). [93] [94] [95] [96] [97] [98] the b-cell immunophenotype is most common in humans; however, in most reported cases in dogs, the origin is either t cell or null cell (neither b nor t cell), although one case of a b-cell phenotype has been reported. lymphomas arise from a clonal expansion of lymphoid cells with distinctive morphologic and immunophenotypic features. many histologic systems have been used to classify non-hodgkin's lymphoma (nhl) in humans, and some of these have been applied to lymphoma in the dog and other species. [99] [100] [101] [102] [103] [104] [105] [106] [107] the national cancer institute (nci) of working formulation and the updated kiel system have been adapted to canine tumors with some success (tables 31-1 and . the world health organization (who) also publishes a histologic classification scheme, which uses the revised european american lymphoma (real) system as a basis for defining histologic categories of hematopoietic tumors in domestic animals. 107 this system incorporates both histologic and immunohistologic criteria (b-and t-cell immunophenotype). the clinical relevance of this system is likely to be high; however, it awaits further investigation. more recently, the oncology committee of the american college of veterinary pathologists (acvp) has established a lymphoma subcommittee to formulate a classification system for lymphoma that has clinical relevance. the subcommittee has obtained information from an international group of veterinary oncologists and pathologists, and its report is expected to be completed by 2008. the working formulation (wf) was developed to allow investigators to "translate" among the numerous classification systems so that clinical trials could be compared in humans. most of the larger compilations agree that most canine lymphomas are intermediate or high grade; however, diffuse immunoblastic forms appear to predominate in the united states, whereas the follicular large cell variations predominate in europe. a comparison of european and american classifications is warranted based on this discrepancy. the wf categorizes tumors according to pattern (diffuse or follicular) and cell type (e.g., small cleaved cell, large cell, immunoblastic), but it does not include information about the immunophenotype of the tumor. 103 the wf subtypes are related to the biology of the tumor and patient survival. the updated kiel classification includes the architectural pattern, morphology (centroblastic, centrocytic, or immunoblastic), and immunophenotype (b cell or t cell) of the tumor cells. 102 in both systems, the tumors then can be categorized as low-grade, intermediate-grade, or high-grade malignancies. low-grade lymphomas composed of small cells with a low mitotic rate typically progress slowly and are associated with long survival times but are incurable. high-grade lymphomas with a high mitotic rate progress rapidly but are more likely to respond to chemotherapy and, in humans, are potentially curable. several features of canine lymphomas become apparent when the wf or updated kiel classification is applied. the most striking difference between canine and human lymphomas is the scarcity of follicular lymphomas in the dog. [108] [109] [110] [111] some diffuse lymphomas in the dog initially may be follicular, but these may progress to the more aggressive, diffuse form by the time of diagnostic biopsy. only a small percentage of canine lymphomas (5.3% to 29%) are considered lowgrade tumors. 60, 104, 105, 112 most low-grade small cell lymphomas are t cell in origin. 105 high-grade lymphomas occur frequently if the diffuse large cell lymphomas, classified as intermediategrade in the wf, are considered high-grade, as in the updated kiel classification (in which they are labeled diffuse centroblastic lymphomas). canine lymphoblastic lymphomas are uncommon. 72, 104 most high-grade lymphomas are of b-cell origin. 105 however, a documented difference exists in the prevalence of the various lymphoma immunophenotypes based on breed. 19 for example, cocker spaniels and doberman pinschers are more likely to develop b-cell lymphoma, boxers are more likely to have t-cell lymphoma, and golden retrievers appear to have an equal likelihood of b-and t-cell tumors. to be clinically useful, these classification systems in the end must yield information about response to therapy, maintenance of remission, and survival. some studies suggest that the subtypes in the wf can be correlated with survival, and the kiel system may be useful for predicting relapse. 113, 114 in most studies, high-grade lymphomas show a complete response to chemotherapy significantly more often than low-grade tumors. however, dogs with low-grade tumors may live a long time without aggressive chemotherapy. dogs with t-cell lymphomas have shown a lower rate of complete response to chemotherapy and shorter remission and survival times than dogs with b-cell tumors. 60, 75, 112, 113 furthermore, t-cell lymphomas tend to be associated with hypercalcemia. [115] [116] [117] in the veterinary literature, 60% to 80% of lymphoma cases in dogs are b-cell lymphoma; t-cell lymphomas account for 10% to 38%; mixed b-and t-cell disease accounts for as many as 22%; and null cell tumors (i.e., neither b-cell nor t-cell immunoreactive) represent fewer than 5%.* the development of monoclonal antibodies to detect specific markers on canine lymphocytes has made immunophenotyping of tumors in dogs routinely available in many commercial laboratories. such techniques also can be performed on paraffin-embedded samples and on cytologic specimens obtained by fine-needle aspiration. [118] [119] [120] [121] [122] the rappaport classification system, proposed in 1956 for human nhl, described the architectural pattern (follicular or diffuse) and the cytologic features (well differentiated, poorly differentiated, or histiocytic) of the tumors. 99, 123 the term histiocytic was applied to tumors in which most of the lymphocytes had larger diameters, more vesicular nuclei, and more prominent nucleoli than those of lymphocytic or undifferentiated lymphomas; it also was used because the malignant cells have some morphologic features of benign histiocytes. however, immunophenotyping has failed to document a biologic relationship between these cells and true histiocytes, therefore the term now is largely considered a misnomer. furthermore, this subgroup of histiocytic lymphomas included tumors with different morphologic and immunophenotypic features. the rappaport classification has not been useful in providing prognostic information or in guiding therapy in dogs with lymphoma because of the low number of follicular tumors in dogs, the problematic "histiocytic" subgroup, and the failure to account for different morphologic and immunologic cell types. 100 one criticism of these classification systems is that they fail to include extranodal lymphomas as a separate category. although differences between nodal and extranodal tumors in biologic behavior and prognosis are well recognized, comparative information about the histogenesis of these tumors has been lacking. for example, in humans, small cell lymphomas arising from malt are composed of cells with a different immunophenotype from that of other small cell lymphomas (i.e., malt lymphomas typically are negative for both cd5 and cd10). 124-126 except for cutaneous lymphoid neoplasms, detailed characterization of extranodal lymphomas in dogs has not been done. although cutaneous lymphoma is a heterogeneous group of neoplasms that includes an epitheliotropic form resembling mycosis fungoides and a nonepitheliotropic form, most cutaneous lymphomas have a t-cell phenotype. 85, 127 to summarize, it is important to determine the histologic grade of canine lymphomas as low (small lymphocytic or centrocytic lymphomas) or intermediate to high (diffuse large cell, centroblastic, and immunoblastic lymphomas). furthermore, determining the immunophenotype of the tumor provides useful information. response rates to chemotherapy are better in animals with b-cell tumors and intermediate-to highgrade lymphomas. dogs with low-grade lymphomas can have long survival times without aggressive therapy. the clinical signs associated with canine lymphoma are variable and depend on the extent and location of the tumor. multicentric lymphoma is the most common form (80%), and generalized painless lymphadenopathy (see figure 31 -1) is the most consistent finding. in addition, hepatosplenomegaly and bone marrow involvement are common. most dogs with multicentric lymphoma do not have dramatic signs of systemic illness (who substage a) (box 31-1), however, a large array of nonspecific signs can occur, such as anorexia, weight loss, vomiting, diarrhea, emaciation, ascites, dyspnea, polydipsia, polyuria, and fever (who substage b). 59, 60, [128] [129] [130] dogs with t-cell lymphoma are more likely to have constitutional signs (i.e., substage b). 118 polydipsia and polyuria are particularly evident in dogs with hypercalcemia of malignancy. dogs also may have a history of or clinical signs related to blood dyscrasias secondary to marked tumor infiltration of the bone marrow (myelophthisis) or paraneoplastic anemia, thrombocytopenia, or neutropenia. these signs could include fever, sepsis, anemia, and hemorrhage. dogs with gastrointestinal or alimentary lymphoma usually have nonspecific gi signs, such as vomiting, diarrhea, weight loss, and malabsorption. [66] [67] [68] the mesenteric lymph nodes, spleen, and liver may be involved. the mediastinal form of lymphoma is characterized by enlargement of the craniomediastinal structures or thymus or both (see figure 31 -3, a), and clinical signs are associated with the extent of disease or polydipsia and polyuria from hypercalcemia. these patients commonly have respiratory distress caused by a space-occupying mass and pleural effusion, exercise intolerance, and possibly regurgitation. dogs with mediastinal lymphoma also may have precaval syndrome, characterized by pitting edema of the head, neck, and forelimbs secondary to tumor compression or invasion of the cranial vena cava (figure 31-4) . signs in dogs with extranodal lymphoma depend on the specific organ involved. cutaneous lymphoma usually is generalized or multifocal. [76] [77] [78] [79] [80] [81] [82] [83] [84] tumors occur as nodules, plaques, ulcers, and erythremic or exfoliative dermatitis. epitheliotropic t-cell lymphoma (e.g., mycosis fungoides) has a chronic clinical course with three apparent clinical stages. initially, scaling, alopecia, and pruritus are seen ( figure 31 -5, a). as the disease progresses, the skin becomes more erythematous, thickened, ulcerated, and exudative. the final stage is characterized by proliferative plaques and nodules with progressive ulceration (figure 31-5, b) . oral involvement also may occur, which can appear as multicentric, erythematous, plaquelike lesions or nodules on the gums and lips ( figure 31 -5, c). 76 dogs with primary cns lymphoma may have either multifocal or solitary involvement. 131-133 seizures, paralysis, and paresis may be noted. ocular lymphoma is characterized by infiltration and thickening of the iris, uveitis, hypopyon, hyphema, posterior synechia, and glaucoma ( figure 31-6 ). 134 in one study of 94 cases of canine multicentric lymphoma, 37% had ocular changes consistent with lymphoma, and in a series of 102 cases of uveitis in dogs, the condition occurred secondary to lymphoma in 17% of the cases. 135, 136 anterior uveitis was most often seen in advanced stage disease (stage v). dogs with intravascular lymphoma usually have signs related to cns, peripheral nervous system (pns), or ocular involvement, [93] [94] [95] [96] [97] [98] including paraparesis, ataxia, hyperesthesia, seizures, blindness, lethargy, anorexia, weight loss, diarrhea, polyuria, polydipsia, and intermittent fever. dogs with pure hepatosplenic lymphoma usually have nonspecific signs such as lethargy, inappetence, and weakness. the differential diagnosis of lymphadenopathy depends on the dog's travel history (i.e., relative to infectious disease) and the size, consistency, and location of the affected lymph nodes. other causes of lymphadenopathy include bacterial and viral infections, parasites (toxoplasma and leishmania spp.), rickettsial organisms (salmon poisoning, ehrlichia sp.), and fungal agents (blastomyces and histoplasma spp.). the potential for hypercalcemia to accompany systemic hormone-like substance, parathyroid hormone-related peptide (pthrp), elaborated by neoplastic cells; however, it also can be related to the production of several other humoral factors, including interleukin-1 (il-1), tumor necrosis factor-alpha (tnf-α), transforming growth factor-beta (tgf-β), and vitamin d analogs (e.g., 1,25-dihydroxyvitamin d). 125,141-144 several investigators have reported that hypercalcemia in dogs with lymphoma is most commonly associated with t-cell lymphoma. 60, 113, 117, 118 other paraneoplastic syndromes that may be encountered include monoclonal gammopathies, neuropathies, and cancer cachexia. 145,146 for most animals suspected of having lymphoma, the diagnostic evaluation should include a thorough physical examination; complete blood count (cbc) with a differential cell count, including a platelet count; serum biochemistry profile; and urinalysis. ultimately, obtaining tissue or cytologic specimens for a definitive diagnosis is essential. a thorough physical examination should include palpation of all assessable lymph nodes, including those palpable by rectal examination; in the authors' experience, a significant proportion of dogs have rectal polyps consisting of aggregates of neoplastic lymphocytes. the mucous membranes should be inspected for pallor, icterus, petechiae, and ulceration, because these signs may indicate anemia or thrombocytopenia secondary to myelophthisis or immune-mediated disease or may be evidence of major organ failure or uremia. abdominal palpation may reveal organomegaly, intestinal wall thickening, or mesenteric lymphadenopathy. thoracic auscultation may reveal the presence of a mediastinal mass or pleural effusion or both. an ocular examination that includes funduscopic assessment may reveal abnormalities such as uveitis, retinal hemorrhage, and ocular infiltration in approximately one third to one half of dogs with lymphoma. 135, 136 complete blood count, biochemistry profile, and urinalysis anemia, the most common lymphoma-related hematologic abnormality, 137,138 usually is normochromic and normocytic (nonregenerative), consistent with anemia of chronic disease. however, hemolytic anemia may also occur, and regenerative anemias may reflect concomitant blood loss or hemolysis. in addition, if significant myelophthisis is present, the anemia may be accompanied by thrombocytopenia and leukopenia. in animals with anemia or evidence of bleeding, a reticulocyte count, platelet count, and coagulation studies also may be indicated. thrombocytopenia may be seen in 30% to 50% of cases, but bleeding is seldom a clinical problem. 138 (i.e., stage v disease) from primary lymphoblastic leukemia (discussed later) is important, because the prognoses are entirely different. hypoproteinemia is observed more often in animals with alimentary lymphoma. if the dogs has a high total protein or evidence of an increased globulin fraction on a chemistry profile, serum proteins should be evaluated by serum electrophoresis. monoclonal gammopathies have been reported to occur in approximately 6% of dogs with lymphoma. 145 serum biochemical abnormalities often reflect the anatomic site involved. in addition, approximately 15% of dogs with lymphoma are hypercalcemic (30% to 40% of those with mediastinal involvement and approximately 35% of those with t-cell lymphomas). 74, 118, 147 in cases of hypercalcemia of unknown origin, lymphoma should always be considered high on the differential disease list, and diagnostics directed at this possibility should be undertaken (see chapter 5) . the presence of hypercalcemia also can serve as a marker for response to therapy. elevations in serum urea nitrogen and creatinine can occur secondary to renal infiltration with tumor, hypercalcemic nephrosis, or prerenal azotemia from dehydration. increases in liver-specific enzyme activity or bilirubin concentrations may result from hepatic parenchymal infiltration. serum globulin elevations, usually monoclonal, occur infrequently with b-cell lymphoma. a urinalysis is part of the minimum database used to assess renal function and the urinary tract. for example, isosthenuria and proteinuria in the absence of an active sediment may indicate renal disease, and hematuria may result from a hemostatic abnormality. it is important to remember that isosthenuria in azotemic dogs with hypercalcemia does not necessarily indicate renal disease, because the high calcium levels interfere with tubular concentration capabilities through disruption of antidiuretic hormone (adh) control. abnormalities in serum levels of alpha fetoprotein, alpha-1 glycoprotein, zinc, chromium, iron, and endostatin also have been investigated in dogs with lymphoma. [149] [150] [151] [152] the clinical and biologic significance of these alterations has yet to be elucidated. morphologic examination of the tissue and cells that comprise the tumor is essential to the diagnosis of lymphoma. care should be taken to avoid lymph nodes from reactive areas (e.g., mandibular lymph nodes); the prescapular or popliteal lymph nodes are preferable. also, lymphoid cells are fragile, and in preparing smears of aspirated material, only gentle pressure should be applied in spreading the material on the slides. in most cases, a diagnosis of lymphoma can be made on evaluation of fine-needle aspirates of affected lymph nodes or other tissues. typically, most of the cells are large lymphoid cells (larger than neutrophils), and they may have visible nucleoli and basophilic cytoplasm ( figure 31-7 , a) or fine chromatin with indistinct nucleoli. because tissue architecture is not maintained in cytologic specimens, effacement of the lymph node or capsular disruption cannot be detected. therefore, marked reactive hyperplasia characterized by increased numbers of large lymphoid cells may be difficult to distinguish from lymphoma, and small cell lymphomas may have few cytologic clues that point to their malignancy. also, classification of lymphoma into the subcategories that comprise the low-, intermediate-, and high-grade forms, which has been attempted using the cytologic appearance and immunophenotypic analysis, 153 is performed most accurately on histologic sections. for accurate histopathologic evaluation, an entire lymph node, including the capsule, should be removed, placed in buffered formalin, and submitted to a pathologist. although needle core biopsies may be satisfactory, it is important to avoid crush artifact or inadequate sample size. most pathologists prefer whole node biopsies because they provide the maximum amount of information. effacement of the normal nodal architecture by neoplastic lymphocytes and capsular disruption are characteristic findings ( figure 31 -7, c and d). diagnostic ultrasonography and ultrasound-guided fineneedle aspiration or needle biopsy have been very useful for evaluation of involvement of the liver, spleen, or abdominal lymph nodes. [154] [155] [156] [157] [158] if possible, the diagnosis should be made by sampling peripheral nodes, avoiding percutaneous biopsies of the liver and spleen. however, if there is no peripheral node involvement, it is appropriate to biopsy affected tissues in the abdominal cavity. with alimentary lymphoma, an open surgical wedge biopsy of the intestine must be obtained, ideally without entering the intestinal lumen, and adequate tissue must be obtained; this is important because of the difficulty involved in differentiating lymphoma from lpe. endoscopic biopsies may be inadequate because only a superficial specimen is obtained; however, more aggressive endoscopic biopsy techniques combined with more accurate histopathologic assessments are improving the diagnostic yield of these less invasive techniques. 159 in many dogs with primary gastrointestinal lymphoma, an inflammatory, nonneoplastic infiltrate (e.g., lpe) may be misdiagnosed on biopsy specimens that are too superficial. cytologic examination of cerebrospinal fluid (csf), thoracic fluid, or aspirates of an intracavitary mass is indicated in animals with cns disease, pleural effusion, or an intrathoracic mass, respectively. in one study of dogs with cns involvement, csf analysis was diagnostic in seven of eight dogs. 131 the characteristics of the csf included an elevated nucleated cell count in the seven dogs, and 95% to 100% of the cells were atypical lymphoid cells. the csf protein concentration was higher in five of seven dogs, ranging from 34 to 310 mg/dl (the reference interval was less than 20 mg/dl). for cutaneous lymphoma, punch biopsies (3 to 4 mm) should be taken from the most representative and infiltrative, but not infected, skin lesions. staging procedures for cutaneous lymphoma vary, and the stage has shown no prognostic importance. 160 molecular techniques can be used to establish a diagnosis of lymphoma or to further characterize a tumor after the initial diagnosis has been made. tissues and cells from peripheral blood, lymph nodes, or other sites can be analyzed by histochemical and cytochemical, immunohistochemical and immunocytochemical, flow cytometric, and pcr techniques. for example, the tumor's immunophenotype (b cell or t cell), proliferation rate (e.g., expression of ki-67, proliferating cell nuclear antigen [pcna], and argyrophilic nucleolar organizer regions [agnors]), and clonality (pcr for antigen receptor gene rearrangement [parr]) can be determined. 60, 105, 113, [119] [120] [121] [122] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] the availability of such analyses is increasing, although currently only the immunophenotype consistently predicts the prognosis in dogs. part iv • specific malignancies in the small animal patient immunophenotyping typically is used to determine the type of cells that make up the lymphoma, but sometimes the technique is helpful for making the diagnosis of lymphoma. when a diverse population of lymphocytes is expected in a tissue, the presence of a homogeneous population of the same immunophenotype is supportive of a neoplastic process. the immunophenotype of a lymphocyte is identified by determining the expression of molecules specific for b cells (e.g., cd79a) and t cells (e.g., cd3). although tumor cells sometimes have morphologic characteristics that typify a particular immunophenotype, exceptions occur, and morphology cannot be used as the sole determinant of the type of lymphocyte. for example, in a series of nine high-grade t-cell lymphomas and leukemias in dogs, the cells had a plasmacytoid appearance, typically associated with b-cell lymphoma. 171 similarly, anatomic location does not always predict the immunophenotype. in a series of 44 cases of gastrointestinal lymphoma in dogs, often considered a neoplasm of b cells, the neoplastic cells were identified as t cells (cd3 positivity) in 75% of the cases. 70 for accurate determination of the immunophenotype, antibodies against lymphocyte markers are applied to tissue sections (immunohistochemistry), cytologic specimens (immunocytochemistry), or individual cells in a fluid medium (flow cytometry). flow cytometric evaluation of cells from needle aspirates also is feasible. 172 for t cells, markers include cd3 (pan t), cd4 (helper t), and cd8 (cytotoxic t); for b cells, the markers are cd79a (see figure 31 -7, b) and cd21. 173 interestingly, aberrant expression of cd molecules has been reported in canine lymphoma. in a study of 59 dogs with lymphoma, tumor cells from six dogs were positive for both t-and b-cell cd markers; however, a clonality assay (discussed later) revealed clonality for either the t-cell or the immunoglobulin receptor but not both. this indicates that in some cases of b-and t-cell lymphoma, the malignant cells may coexpress b-and t-cell cd markers. 119 antibodies against these molecules are used to determine the immunophenotype; however, they also have a potential use as a therapeutic modality if tumor cells could be targeted using these antibodies. histologic assessment of markers of multidrug resistance and apoptotic pathways (e.g., p-glycoprotein, p53, and bcl-2 proteins) currently are being evaluated in dogs with lymphoma. however, their significance requires further evaluation. 16, 163, [174] [175] [176] [177] clonality assay occasionally the diagnosis of lymphoma and the differentiation of malignant verses benign proliferation of lymphocytes is not possible based on standard histologic and cytologic criteria. in these cases, advanced molecular analyses are necessary to help confirm a diagnosis. clonality is the hallmark of malignancy; that is, the malignant cell population theoretically should be derived from expansion of a single malignant clone characterized by a particular dna region unique to that tumor. for example, in a dog with t-cell lymphoma, all the malignant cells should have the same dna sequence for the variable region of the t-cell receptor (tcr) gene; likewise, in a dog with b-cell lymphoma, the tumor cells should have identical dna sequences in the variable region of the immunoglobulin receptor gene. conversely, in reactive lymphocytosis, the cells are polyclonal for their antigen receptors. using this knowledge, investigators have used polymerase chain reaction (pcr) pcr techniques to amplify the variable regions of the t-cell and immunoglobulin receptor genes to detect clonal lymphocyte populations in dogs ( figure 31-8) . [167] [168] [169] [170] uniform size of polymerase chain reaction (pcr) products as an indicator of clonality. each panel shows four pcr reactions on a single dna sample. the first lane (left) in each panel is a positive control that indicates that dna is present (any nonrearranged gene would be an appropriate target for this reaction). the middle two lanes represent two different reactions that amplify immunoglobulin cdr3, and the fourth lane shows tcrγ cdr3 amplification. the samples are separated on a polyacrylamide gel. a, lymph node aspirate from a normal dog. b, lymph node aspirate from a dog with histologically confirmed multicentric b-cell lymphoma. c, lymph node aspirate from a dog with histologically confirmed t-cell lymphoma. (from avery pr, avery ac: vet clin pathol 33:196-207, 2004.) in physician-based medicine, such assays of clonality are approximately 70% to 90% sensitive and have a false positive rate of approximately 5%, and recent studies report similar rates in the dog. 169 false negative and false positive results can occur with clonality assays. for example, cells from a dog with lymphoma may be negative for clonality if the clonal segment of dna is not detected with the primers used, if the malignant cells are natural killer (nk) cells (rare), or if the malignant cells are present in too low a frequency to be detected. 169 false positive results occur rarely with some infectious diseases, such as ehrlichiosis and lyme disease. in these cases a diagnosis should be made only after the results of all the diagnostic tests are considered, including histologic and cytologic evaluation, immunophenotyping, and clonality studies, in conjunction with the signalment and physical findings. molecular techniques, in addition to aiding the diagnosis, could also be useful in determining early recurrence, more accurate clinical staging, and so-called molecular remission rates, because they are more sensitive than standard cytologic assessment for the peripheral blood, bone marrow, and lymph nodes. after a diagnosis has been established, the extent of disease should be determined and correlated to the clinical stage of disease. the who staging system routinely used to stage dogs with lymphoma is presented in box 31-1. most dogs (more than 80%) are presented in advanced stages (stage iii or iv). some type of imaging and an assessment of bone marrow involvement may be indicated for staging. the degree to which thorough staging is implemented depends on three factors: whether the result will alter the treatment plan; whether relevant prognostic information will be gleamed; and whether the client needs to know. in addition, when different protocols are compared with respect to efficacy, consistent and similar staging systems should be used to avoid so-called stage migration, which results when one staging methodology is more accurate than another. 178 the effect of stage migration currently is being evaluated in veterinary patients with lymphoma, and until the concept has been thoroughly explored, the impact on the prognosis should be considered when different published outcomes are compared. a bone marrow aspirate or biopsy (from the proximal humerus or iliac crest) is indicated for complete staging and in dogs with anemia, lymphocytosis, peripheral lymphocyte atypia, or other peripheral cytopenia. in one study of 53 dogs with lymphoma, 28% had circulating malignant cells and were considered leukemic, whereas bone marrow examination indicated involvement in 57% of the dogs. 179 the presence of a few prolymphocytes and large lymphocytes with nucleoli in the circulation of dogs with lymphoma may indicate bone marrow involvement. it is important to remember that these cells also can be seen with gi parasitism, immune-mediated hemolytic anemia, and other immune-mediated diseases. recently, circulating tumor cells in dogs with stage iii lymphoma were identified using a clonality assay (parr). 168 the assay is more sensitive than routine microscopy in detecting malignant cells in circulation, but the correlation of these results with staging and prognosis has not been determined. bone marrow evaluation offers prognostically valuable information, but if the client is committed to treatment regardless of the stage of disease, it is not necessary. evaluation of thoracic and abdominal radiographs may be important in determining the extent of internal involvement. approximately 60% to 75% of dogs with multicentric lymphoma have abnormalities on thoracic radiographs; one third have evidence of pulmonary infiltrates, and two thirds have thoracic lymphadenopathy (sternal and tracheobronchial lymph nodes) and widening of the cranial mediastinum (see . [61] [62] [63] [64] craniomediastinal lymphadenopathy is detected in 20% of dogs with lymphoma. 63 abdominal radiographs reveal evidence of sublumbar (iliac) and/or mesenteric lymph node, spleen, or liver involvement in approximately 50% of cases. 63, 64 in the authors' practice, in typical cases of canine multicentric lymphoma, imaging is limited to thoracic radiographs because no prognostic difference exists between dogs with stage iii disease and those with stage iv disease (i.e., liver or spleen involvement); however, the presence of craniomediastinal lymphadenopathy is prognostically significant (see prognosis later in this section). if clinical signs attributable to abdominal disease are present, further imaging of the abdomen is warranted. in addition, as stated previously, abdominal ultrasonography can be important for obtaining ultrasound-guided intraabdominal samples for diagnosis. it is also useful for the diagnosis of gastrointestinal and hepatosplenic lymphoma. [90] [91] [92] 180, 181 advanced imaging modalities, including computed tomography (ct), magnetic resonance imaging (mri), and positron emission/computed tomography (pet/ct), are becoming more commonplace in veterinary practice, and their usefulness is only now being fully determined. [182] [183] [184] [185] [186] the therapeutic approach to a particular patient with lymphoma is determined by the stage and substage of disease, the presence or absence of paraneoplastic disease, the patient's overall physiologic status, the financial and time commitments of the client, and the client's level of comfort with regard to the likelihood of treatment-related side effects. because most canine lymphomas are intermediate-to high-grade tumors, histopathologic characterization has played a less important role in determining the optimal treatment. without treatment, most dogs with lymphoma die of the disease in 4 to 6 weeks. 145 with few exceptions, canine lymphoma is considered a systemic disease and therefore requires systemic therapy to achieve remission and prolonged survival. systemic chemotherapy continues to be the therapy of choice for canine lymphoma. in general, combination chemotherapy protocols are superior in efficacy to single agent protocols. single agent protocols, except for doxorubicin, have a lower response rate that is not as durable as combination chemotherapy. in rare cases in which lymphoma is limited to one site (especially an extranodal site), the animal can be treated with a local modality, such as surgery or radiation therapy, as long as the caregiver and clinician are committed to diligent re-evaluation to document subsequent systemic involvement. many chemotherapy protocols for dogs with lymphoma have been developed over the past 15 to 20 years (table 31-3) . 59, 61, 72, 128, [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] most complex combination protocols are modifications of chop protocols initially designed for human oncologic use. chop represents combinations of cyclophosphamide (c), doxorubicin (represented by the h, for hydroxydaunorubicin), vincristine (o, oncovin) and prednisone (p). conventional chemotherapy induces complete remission (cr) in approximately 60% to 90% of dogs, with median survival times of 6 to 12 months depending on the protocol used. approximately 20% to 25% of dogs live 2 years or longer after initiation of these protocols (figure 31-9) . response rates and the duration of response vary according to the presence or absence of prognostic factors discussed in the prognosis section. the cost to the owner depends on the drug or drugs selected, the size of the animal, the frequency of administration, and the laboratory tests needed to monitor toxicity. dogs that respond to chemotherapy and achieve complete remission usually are free of clinical signs associated with lymphoma and subsequently return to a very good quality of life. treating dogs with lymphoma is gratifying, because a high percentage have a complete response. most dogs tolerate chemotherapy well, and in our experience only a minority of dogs develop significant toxicity. studies assessing clients' perceptions of medical treatment for cancer in general and lymphoma in particular generally report a positive experience; most owners felt that the treatment was worthwhile, that it resulted in improvement in their pet's well-being, and that the animal's quality of life during treatment was good. 202,203 very few clients expressed regret about having the lymphoma treated with a multidrug protocol. with lymphoma, the fundamental goals of chemotherapy are to induce a complete and durable (longer than 6 months) first remission (induction), to reinduce remission when the tumor recrudesces (or the patient relapses) after achievement of a remission (reinduction), and finally to induce remissions when the cancer fails to respond to induction or reinduction using drugs not present in standard protocols (rescue). a previously unanswered question in the treatment of lymphoma was whether long-term maintenance chemotherapy was useful after an initial course of aggressive induction chemotherapy that lasted 6 months or less. long-term maintenance chemotherapy has been shown to be ineffective in humans with hodgkin's disease, nhl, and multiple myeloma. however, the initial induction course of chemotherapy in humans is much more aggressive than that used in veterinary patients. although no randomized studies have been performed to address the therapeutic benefit of long-term maintenance chemotherapy in dogs, comparisons of dogs treated with chop-based protocols in which all treatment was stopped after 6 months of induction therapy 129 were compared with sequentially treated, historical controls that received a nearly identical protocol that included long-term maintenance therapy. 60 the dogs that received the shortened, less expensive, no-maintenance protocol had comparable remission and survival durations and were more likely to achieve second remissions when they relapsed after completion of chemotherapy than their counterparts that received long-term maintenance therapy. other studies, although not prospective randomized trials, suggest that aggressive induction or discontinuous therapy (i.e., induction without maintenance) is as good as or superior to protocols that use an extended maintenance phase. 190, 193, 194, 200 these data, taken together, suggest that maintenance therapy is not necessary and indeed may be inappropriate for dogs with lymphoma that are treated with similar combination chemotherapy protocols. single agent chemotherapy with known activity for dogs with lymphoma agents include vinblastine, actinomycin-d, mitoxantrone, chlorambucil, methotrexate, dtic, 9-aminocamptothecin, ifosfamide, cytosine arabinoside, gemcitabine, lomustine, and dolastatin-10. 206-218 of these, cytosine arabinoside, ifosfamide, dolastatin-10, and gemcitabine appear to have only minimal activity. except for doxorubicin, induction with single agent chemotherapy does not result in durable remission durations compared with standard combination protocols. the efficacy of incorporating these newer drugs with single agent activity into standard combination protocols awaits further investigation. providing precise treatment recommendations for the wide variety of clinical settings of dogs with lymphoma is difficult, especially in light of the plethora of published combination drug protocols (see table 31 -3). because of the large and ever increasing number of protocols available, several factors should be considered and discussed with caregivers on a case-by-case basis in making the choice of protocol. these factors include the cost, time commitment, efficacy, toxicity, and experience of the clinician with the protocols in question. with the increased availability of generic drugs, protocols are becoming affordable to a larger segment of veterinary clients. in general, more complex combination chemotherapy protocols are more expensive, more time-consuming (i.e., requiring repeated office visits and closer monitoring), and more likely to result in toxicity than simpler, single agent protocols. however, as a general rule, more complex combination protocols result in longer remission and survival durations than single agent protocols. the earliest treatment protocol used in veterinary patients was a non-doxorubicin-based combination chemotherapy (i.e., cyclophosphamide, vincristine, and prednisone [cvp]), a relatively simple, easy protocol that is well-tolerated and results in a 60% to 70% cr rate and a median survival time of 6 to 7 months. 187,188 however, it has been clearly established that the standard of care combination protocols used in dogs with lymphoma include doxorubicin, and all are essentially variations on the chop protocols previously discussed and listed in table 31 -3. although many of these chop protocols include l-asparaginase, which is added either at initiation or at varying times throughout the protocol, several studies have confirmed that the addition of l-asparaginase in induction protocols does not result in clinically relevant increases in remission rate, speed of attaining remission, or first-remission duration; therefore its use is best reserved for rescue situations (discussed later). 128,190,219,220 regardless of the veterinary chop-based combination protocol used, they all generally result in an 80% to 90% cr rate with median survival times of 12 months. about 25% of dogs are long-term survivors (longer than 2 years), and some are cured. the chop protocol used by the authors at the time of publication (box 31-3) generally is well tolerated by dogs. this protocol does not have a maintenance therapy arm, and all treatments stop at 19 weeks if the animal is in complete remission. if client factors or other considerations preclude a chop-based protocol, single agent doxorubicin can be offered as an alternative, with the patient receiving five doxorubicin treatments (30 mg/m 2 given intravenously every 3 weeks). the expected complete response rate will range from 50% to 75% with an anticipated median survival time of 6 to 8 months. 128, 188, 189, 197 if financial or other client concerns preclude the use of more aggressive systemic chemotherapy, prednisone therapy alone (2 mg/kg given orally daily) often results in a short-lived remission of approximately 1 to 2 months. in these cases, it is important to inform clients that should they decide to pursue more aggressive therapy later, dogs with previous prednisone therapy are more likely to develop multiple drug resistance (mdr) while receiving single agent prednisone and to have shorter remission and survival durations with subsequent combination protocols. this is especially true after long-term prednisone therapy and in dogs that have experienced a recurrence while receiving prednisone. 195,221 therefore, the earlier a client opts for more aggressive therapy, the more likely it is that a durable response will result. a cbc should be performed before each chemotherapy treatment. a neutrophil count of at least 2000/ml and a platelet count of 50,000/ml should be present before the chemotherapeutic drugs are administered. if the neutrophil count is below 2000/ml, it is best to wait 5 to 7 days and repeat the cbc. if the count has risen above 2000 cells/ml, the drug can be safely administered. a caveat to these restrictions: in dogs presented before initiation of chemotherapy that have low neutrophil and platelet counts because of bone marrow effacement, myelosuppressive chemotherapy is instituted in the face of cytopenias to "open" the bone marrow and allow counts to normalize. with regard to breeds likely to have mdr-1 gene mutations (e.g., collies; see chapter 11) and which therefore are at risk for serious, unexpected chemotherapy toxicity, the authors initiate a chop protocol out of sequence, beginning with non-mdr-1-associated drugs, such as cyclophosphamide. this ensures treatment of the lymphoma while allowing sufficient time for analysis of mdr-1 gene mutations (see chapter 11) before mdr-1-associated drugs are initiated. eventually, most dogs that achieve a remission relapse or experience a recrudescence of lymphoma. this usually represents the emergence of tumor clones that are inherently more resistant to chemotherapy than the original tumor; so-called mdr clones that either were initially drug resistant or became so after exposure to selected chemotherapeutic agents. 175, [222] [223] [224] evidence suggests that in recurrent lymphoma in dogs, tumor cells are more likely to express the mdr-1 gene that encodes the protein transmembrane drug pump often associated with multiple drug resistance. 174, 175, 224 other causes of relapse after chemotherapy include inadequate dosing and frequency of administration of chemotherapy and failure to achieve high concentrations of chemotherapeutic drugs in certain sites, such as the central nervous system. at the first recurrence of lymphoma, reinduction should be attempted first by reintroducing the induction protocol that was successful initially. special attention must be given to the cumulative dose of doxorubicin that will result from reinduction; also, a baseline cardiac assessment, the use of cardioprotectants, alternative drug choices, and client education should all be considered. in general, the likelihood of a response and the length of the reinduction are half those seen in the initial therapy; however, some animals enjoy long-term reinductions, especially if the patient had completed the initial induction regimen and was off chemotherapy when the relapse occurred. a reinduction rate of nearly 90% can be expected in dogs that have completed chop-based protocols and then relapse while off therapy. 129 if reinduction fails or the dog does not respond to the initial induction, use of so-called rescue agents or rescue protocols can be attempted. these are drugs or drug combinations that typically are not found in the standard chop protocol and are withheld for use in cases of drug resistance. the most common rescue protocols most commonly used in dogs include single agent or combination use of actinomycin d, mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), a doxorubicin/dacarbazine combination, lomustine (ccnu), l-asparaginase, and the combination mechlorethamine, vincristine (oncovin) procarbazine, and prednisone (mopp). 176, 206, [209] [210] [211] [225] [226] [227] [228] [229] overall rescue response rates of 40% to 50% are reported, but these responses usually are not durable, and median response times of 1.5 to 2.5 months are typical. a small number of animals will enjoy longer rescue durations. despite the plethora of published chemotherapy protocols for dogs with lymphoma, it appears that veterinary medicine has achieved about as much as it can from the currently available chemotherapeutic drugs in standard settings, because no dramatic improvement has been made in the 12-month median survival "wall" and the 25% 2-year survival rate. advances in remission and survival durations await the development of new methods of delivering or targeting old chemotherapeutic drugs and the development of new generation chemotherapeutics or novel nonchemotherapeutic treatment modalities. mechanisms of avoiding or abrogating mdr, enhancing tumor apoptosis (programmed cell death), and targeting treatments with immunoconjugates (i.e., antibody-directed therapies), as well as novel immunomodulatory therapies, are all active areas of investigation in both human and veterinary medicine. drug resistance can develop in cancer patients after exposure to selected chemotherapeutic agents and often is associated with expression of p-glycoprotein (see chapter 11) . p-glycoprotein acts as a drug efflux pump that actively extrudes drugs from tumor cells, preventing a cytotoxic drug from reaching the cellular site of action. mdr and p-glycoprotein are controlled by the mdr-1 gene. 222,223,230 mdr has been reported in canine lymphoma after treatment with chemotherapy. 174, 224, 231 in one study, expression levels of messenger ribonucleic acid (mrna) that encodes the canine mdr-1 gene was characterized in canine cell lines and lymphomas. 231 although expression of mdr-1 mrna correlated with in vitro drug sensitivity, it did not correlate with in vivo doxorubicin sensitivity in dogs with lymphoma in this study. methods of increasing the time that tumor cells are exposed to chemotherapeutic drugs theoretically should enhance tumor killing. these methods could intralymphatic (il) administration of an autologous killed lymphoma tumor cell vaccine has been done in dogs in which remission was achieved with a combination chemotherapy protocol. in a study that compared 28 dogs that received chemotherapy and then il vaccination with 30 dogs that received chemotherapy alone, the median remission times were 98 and 28 days, respectively (p < 0.024). 238 unfortunately, the survival times for the two groups were not significantly different (305 days for chemotherapy plus il vaccine and 184 days for chemotherapy alone). dogs that responded had significant increases in specific antibody to lymphoma antigens compared to those that did not respond. another immunotherapy approach involved mab-231, a murine-derived anticanine monoclonal antibody (igg2a). this antibody mediates antibody-dependent cellular cytotoxicity (addc) and complement-mediated cellular cytotoxicity (cmcc), 239,240 and it prevented outgrowth of canine lymphoma xenografts in nude mice. 241 in a noncontrolled clinical study, 215 dogs were treated with chemotherapy (l-asparaginase, vincristine, cyclophosphamide, and doxorubicin). 242 after two cycles of chemotherapy, 174 dogs had achieved complete remission and were treated with an intravenous infusion of mab-231 daily for 5 days. the median survival time of the dogs treated with mab-231 was 493 days. the 2-year survival rate was 15.6%. the median number of chemotherapy cycles in the first year was three, and the median number of mab-231 cycles was 1.5. the mab-231 antibody went off the commercial market in the mid-1990s. definitive randomized trials to determine its effectiveness are still lacking. most dogs with lymphoma have the multicentric form and need systemic chemotherapy for effective treatment of the disease. however, surgery has been used to treat solitary lymphoma (early stage i) or solitary extranodal disease. in such cases, careful staging is necessary to rule out multicentric involvement before the local disease is treated. the benefit of surgical removal of the spleen in dogs with massive splenomegaly remains unclear. 243,244 in a published report, 16 dogs with lymphoma underwent splenectomy for a massive spleen and subsequently were treated with chemotherapy. within 6 weeks of splenectomy, five of the 16 dogs died of disseminated intravascular coagulation (dic) and sepsis. the remaining 11 dogs had a complete response rate of 66%, and seven of these, which were followed until their death, had a median survival time of 14 months. splenectomy should be considered only if the lymphoma is in remission in other sites and if the splenic enlargement is caused by lymphoma that is not responsive to chemotherapy. in dogs with lymphoma, splenectomy also can be considered as a treatment for uncontrolled hemolytic anemia and persistent thrombocytopenia. the role of radiotherapy for the management of lymphoma in dogs currently is under investigation. the use of whole body irradiation without bone marrow transplantation has yielded poor results. however, radiation therapy may be indicated in selected cases. 245-248 the indications are: • local stage i or stage ii disease (i.e., nasal lymphoma, cns lymphoma) • palliation of local disease (e.g., mandibular lymphadenopathy, rectal lymphoma, mediastinal lymphoma accompanied by precaval syndrome, localized bone involvement) • whole body irradiation combined with bone marrow or stem cell transplantation • staged half-body irradiation after chemotherapyinduced remission the use of staged half-body irradiation after achieving remission with induction chemotherapy has undergone preliminary investigation as a form of consolidation or maintenance. 247, 248 in these investigations, radiation therapy is delivered either to the cranial or the caudal half of the dog's body in two consecutive 4 gray daily fractions; then, after a rest of 3 or 4 weeks, the other half of the body is irradiated in a similar fashion. although these preliminary investigations were not randomized, their results suggest that radiation therapy, either after completion of chemotherapy or sandwiched between chemotherapy sessions, when dogs are in either complete or partial remission, is safe and should be investigated more extensively to determine whether a significant therapeutic gain could be realized. in general, the veterinary literature offers little information on the treatment of the various extranodal forms of lymphoma in dogs, therefore the ability to predict outcome is limited. the authors recommend that, after extensive staging, local therapies (e.g., surgery, local radiation therapy) should be used in cases in which disease is localized to a solitary site. if multiple extranodal sites are involved or if they are part of a more generalized process, systemic chemotherapy should be given. most dogs with alimentary lymphomas have diffuse involvement of the intestinal tract. involvement of local lymph nodes and the liver is common. chemotherapy in dogs with diffuse disease has been reported to be unrewarding for the most part. 67 however, in the authors' experience, more aggressive, chop-based protocols (which are used extensively for multicentric lymphoma in dogs) have resulted in several cases of durable remission for alimentary lymphoma. solitary alimentary lymphomas are rare in the dog, but if the tumor is localized and can be removed surgically, the results with or without follow-up chemotherapy can be encouraging. most cns lymphoma in dogs results from metastasis of multicentric lymphoma. however, primary central nervous system lymphoma (pcnsl) has been reported. 131, 132 if tumors are localized, local radiation therapy should be considered. few studies have reported on the use of chemotherapy. in one study, cytosine arabinoside (ara-c) at a dosage of 20 mg/m 2 was given intrathecally by bolus injection after withdrawal of an equal volume of csf. 131 the dose was diluted in 2 to 4 ml of lactated ringer's solution and was injected twice weekly for a total of six treatments. this treatment was combined with systemic chemotherapy and cns irradiation. overall, the response rates were low and of short duration (several weeks to months). the treatment of cutaneous lymphoma depends on the extent of disease. solitary lesions may be treated with surgical excision or radiation therapy. fractionated radiation therapy (to a total dose of 30 to 45 gy) has been associated with long-term control. 80 diffuse non-t-cell lymphoma is best managed with combination chemotherapy, although the response rate is less than in multicentric lymphoma. in one study, investigators reported that combination chemotherapy with cyclophosphamide, vincristine (oncovin), cytosine arabinoside, and prednisone (coap) induced longterm remission in some cases. 249 five of six dogs with diffuse non-t-cell cutaneous lymphoma attained a complete or partial remission, with a median remission duration of longer than 250 days and a median survival of longer than 399 days. 249 retinoids, such as isotretinoin (accutane) and etretinate (tegison), have been used successfully in canine and human t-cell cutaneous lymphoma. 82, 250 in one study, 12 dogs with cutaneous lymphoma were treated with isotretinoin (3 to 4 mg/kg given orally daily), and two were treated with etretinate (1.25 to 1.45 mg/kg given orally daily, continuously). eleven of these 14 dogs had t-cell cutaneous lymphoma, and six of the 14 dogs achieved remission. in another study, four dogs with t-cell lymphoma were treated successfully with isotretinoin for 13, 11, 10, and 5 months. 82 in the authors' experience, retinoid treatment must be given for at least 2 months to note a response. polyethylene glycol (peg)-l-asparaginase (30 mg/kg given intramuscularly weekly) induces responses in dogs with cutaneous t-cell lymphomas, although remissions are not durable, and no cures have been noted. 251 prednisone may also be necessary to control pruritus. in the authors' experience, pegylated-liposomal doxorubicin (doxil) has produced remissions in approximately 40% of cases. although most of these were short-lived responses, remissions of 1 year or longer occasionally have occurred. a preliminary abstract reported activity for oral ccnu with cutaneous lymphoma in dogs. 252 all seven dogs (five with mycosis fungoides [mf] and two with nonepitheliotropic disease) that were treated with ccnu (50 mg/m 2 given orally every 3 weeks) achieved a complete response, and two of those responses were relatively durable (7 and 15 months). a larger cohort needs to be treated before definitive response rates and durations can be given. topical chemotherapy is another strategy for treating cutaneous t-cell lymphoma, although it is rarely used in veterinary medicine because of "patient compliance" problems. mechlorethamine (mustargen) can be applied topically as an aqueous solution or an ointment base. the aqueous solution is prepared by combining 10 mg of mechlorethamine with 50 ml of tap water. the ointment is prepared by mixing 90 mg of mechlorethamine with 10 ml of absolute alcohol and further combining enough xipamide (aquaphor) to prepare 900 g of ointment. hair must be removed before application. gloves must be used when the drug is applied, because mechlorethamine is carcinogenic and can cause contact hypersensitivity in humans. the response to therapy varies, and the treatment often is only palliative. 253 the prognosis for canine lymphoma varies and depends on a number of factors, such as the location of disease, the extent of disease (the clinical stage), the presence or absence of clinical signs (the substage), the histologic grade, the immunophenotype (t cell or b cell), exposure to previous chemotherapy or corticosteroids and subsequent development of mdr (see chapter 11), altered cell death processes (apoptosis), the proliferation rate of the tumor, the presence of concurrent medical problems or paraneoplastic conditions (e.g., hypercalcemia, weight loss, and liver insufficiency), and possibly gender.* although canine lymphoma is rarely curable (fewer than 10% of cases), complete responses and a good quality of life during extended remissions and survival are typical. factors that have been shown to influence survival are shown in table 31 -5. the two factors most consistently identified as being prognostically important in dogs with lymphoma are the immunophenotype and who substage (see figure 31 -9). many reports have confirmed that dogs with cd3-immunoreactive tumors (i.e., t-cell derivation) are associated with significantly shorter remission and survival durations. † this holds true primarily for dogs with multicentric lymphoma, because the immunophenotype of solitary or extranodal forms of lymphoma has not been thoroughly investigated with respect to the prognosis. in addition, it has been shown that dogs with b-cell lymphomas that express lower than normal levels of b5 antigen (expressed in 95% of nonneoplastic lymphocytes) also have shorter remission and survival durations. 75 dogs with who substage b disease (i.e., clinically ill) also do poorly compared to dogs with substage adisease (see figure 31 -9). ‡ dogs with stage i or stage ii disease have a better prognosis than dogs with more advanced disease (stage iii, iv, or v). 187, 188 in some studies, an elevated serum calcium (over 11.9 mg/dl) has been shown to be a negative prognostic factor 72, 257 ; however, this rarely holds true with multivariate analysis because hypercalcemia is associated with the t-cell phenotype. the histologic grade (subtype) has been found to influence the prognosis in some studies; however, our ability to predict outcome based on subtype is still quite limited. dogs with lymphoma classified as intermediate-or high-grade (large cell, centroblastic, and immunoblastic) tend to respond to chemotherapy but can relapse early. dogs with low-grade lymphomas (small lymphocytic or centrocytic) have a lower response rate to chemotherapy, yet have a survival advantage over dogs with intermediate-or high-grade lymphomas ( figure 31 -10) in that the course of disease may be more indolent. 113 using the working formulation, dogs with low-grade lymphomas have a survival advantage compared to dogs with intermediate-or high-grade tumors. 78 recently, proliferative assays (e.g., analysis of bromodeoxyuridine [brdu] uptake, ki-67 antibody reactivity, and agnor indices) to measure the proliferative activity of tumor cells have been shown to provide significant prognostic information in dogs treated with combination chemotherapy. 60, 163, 254, [258] [259] [260] however, the results of different studies are contradictory. in two trials, dogs that had tumors with short doubling times, high agnor frequencies, or high ki-67 immunoreactivity had a better prognosis than dogs with long doubling times or low agnor frequencies. 60, 163 in other trials, the low-proliferating tumor groups were associated with a better prognosis. 254, 259 in one trial, the proportion of tumor cells undergoing apoptosis was modestly predictive of remission duration. 163 the anatomic site of disease also has considerable prognostic importance. primary diffuse cutaneous, diffuse gastrointestinal, hepatosplenic, and primary cns lymphomas tend to be associated with a poor prognosis. cutaneous lymphoma tends to progress slowly, and in our experience the responses to systemic chemotherapy are less durable. localized lymphomas in the skin can be managed with radiation therapy or surgery or both, and these tumors have a better prognosis. in some dogs with lymphoma, significant involvement in the bone marrow may be present (i.e., tumor cells comprise more than 50% of all nucleated cells) and circulating malignant lymphocytes may be present in the peripheral blood. these dogs tend to have an overall poor prognosis. in some cases it is difficult to determine whether the disease arises from the bone marrow (e.g., acute lymphoblastic leukemia [all]) or is a diffuse lymphoma with extensive involvement in the marrow. immunophenotyping is helpful in these cases, because the tumor cells in all typically are cd34 positive. gender has been shown to influence the prognosis in some studies. 59, 191 neutered females tend to have a better prognosis. males may have a higher incidence of the t-cell phenotype, which may account for the poorer prognosis. 60 other reported potential biomarkers of the prognosis include circulating levels of glutathione-s-transferase, thymidine kinase, and vascular endothelial growth factor (vegf). 261-263 one report suggests that a history of chronic inflammatory disease of several types predicts a likelihood of early relapse. 264 these putative prognostic indicators require confirmation in larger trials. lymphoid leukemia is the proliferation of neoplastic lymphocytes, which usually originate in the bone marrow but occasionally may originate in the spleen. the neoplastic cells may or may not be circulating in the peripheral blood. lymphocytic leukemia is more common than nonlymphocytic leukemia and other myeloproliferative disorders (mpds). the true incidence is not known. in a series of 30 cases of all, german shepherds accounted for 27% of the cases, and the male to female (m:f) ratio was 3:2. 265 in this study, the median age was 5.5 years (range, 1 to 12 years), and eight dogs were younger than 4 years of age. recently, all was reported in a 12-week-old greyhound. 266 well-differentiated or chronic lymphocytic leukemia (cll) is seen less frequently than all but more commonly than mpd. the median age is 10 to 12 years. the m:f ratio has been reported as 1.8:1 (22 dogs) and approximately 2.3:1 in 15 dogs with granular lymphocytic (gl) t-cell cll. 267 in a large study of 73 dogs with cll, no gender predilection was found in dogs with b-cell or non-gl cll; female dogs were overrepresented among dogs with gl cll, (m:f ratio, 1:1.7). 268,269 as with lymphoma, the etiology of lymphoid leukemia is unknown. retroviruses have been implicated in diverse animal species such as cats, cattle, fish, snakes, birds, rodents, and nonhuman primates. a retroviral cause in dogs has not been proved. however, a retrovirus with morphology typical of lentiviruses has been isolated from mononuclear cells obtained from the peripheral blood of a dog with all. 270 in humans, acute leukemia has been associated with exposure to radiation, benzene, phenylbutazone, and antineoplastic agents. [271] [272] [273] [274] [275] [276] the alkylating agents can cause chromosomal damage and are clearly carcinogenic. 276 human t-lymphotropic virus type 1 (htlv-1) is a proven cause of leukemia in a large cohort of human patients from the southern islands of japan. 277,278 the etiology of cll is less clear, but genetic factors likely are important. extrapolation of predisposing factors across species is not warranted, and the etiologic factors for dogs may be quite different from those for humans, given the difference in the predominant immunophenotype of the neoplastic cells (discussed later). in all, the blast cells infiltrate the bone marrow, and the result is variable degrees of anemia, thrombocytopenia, and neutropenia. infiltration of the spleen and liver is common, and extramedullary sites (e.g., the nervous system, bone, and gi tract) also may be involved. some animals may have lymph node involvement and develop generalized lymphadenopathy. 265 the lymphocytes of cll are virtually indistinguishable morphologically from normal small lymphocytes, and they have a low proliferation rate. the accumulation of lymphocytes likely results from their prolonged life span. in b-cell cll, the marrow is infiltrated with mature lymphocytes, and the extent of infiltration is less than that seen with all or mpd. the neoplastic cells in gl t-cell cll originate in the spleen, and bone marrow involvement may or may not occur. 173 dogs with cll tend to have a mild anemia, and granulocytes and platelets are only mildly reduced. splenomegaly is common, and lymph nodes can be slightly to moderately enlarged. 269 despite the well-differentiated appearance of the lymphocytes in cll, these cells may function abnormally. part iv • specific malignancies in the small animal patient paraneoplastic syndromes include monoclonal gammopathies, immune-mediated hemolytic anemia, pure red cell aplasia and, rarely, hypercalcemia. 173, 279 hypercalcemia was reported in a giant schnauzer with b-cell cll, which is highly unusual given that hypercalcemia is associated with t-cell lymphoproliferative disorders. in one study of 22 dogs with cll, 68% had monoclonal gammopathies. 269 the immunophenotypes were not reported, but a monoclonal gammopathy is associated with production of immunoglobulins by the leukemic cells (b cells). the immunoglobulin is usually igm or iga. the term macrogammaglobulinemia is used to describe igm gammopathy (see chapter 31, section d). dogs with cll and an igm monoclonal gammopathy are said to have waldenström's macroglobulinemia. dogs can also develop hyperviscosity syndrome (see chapter 31, section d). reports of the immunophenotype of neoplastic cells in canine leukemias are increasing in frequency. one consistent finding in acute leukemias (either all or acute myeloid leukemia) is the presence of cd34 on the blast cells. this marker is useful for distinguishing all (cd34-positive) from cll and lymphoma with bone marrow infiltration (cd34-negative). in one study of 12 cases of cll, eight dogs were associated with a cd8-positive t-cell phenotype. 75 in a larger study of 73 cases, only 26% of the cases had a b-cell immunophenotype, whereas 73% were identified as t-cell cll (cd3-positive). in 54% of these t-cell leukemias, the cells had the morphology of gls, and most of them were cd8-positive. these findings are difficult to reconcile with the high frequency of monoclonal gammopathies reported in the earlier study. it also should be noted that this differs from human clls, which typically are lymphoproliferative diseases of b cells. dogs with all usually have a history of anorexia, weight loss, polyuria, polydipsia, and lethargy. splenomegaly is typical, and other physical abnormalities may include hemorrhages, lymphadenopathy, and hepatomegaly. anemia, thrombocytopenia, and an elevated white blood cell (wbc) count are common. the anemia may be severe and usually is characterized as normocytic and normochromic (nonregenerative). wbc counts usually are increased despite neutropenia because of an increased number of circulating lymphoblasts (more than 14,000 cells/ml). however, some dogs may be leukopenic. 265 neoplastic lymphoblasts may infiltrate the bone marrow extensively, resulting in depression of normal hematopoietic elements. dogs with cll often are asymptomatic, although some owners report lethargy and a decreased appetite. mild lymphadenopathy and splenomegaly may be present. most dogs tend to be mildly anemic (packed cell volume [pcv] less than 35%) and thrombocytopenic (110,000 to 190,000 platelets/ml). the wbc count usually exceeds 30,000 cells/ml but can vary from normal to more than 100,000 cells/ml, owing to an increase in circulating mature lymphocytes. 269 lymphocytosis is persistent, and granulocytes usually are present in normal numbers. in some dogs the disease is identified incidentally when the patient undergoes evaluation for an unrelated problem. the clinician must consider the signalment, history, physical findings, cell morphology, and immunophenotype to diagnose the lymphoid leukemias accurately. a knowledge of the profile of lymphocyte subsets in the peripheral blood of normal dogs is helpful for determining whether a particular subset has expanded. approximately 80% of circulating lymphocytes in dogs are t cells, and about 15% are b cells. nk cells and double-negative (cd4−cd8−) t cells account for the remaining fraction. in the t-cell fraction, helper t cells (cd4+) outnumber cytotoxic t cells (cd8+). 173 lymphocytic leukemia should be a consideration if atypical lymphocytes are in circulation, if the immunophenotype of the lymphocytes in circulation is homogenous, or if a phenotype typically present in low numbers has increased. differential diagnoses for lymphocytosis include certain infectious diseases (e.g., chronic ehrlichiosis), postvaccinal responses in young dogs, il-2 administration, and transient physiologic or epinephrine-induced lymphocytosis. reactive and neoplastic lymphocytoses sometimes are difficult to differentiate. in these cases, pcr assays are used to determine the clonality of the t cell or immunoglobulin receptor genes in a population of lymphocytes (see earlier discussion). 167, 169, 268 infiltration of the bone marrow by neoplastic lymphoid cells is the hallmark of all and is seen in most cases of cll; careful examination of peripheral blood and bone marrow by experienced cytopathologists is essential for establishing a diagnosis of lymphocytic leukemia. if adequate diagnostic bone marrow cannot be obtained by aspiration, a bone marrow core biopsy should be performed. in all, lymphoblasts predominate in the bone marrow and also are present in peripheral blood. infiltration of bone marrow by lymphoblasts is accompanied by a decrease in the granulocytic, erythroid, and megakaryocytic cell lines. perhaps the distinguishing feature of lymphoblasts is the nuclear chromatin pattern, which is more condensed than the chromatin in myeloblasts. 280 lymphoblasts are larger than neutrophils, have a high nucleus-to-cytoplasm ratio, and blue cytoplasm, which in some cases is intensely basophilic ( figure 31-11, a) . nucleoli, although present, are less prominent in lymphoblasts than in myeloblasts. nevertheless, these cells cannot be easily distinguished from blast cells of other hematopoietic lineages, and lineage-specific markers on the cells must be identified by immunocytochemistry or flow cytometry. 278 the most frequently used markers are cd3 for t cells and cd79a for b cells. as mentioned earlier, cd34 is found on blast cells of both lymphoid and myeloid lineages, and positivity for cd34 helps to confirm leukemia of immature cells (acute leukemia) and to distinguish all from stage v lymphoma. immature and differentiating lymphocytes may stain strongly for alkaline phosphatase activity, which suggests that this cytochemical staining procedure is not specific for myeloid leukemia. 278, 280, 281 in b-cell and non-gl t-cell cll, the lymphocytes are small mature cells ( figure 31-11 , b) that appear in excessive numbers in bone marrow (30% or more of all nucleated cells) early in the disease. 269 infiltration becomes more extensive as the disease slowly progresses, and eventually the neoplastic cells replace normal marrow. the lymphoid lineage of the cells in cll typically is easy to identify, and immunophenotyping is done to determine the lymphocyte subset. in dogs, most cases of cll are t-cell (cd3+) proliferations. in gl cll, the neoplastic cells originate in the spleen. 173 a separate clinical staging system has not been developed for lymphocytic leukemia. currently, all dogs with leukemia are classified as stage v based on the who staging system for lymphoma (see box 31-1). although a specific clinical staging system for cll has been used in humans, it has not been evaluated in the dog. 282 similar to other infiltrative bone marrow malignancies, all causes morbidity by suppressing bone marrow function. neutropenia, thrombocytopenia, and anemia may be severe. therapy must be aggressive; to restore hematopoiesis, a 1.5 to 2 logarithmic reduction in leukemic cell numbers (to less than 100 million cells) must be achieved. patients need supportive therapy, such as fresh whole blood, broad-spectrum antibiotics, fluid therapy, and nutritional support. patients must be monitored carefully for bleeding and thrombosis, which may signal the development of dic. all requires aggressive chemotherapy. consistently efficacious protocols for all have not been developed in veterinary medicine. chop-based protocols, similar to those used for lymphoma (see , tend to be used as treatment protocols for dogs with all. in one report on the use of vincristine and prednisone in dogs with all, 40% of the dogs responded to vincristine and prednisone, 20% with a complete remission and 20% with a partial remission. 265 with the addition of doxorubicin and l-asparaginase, it is anticipated that response rates will increase over those previously reported using vincristine and prednisone alone; however, the relative rarity of all limits the ability to identify effective protocols. a b because of the indolent nature of cll in many animals, the question of whether all dogs with the disease should be treated is controversial. 283,284 most oncologists recommend observation over active therapy when the discovery of cll is incidental, when no physical or clinical signs are present, and when no significant hematologic abnormalities are identified. therapy should be instituted if the animal is anemic or thrombocytopenic, is showing evidence of significant lymphadenopathy or hepatosplenomegaly, or has an excessively high wbc count (over 60,000 lymphocytes/ml) (box 31-4). the most effective drug evaluated thus far is chlorambucil. 269 chlorambucil is administered at a dosage of 0.2 mg/kg or 6 mg/m 2 given orally once daily for 7 to 14 days. the dosage then can be reduced to 0.1 mg/kg or 3 mg/m 2 given orally once daily. for long-term maintenance, a dosage of 2 mg/m 2 given orally every other day can be used. the dosage is adjusted according to the clinical response and bone marrow tolerance. chlorambucil should be administered without food to increase the rate of absorption. 285 corticosteroids are lymphocytolytic and lead to cell death by apoptosis. studies in humans have shown that the antitumor activity of chlorambucil combined with prednisone is better than that of chlorambucil alone. 286 when the bone marrow is heavily infiltrated with cll cells, and neutropenia, thrombocytopenia, and anemia occur, use of a more aggressive alkylating agent (usually cyclophosphamide [250 mg/m 2 given once]) when initiating oral chlorambucil and prednisone therapy can be considered to increase the speed of initial remission; however, this modification of the protocol has not been scrutinized in clinical trials. if chlorambucil or cyclophosphamide fails, the choice of treatment is combination chemotherapy similar to that presented in box 31-3. the treatment of cll is primarily palliative, and complete remissions are rare. because of the indolent nature of this disease, however, survival times have been in the range of 1 to 3 years with a good quality of life. 269, 284 in humans, splenectomy has been shown to increase survival significantly in individuals with aggressive forms of cll; however, splenectomy in dogs with cll has not been evaluated. 287 the phenotypic expression of cll usually is stable over months to years. however, the disease may evolve into an acute phase, and some dogs develop a rapidly progressive, pleomorphic (immunoblast) lymphoma. 269 in humans, this is called richter's syndrome. 288 the prognosis for response to treatment is poor for this form. in general, the prognosis for all in the dog is very poor. in a study of 21 dogs treated with vincristine and prednisone, the dogs achieving complete or partial remission (29%) had a median survival time of 120 days, and few dogs survived longer than 8 months with that protocol. 265 in one case report, a dog with all was treated with an infusion of a large volume of fresh canine plasma and whole blood, and a complete remission was maintained for 19 months without additional therapy. this is a very unusual response, which indicates that normal blood contains some antileukemic factor or factors. 289 as stated before, cll is a slowly progressive disease, and some animals do not require therapy. one dog was observed for almost 2 years without treatment. 283 for dogs that are treated, normalization of wbc counts can be expected in 70% of cases. in one report of 17 dogs treated with vincristine, chlorambucil, and prednisone, the median survival time was approximately 12 months, with an expected 30% survival at 2 years. 269 in other studies, dogs treated intermittently with chlorambucil and prednisone have had remissions of 10 to 30 months. 284 david m. vail the lymphomas (malignant lymphoma and lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. they usually arise in lymphoid tissues, such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. lymphoma is one of the most common neoplasms seen in the cat. the feline leukemia virus (felv) was the most common cause of hematopoietic tumors in the cat during the so-called felv era of the 1960s through the 1980s, when 60% to 70% of lymphoma cases were associated with felv antigenemia. [1] [2] [3] [4] [5] [6] [7] several studies have documented the potential molecular means by which felv can result in lymphoid neoplasia. 8, 9 however, over the past 20 years in north america, a profound change has occurred in the viral status, presentation, signalment, and frequency of anatomic sites in cats with lymphoma, as documented by two large studies of feline lymphoma involving more than 700 cats. 10, 11 the change in the epidemiology and characteristics of lymphoma in cats appears to coincide with the widespread integration of clinically relevant felv diagnostic assays (indirect immunofluorescence assay) and the affected animal elimination regimens of the late 1970s; it was further enhanced by the appearance of commercially available felv vaccines in the late 1980s. the decline in felv-associated lymphoma was mirrored by a decline in the overall prevalence per year of felv positivity in cats tested, as characterized by reports from the tufts veterinary diagnostic laboratory from 1989 to 1997 10,12 and by louwerens' group, 11 which reported a decline in felv association in more than 500 cases of lymphoma in cats brought to the university of california-davis veterinary teaching hospital. in these reports, felv antigenicity represented only 14% to 25% of the cats with lymphoma. the incidence figures for feline lymphoma generated before the use of felv vaccination and testing became widespread suggest that lymphoma accounted for 50% to 90% of all hematopoietic tumors in the cat 13, 14 ; because hematopoietic tumors (lymphoid and myeloid) represent approximately one third of all feline tumors, the estimated incidence of lymphoid neoplasia was 200 per 100,000 cats at risk. 15 in one series of 400 cats with hematopoietic tumors, 61% had lymphoma and 39% had leukemias and mpds (21% of the mpds were categorized as undifferentiated leukemias, most likely myeloid in origin). 16 an important fact that louwerens' study revealed is that despite a sharp drop in felv-associated lymphoma, the overall prevalence of lymphoma in cats is increasing. 11 this appears to be due to an increase in the number of affected cats and the relative frequency of the abdominal (particularly the intestinal) anatomic form of lymphoma in the species (figure 31-12) . as might be expected, along with a shift away from felv antigen-associated tumors came a shift away from the traditional signalment and relative frequency of anatomic sites. 10, 11 this observation is supported outside north america by the similar signalment and anatomic frequency data obtained in australia, where felv infection is quite rare. [17] [18] [19] [20] the median age of approximately 11 years now reported in north america for lymphoma in cats is considerably higher than the median age of 4 to 6 years reported during the felv era. 1,3-6,10,11,21 the median age of cats within various anatomic tumor groupings has not changed, and anatomic forms traditionally associated with felv, such as the mediastinal form, still occur in younger, felv-antigenemic cats. similarly, the alimentary form occurs most often in older, felv-negative cats. 10, 11, [18] [19] [20] table 31 -6 presents an overview of the characteristics of the various anatomic sites of lymphoma in cats. younger cats with lymphoma tend to be felv antigenemic and are more likely to have mediastinal or multicentric lymphoma. most cats with spinal lymphoma (85% to 90%) also are felv antigenemic. 22, 23 older cats usually are felv negative and develop alimentary lymphoma. 10, 17, 19, 20, [24] [25] [26] [27] in a large compilation of australian cases, male cats and the siamese/oriental breeds were overrepresented. 17 similar breed findings have been observed in north america, 11 but similar gender findings have not emerged. 28, 29 the siamese/oriental breeds appear to have a predisposition for the mediastinal form that affects a younger population (median age, 2 years). 11 evidence also indicates that feline immunodeficiency virus (fiv) infection can increase the incidence of lymphoma in cats. [30] [31] [32] [33] [34] [35] [36] [37] in contrast to felv, which plays a direct role in tumorigenesis, fiv appears to have an indirect role, likely secondary to the immunosuppressive affects of the virus. 35 shelton and colleagues 30 determined that fiv infection alone in cats was associated with a fivefold increased risk for the development of lymphomas. coinfection with felv further potentiates the development of lymphoproliferative disorders. experimentally, cats infected with fiv have developed lymphoma in the kidney, alimentary tract, and liver and in multicentric sites. fiv-associated lymphoma is more likely to have a b-cell immunophenotype whereas t-cell type is predominantly associated with felv. 32, 33, 35, 36, [38] [39] [40] [41] some have suggested that fiv infection may be associated more often with alimentary lymphoma of b-cell origin, 40, 41 which may be related to chronic dysregulation of the immune system or activation of oncogenic pathways. however, in another large compilation of cases, fiv antigenemia only rarely was associated with alimentary lymphoma. 10, [24] [25] [26] [27] genetic and molecular factors as discussed in chapter 31, section a, recent advances in molecular cytogenetics (see chapter 1, section a), including gene microarray techniques, are being used in investigations of chromosomal aberrations in veterinary species with lymphoma. the previously mentioned predisposition of the oriental cat breeds to the development of lymphoma suggests a genetic predisposition and indicates heritable risks. 11, 17 as our knowledge of molecular events and tumorigenesis has expanded, several molecular aberrations have been implicated in various feline tumor types, and some associated with lymphoma have been identified. altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and deathpathway alterations are common in human lymphomas and likely are also involved in the cat. n-ras aberrations, although rare in cats, have been implicated. 42 telomerase activity (see chapter 14, section d) also has been documented in feline lymphoma tissues. 43, 44 other factors implicated in feline lymphoma include alterations in cellular proliferation and in cell cycle and death (apoptosis) pathways, particularly the cyclindependent kinase cell cycle regulators and the bcl-2 family of proapoptotic and antiapoptotic governing molecules, which have been implicated in human nhl. 45, 46 environmental factors evidence that exposure to environmental tobacco smoke (ets) is a risk factor for lymphoma in humans has prompted investigations in cats. in one report, cats with any exposure to ets had a relative risk of developing lymphoma of 2.4; the relative risk for cats with 5 or more years of exposure was 3.2. 47 immune system alterations in the cat, such as those brought on by fiv infection, have been implicated in the development of lymphoma. [30] [31] [32] [33] [35] [36] [37] in a report on 95 feline renal transplant recipients, nearly 10% developed de novo malignant lymphoma, which bolsters support for a link to immunosuppression in the species. these findings are similar to those seen in immunosuppressed human organ transplant patients. 48 although definitive proof is lacking, a growing body of indirect evidence suggests that lymphoma may be associated with a state of chronic inflammation, such as in intestinal and nasal lymphoma. in particular, an association has been suggested between intestinal lymphoma and inflammatory bowel disease 11,49-51 ; however, others have not found support for this concept. 52 additional support for this association is provided by a recent report that suggests that cats with vaccine site-associated sarcoma, a syndrome directly linked to inflammation, are also at risk for the development of lymphoma. 53 although no direct evidence exists, a link between diet and the development of intestinal lymphoma in cats has been suggested. 11 this association is supported by the relative and absolute increase in the intestinal form of lymphoma over the past 20 years and by the fact that several dietary modifications in cat food have occurred in a similar time frame in response to conditions such as urinary tract disease. further investigation is warranted to prove or disprove such assertions. lymphoma can be classified on the basis of anatomic location and histologic criteria. several systems of anatomic classification exist for lymphoma in the cat. some categorize the disease into mediastinal, alimentary, multicentric, nodal, leukemic, and individual extranodal forms. others have combined various nodal and extranodal forms into categories of atypical, unclassified, and mixed, while still others have combined intestinal, splenic, hepatic, and mesenteric nodal forms into one category (intra-abdominal). for the purposes of this discussion, the "mixed" form is defined as cases involving multiple sites in which no primary site categorization was possible. some discrepancies in the discussion of frequency inevitably will result from the variations in classification used in the literature. the relative frequency of anatomic forms and the immunophenotype may vary with geographic distribution and may be related to genetic and felv strain differences, as well as to the prevalence of felv vaccine use (see table 31 -6). in most larger studies, most cases of lymphoma in cats (approximately 70% to 75%) are of the b-cell immunophenotype; however, the mediastinal, leukemic, and purely hepatic forms are more likely to be of t-cell derivation. 10, 18 cats with t cell-rich b-cell lymphoma (hodgkin's-like lymphoma) and nk-like t-cell lymphoma (non-t, non-b-cell) have been described. 54, 55 as with dogs, most lymphomas in cats have an intermediate-grade or a high-grade histology according to the working formulation (wf) criteria. 18, 56 alimentary/intestinal lymphoma alimentary/intestinal lymphoma can manifest as a purely intestinal infiltration or as a combination of intestinal, mesenteric lymph nodes, and liver involvement. 11 some reports limit the alimentary form to gi involvement, with or without extension to the liver. approximately two thirds to three fourths of reported cases are of the b-cell immunophenotype. most patients are older cats, and very few cases appear to be associated with felv antigenemia. 10, 11, 18, [24] [25] [26] [57] [58] [59] however, some discordance on these points is seen in the literature. in one smaller report, most alimentary cases sampled were found to be of the t-cell immunophenotype. 24 the population size was relatively small in comparison and may represent a geographic variation. the epitheliotropic form of intestinal lymphoma also has been reported to be more commonly t-cell in origin. 49, 57 a canadian group investigated formalin-fixed archival lymphoma tissue in cats and found a nearly even distribution between the b-cell and t-cell immunophenotype; they also found that approximately two thirds of the samples were felv positive by pcr techniques. 2,60-62 some felv-negative tumors may be derived from transformation of multipotent lymphoid or monocyte precursors 61 or fiv-transformed b lymphocytes. 32, 33, 38, 39 the most common site of involvement in the alimentary tract is the small intestine (50% to 80% of cases), followed by the stomach (approximately 25%), ileocecocolic junction, and colon. 25, 26, 62 the tumor can be solitary or diffuse throughout the intestines (figure 31-13) , muscle layers, and intestinal submucosa, resulting in annular thickening that leads to partial or complete intestinal obstruction. in a series of colonic neoplasias part iv • specific malignancies in the small animal patient necropsy specimen from a cat with intestinal lymphoma. note the generalized thickening of the small intestine and the associated mesenteric lymph node involvement (arrows). in cats, lymphoma was the second most common malignancy (41%), exceeded only by adenocarcinoma. 27 an abstract report has described chronic lymphocytic-plasmacytic enteritis in cats that progressed to overt lymphoma after 6 to 18 months of conservative therapy. 63 the mediastinal form can involve the thymus and the mediastinal and sternal lymph nodes. pleural effusion is common. in two large compilations, 63% of cats with thymic disease and 17% of cats with pleural effusion were documented as having lymphoma. 64, 65 occasionally the tumor extends from the thoracic inlet and can be palpated in the ventral neck region. hypercalcemia is common with mediastinal lymphoma in dogs but is rare in cats with lymphoma. [66] [67] [68] pthrp has been detected through immunoradiometric assays in cats with hypercalcemia of malignancy, including one cat with lymphoma. 69 most cats with mediastinal lymphoma are young and felv positive, and their tumors are of the t-cell immunophenotype.* involvement of peripheral lymph nodes alone is very unusual in cats with lymphoma, representing approximately 4% to 10% of cases. 10, 11 in contrast, approximately one fourth of all other anatomic forms of lymphoma have some degree of lymph node involvement. 11 one third of cats with nodal lymphoma are of the t-cell immunophenotype and are felv antigenemic. 10, 18 as lymphoma progresses, bone marrow infiltration with malignant cells and hepatosplenomegaly may develop. recently an uncommon and distinct form of nodal lymphoma in cats, referred to as hodgkin's-like lymphoma, has been reported. this form typically involves solitary or regional nodes of the head and neck and histologically resembles hodgkin's lymphoma in humans. 17, 54, 70 these tumors generally manifest as an enlargement of a single mandibular or cervical node and immunophenotypically are classified as t cell-rich b-cell lymphoma by immunohistochemical analysis. 54, 70 none have been associated with either felv or fiv. several reports of nonneoplastic peripheral lymphadenopathy in cats have been published. [71] [72] [73] [74] [75] this condition resembles lymphoma clinically, and it has histologic features that also may resemble those of lymphoma. [71] [72] [73] [74] [75] affected lymph nodes may be two or three times normal size. in one report, the syndrome was called distinctive peripheral lymph node hyperplasia (dplh) of young cats. 72 these cats tend to be young (2 to 4 years old), and many have had episodes of fever or previous viral infections, or they may have hypergammaglobulinemia (polyclonal gammopathy); most are felv negative. 71 histopathologically, the nodal architecture is severely distorted, showing loss of subcapsular and medullary sinuses. the cell population shows an admixture of histiocytes, lymphocytes, plasma cells, and immunoblasts and occasionally effaced lymphoid follicles. the lymph nodes regress spontaneously in most of these cats. the histologic changes noted in these lymph nodes resemble the histologic features of acquired immunodeficiency syndrome (aids)-related lymphadenopathy in humans. 72 in another report, benign lymphadenopathy in cats was associated with argyrophilic intracellular bacteria. 75 the most common extranodal sites for lymphoma are the kidneys, nasal cavity, eyes, retrobulbar space, cns, and skin. renal lymphoma can be primary or associated with alimentary lymphoma. based on several studies, the median age for cats with renal lymphoma is approximately 7.5 years. one fourth of cases are felv antigenemic, and most are of the b-cell immunophenotype. 10, 11, 18, 76 the frequency of renal lymphoma is reported to be approximately 5% of all lymphomas. extension to the cns is a common sequela to renal lymphoma and occurs in 40% to 50% of treated cats. 76 nasal/paranasal lymphoma usually is a localized disease; however, systemic extension occasionally is seen. 10, 77, 78 neoplasia accounts for most nonviral nasal/paranasal disease in cats, and lymphoma has been reported to represent nearly one third to one half of these cases. [79] [80] [81] it occurs primarily in older, felvnegative cats (median age, 9 to 12 years). at least three fourths of cases are b cell in origin, and most are intermediate grade or high grade histologically. 10,79-81 a subset of epitheliotropic t-cell nasal lymphomas has been reported. 81 cats that are concurrently felv positive are more likely to have concurrent systemic disease. in the older literature, cns lymphoma was most often seen extradurally in the spinal canal of felv-positive cats (85% to 90%). 20, 21, 82 however, in more recent reviews, both spinal cord and intracranial lymphoma occurs mostly in older, felv-negative cats, and spinal cord sites are more commonly intradural than extradural. 83, 84 after meningioma, lymphoma is the second most common tumor involving the cns in cats. 84 feline cns lymphoma may be primary or may occur secondary to multicentric involvement (especially of the renal system or bone marrow). [83] [84] [85] [86] bone involvement is rarely seen radiographically. 87 multiple cord regions and the brain are involved in nearly 50% of cats with spinal lymphoma, and more than 80% of these patients have other organ (e.g., renal) and bone marrow involvement. 20, 21, 83 only one third of cases of intracranial lymphoma are primary and confined to the cns, and all cats tested have been fiv negative. 84 cutaneous lymphoma generally is primary but can be seen secondary to multicentric involvement. it commonly is seen in older cats (median age, 10 to 12 years) and gender sex or breed predominance has not been found. [88] [89] [90] [91] [92] although patients usually are felv negative, one case report using pcr techniques found evidence of felv provirus in tumor dna. 92 cutaneous lymphoma can be solitary or generalized. two forms of cutaneous lymphoma have been distinguished histologically and immunohistochemically. [88] [89] [90] [91] [92] in most species, the epitheliotropic form, sometimes referred to as mycosis fungoides, is composed of t lymphocytes, whereas the nonepitheliotropic form usually is composed of b lymphocytes. in contrast, one report of nonepitheliotropic cutaneous lymphoma in cats found that five of six cases were of t-cell derivation. 93 neoplastic t lymphocytes are large and have abundant cytoplasm and convoluted nuclei (mycosis cells). they usually form intraepidermal nests of five to 10 cells, which are separated from surrounding keratinocytes by a clear space (pautrier's microabscesses). the b-cell lymphomas show lymphocytes deep in the epidermis, with sparing of the papillary dermis and epidermis. a recent report described 23 cases of cutaneous lymphocytosis, an uncommon disease histologically resembling well-differentiated lymphoma. 94 solitary lesions were most common, and all were composed primarily of t-cells, although two thirds had some b-cell aggregates. cutaneous lymphocytosis was characterized as a slowly progressive disorder, but internal organ infiltration developed in a few cases. a cat with cutaneous t-cell lymphoma and circulating atypical lymphocytes has been reported. 91 the circulating cells were lymphocytes with large, hyperchromatic, grooved nuclei. in humans, cutaneous t-cell lymphoma with circulating malignant cells is called sézary syndrome, 95 which also has been reported in dogs. [96] [97] [98] [99] a number of histopathologic grading systems have been used to classify human nhl, including the wf (discussed earlier in the chapter). most recently, the wf was used to classify more than 600 cases of feline lymphoma. 56 low-grade lymphoma was found in 11% of the cases, intermediate-grade disease in 35%, and high-grade lymphoma in 54%. about 1.1% were plasmacytomas. more than one third of the tumors were the immunoblastic type. lymphoblastic lymphoma, a subtype of the high-grade tumor, accounted for less than 3%. similarly, in a large group of cases compiled in australia (n = 118), 90% of the cases were mediumto high-grade disease as classified by the wf. 18 also as discussed earlier in the chapter, who has published a histologic classification scheme that uses the revised european american lymphoma (real) system as a basis for defining histologic categories of hematopoietic tumors of domestic animals. 100 this system incorporates both histologic criteria and immunohistologic criteria (b-and t-cell immunophenotype). the clinical relevance of this system is likely to be high; however, it awaits further investigation and evaluation. in a series of 28 cases of alimentary lymphoma, 89% were the high-grade lymphoblastic type. 25 however, in another report, many of the intestinal lymphomas had several characteristics of malt, as described in humans; that is, a tendency to remain localized in the lamina propria, a relatively indolent clinical behavior, and an excess of low-grade tumors. 56 the age of affected animals varied considerably with various subtypes, but in general, low-grade tumors tended to develop in older cats (over 10 years of age), and high-grade tumors tended to develop in younger cats (under 6 years of age). a less commonly reported, distinct form of alimentary lymphoma has been described and classified as large granular lymphoma. [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] these lesions are granulated, round cell tumors that also have been called either globule leukocyte tumors or large granular lymphocyte lymphoma, although they probably are variations of the same disease. large granular lymphocytes are characterized by abundant cytoplasm with prominent azurophilic granules. this population of cells includes nk cells and cytotoxic t cells. several reports have identified their immunophenotype as cd3, cd57-like, perforin positive, and cd20 negative; these cells also have been described as having a t-cell receptor gene rearrangement. these tumors commonly originate in the small intestine, especially the jejunum or mesenteric lymph nodes; however, most cases show widespread metastasis to the lung, myocardium, salivary gland, and spinal cord. leukemia also has been reported with this disease. affected cats generally are felv negative. large granular lymphocytes must be differentiated from several other granular cell types that may be found in the small intestine, including enterochromaffin cells, mast cells, and eosinophils. the clinical signs associated with feline lymphoma vary and depend on the location and extent of disease. the alimentary form is most commonly associated with an abdominal mass that originates in the gi tract. the condition often is associated with enlarged mesenteric lymph nodes or other organ involvement. in 50% to 85% of cases, a palpable abdominal mass or thickened bowel loops are present. 24, 25, 27, 62, 111 clinical signs may consist of weight loss, anorexia, diarrhea, and occasional vomiting. in approximately half of cases, the only historical finding is anorexia and weight loss. 25 other reported presentations include abdominal distention, splenomegaly, persistent thrombocytopenia, and pica. hematochezia and tenesmus may be present if the lymphoma involves the colon. 27 polyuria and polydipsia have been reported in approximately 10% of cases. 62 in a small percentage of cases, the patient may have signs consistent with an acute abdomen as a result of intestinal perforation and concurrent peritonitis. 24 clinical signs of the mediastinal form of lymphoma include dyspnea, tachypnea, and a noncompressible anterior mediastinum with dull heart and lung sounds. 20, [111] [112] [113] [114] in rare cases, horner's syndrome may be present as a result of involvement of the sympathetic nerve as it ascends around the first rib, and edema of the head may be caused by pressure on the cranial vena cava. 111 pleural effusion is common; the pleural fluid is characterized by serohemorrhagic to chylous effusion, and in most cases neoplastic cells (lymphoblasts) are identified. 65, 114 cats with the nodal form of lymphoma have variable clinical signs, depending on the location and extent of disease; however, these cats often are depressed and lethargic. peripheral lymphadenopathy as the only physical finding is a very uncommon presentation for cats. as stated earlier, an uncommon and distinct form of nodal lymphoma in cats is hodgkin's-like lymphoma. this form typically involves a single mandibular or cervical node, and the cat usually does not have any overt clinical signs. 54, 70 in cats, unlike in dogs, peripheral lymphadenopathy without organomegaly is more often hyperplastic or reactive and does not represent lymphoma. 73 the extranodal sites include the kidneys, skin, eyes, nasal area, and cns. renal lymphoma is most consistently bilateral, even in cats that appear to have unilateral disease. 76 in general, the kidneys are uniformly enlarged; however, they may also feel lumpy and irregular on palpation. more than 50% cats have signs consistent with renal insufficiency. 76 cats with cns lymphoma most often have signs associated with thoracolumbar involvement. 22, 23 the most common sites are between the second thoracic and fourth lumbar vertebrae. signs include gradual or sudden onset of weakness, upper motor neuron paralysis to the bladder, tail flaccidity, hyperpathia in the region of the lesion, and progressing ataxia. the neurologic dysfunction may be insidious or may progress rapidly. 23 cats with cervical spinal cord or nerve root involvement generally show peracute tetraparesis and diminished sensation in the thoracic limbs. those with cervical root involvement may show root lesions (root signature), such as lameness and hyperesthesia upon shoulder extension. common presenting signs for intracranial lymphoma, in decreasing order of frequency, include anorexia, ataxia, lethargy, altered consciousness, and aggression. 84 cats with nasal lymphoma usually have signs localized to the nasal passage. in a large compilation of cases, the presenting complaints, in decreasing order of frequency, were unilateral nasal discharge (bilateral is less common), facial deformity, dyspnea, and epistaxis. 79-81 stertor, anorexia, epiphora, exophthalmia, sneezing, and regional lymphadenopathy also can occur. cutaneous lymphoma may be solitary or diffuse and manifest with alopecia, erythema, and crusted papules. minimal peripheral lymphadenopathy may also be present. in most cats the signs have a prolonged duration (i.e., several months). [88] [89] [90] [91] [92] 94 nonspecific signs all cats with lymphoma, regardless of the site, may have secondary bone marrow infiltration that leads to anemia and a leukemic blood profile. anemia is a common condition in cats with lymphoma, and at least 50% have moderate to severe nonregenerative anemia. signs related to paraneoplastic hypercalcemia (pu/pd) can occur in cats, but the syndrome is much less common than in the dog. in one survey of hypercalcemia in cats, approximately 10% were diagnosed with lymphoma of various anatomic types. 115 one case of hypereosinophilic paraneoplastic syndrome and one case of symmetric cutaneous necrosis have been reported in cats with lymphoma. 116,117 a number of disease conditions can be confused with lymphoma in cats (table 31-7) . for most cats suspected of having lymphoma or leukemia, the diagnostic evaluation should include a cbc with differential cell count, platelet count, serum chemistry profile, and a test for felv and fiv. bone marrow aspiration or biopsy may be indicated to evaluate for possible involvement and complete staging of the extent of disease. bone marrow evaluation is particularly indicated if anemia, cellular atypia, and leukopenia are present. histopathologic evaluation of lymph nodes or involved organ tissue (procured by means of surgical incision, endoscopy, or needle core biopsy) is essential for a definitive diagnosis. in the cat, lymph node fine-needle aspiration alone is not sufficient in most cases because of the difficulty involved in distinguishing lymphoma from benign hyperplastic lymph node syndromes unique to the species; these include idiopathic peripheral lymphadenopathy, plexiform vascularization of lymph nodes, and peripheral lymph node hyperplasia of young cats. [71] [72] [73] [74] [75] whole lymph node excision is preferred in these cases, because determination of the orientation, invasiveness, and architectural abnormalities may be necessary for diagnosis. additional site-specific cytologic or histologic assessments may be warranted when extranodal sites are suspected. histologic and cytologic samples also can be analyzed by various histochemical and immunohistochemical techniques to determine the immunophenotype (b cell or t cell), tumor proliferation rate (e.g., ki-67, pcna, agnors), and telomerase activity (see chapter 14, section d), and histologic subtype (low, intermediate, or high grade). 10, 18, 26, 34, 43, 44, 46 the availability of such analysis is increasing, but currently none has proved to be predictive of outcome in cats. serum chemistry profiles can help establish the overall health and clinical staging of cats, but the results are not specific for a diagnosis. elevated liver enzymes may indicate hepatic infiltration with lymphoma, and elevated blood urea nitrogen (bun) and creatinine levels may indicate renal lymphoma. for cats with alimentary lymphoma, hypoproteinemia and anemia are reported to occur in up to 23% and 76% of cases, respectively. 25, 118 hypercalcemia is rare in cats but has been reported in patients with lymphoma at various anatomic sites. 25,66-68 elevated globulin levels may indicate the presence of a monoclonal gammopathy with or without serum hyperviscosity (this is a rarely reported paraneoplastic syndrome in cats with lymphoma). 119,120 hypoglycemia was reported in approximately one third of cats with lymphoma in an australian study. 118 serum alpha 1-acid glycoprotein concentrations have been reported to be elevated in cats with lymphoma, but no clinical relevance has been associated with this finding. 121 most cats with alimentary lymphoma develop a palpable abdominal mass or thickened intestinal loops. approximately one third of patients have a mass that can be visualized on abdominal radiographs, and about 90% have ultrasound abnormalities. 24, 25 the ultrasound abnormalities may include mesenteric lymphadenopathy in 33% to 50% of cases, an intestinal mass or thickening in approximately 40% of cases, and possibly splenomegaly (approximately 33% of cats with ultrasonographic changes in the spleen have lymphoma 122 ), hepatomegaly, or effusion. 24, 25 in one compilation of 28 cases, the lesions appeared ultrasonographically to be localized in 70% of cases and diffuse in 30%; however, at surgery only 33% were resectable. 25 for alimentary lymphoma, especially if primary gi lymphoma is suspected, caution must be exercised when endoscopically obtained tissue is used because of the difficulty in differentiating lymphoplasmacytic gastroenteritis from primary, diffuse, intestinal lymphoma. 123 however, diagnosis of this disease by means of endoscopically derived biopsies is improving with more advanced techniques and instrumentation. a wedge biopsy through serosa and muscularis, avoiding the mucosa, may be necessary to establish a diagnosis in cases in which endoscopic samples are equivocal. as an alternative, because nearly half of alimentary lymphomas have secondary mesenteric lymph node involvement, ultrasound-guided biopsy specimens or fine-needle aspirates may be adequate for a diagnosis. for cats with mediastinal lymphoma, diagnostic suspicion may begin with a noncompressible cranial thorax on physical examination and conformation of a mediastinal mass or pleural effusion on a thoracic radiograph (figure 31-14) . fine-needle aspiration of any suspected mass or cytologic evaluation of pleural fluid may be sufficient to establish a diagnosis. in most cats the finding of a monotonous population of lymphoblasts establishes a diagnosis. 65, 124, 125 however, definitive diagnosis of lymphoma in cats with a mediastinal mass and concurrent chylothorax can be challenging. 114 the ct appearance of these lesions has been 127 in chylous effusions, the pleural fluid triglyceride concentration is greater than in the serum; however, anorectic cats have lower triglyceride levels in the pleural fluid. a major differential diagnosis for mediastinal lymphoma is thymoma. the cytologic features of thymoma were recently described, and although these were found to be distinct from lymphoma in many cases, the diagnosis was challenging because of a preponderance of small lymphocytes in thymoma. 128 mast cells can also be seen in up to 50% of aspirate samples from thymomas. diagnostics for most extranodal forms of lymphoma are site specific. in cats suspected of having spinal lymphoma, survey radiographs of the spine rarely reveal osseous lesions. myelograms, ct, or mri is indicated, and in approximately 75% of cases, an extradural or intradural mass is detected. 22, 23, 83, 84 most lesions occur at a thoracolumbar or lumbosacral location. 83 fluoroscopic-guided, fine-needle aspiration of epidural lesions may yield enough tissue for a cytologic diagnosis. in most of the cats evaluated in one study, csf analysis revealed a clear, colorless fluid with a mixed pleocytosis (mean, 140 cells/µl; range, 0 to 1625 cells/ul) and an elevated protein content (mean, 140.7 mg/dl; range, 12 to 405 mg/dl). 23 malignant lymphocytes were identified in six of 17 cats evaluated with csf analysis. in a report of intracranial neoplasia, one of two cats with lymphoma had lymphoblasts in the csf fluid. 84 bone marrow and renal involvement are common in cats suspected of having cns lymphoma, and cytologic assessment of these organs generally is easier than in spinal sites. if nasal lymphoma is suspected, a biopsy specimen can be obtained either by intranasal procurement or by flushing one hemicavity with a bulb syringe and saline while occluding the contralateral cavity and collecting samples flushed out the nasopharynx (figure 31-15 ). thorough staging to make sure that the disease is confined to the nasal passages is recommended, because this presentation can be treated locally with radiotherapy if systemic involvement is ruled out. a ct scan is indicated to determine local involvement and to plan radiotherapy if it is to be pursued. for cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative sites, although overtly infected skin lesions should be avoided. complete staging to rule out systemic disease is also recommended for cats with cutaneous lymphoma, because local therapies can be applied in cases of solitary disease. in the case of renal lymphoma, physical examination findings of massive and often bilateral renomegaly raise the index of suspicion. the radiographic appearance is a smooth to irregular renomegaly ( figure 31-16, a) that is most consistent with either renal lymphoma or polycystic kidney disease. the disease usually is diffuse throughout the renal cortex ( figure 31-16, b) , and transabdominal needle aspiration or core biopsy is diagnostic in most cases. a who staging system exists for the cat that is similar to the one used in the dog (see box 31-1). however, because of the high incidence of visceral involvement in the feline species, another staging system is used more often (box 31-5). 21 because lymphoma in cats is more varied with respect to location and anatomic types, staging systems generally are less helpful for predicting response. occasionally the diagnosis of lymphoma and the differentiation of malignant and benign proliferation of lymphocytes is not possible based on standard histologic and cytologic criteria. in these cases, advanced molecular analyses may be helpful for confirming a diagnosis. clonality is the hallmark of malignancy; that is, the malignant cell population theoretically is derived from expansion of a single malignant clone. this was discussed earlier in the chapter for lymphoma in the dog (see section a and figure 31-8) , and the same assays can be applied for advanced diagnosis of lymphoma in cats. 85 investigators have used pcr techniques to amplify the variable regions of the t-cell and immunoglobulin receptor genes to detect clonal lymphocyte populations in cats, and these techniques appear to be a useful adjunctive diagnostic tool, although they are undergoing a more thorough assessment. molecular techniques may also prove useful for assessing early recurrence, for more accurate clinical staging, and for providing so-called molecular remission rates, because they are more sensitive than the standard cytologic assessment of peripheral blood, bone marrow, or lymph nodes. our knowledge base for treating cats with lymphoma is less well established and less predictable than that for dogs, primarily because of the greater variation in histologic type and anatomic locations observed in felines. the chemotherapeutic agents most often used to treat feline lymphoma are similar to those used for dogs and humans with lymphoma (see section a); they include doxorubicin, vincristine, cyclophosphamide, methotrexate, l-asparaginase, ccnu, and prednisone.* in general, cats tolerate chemotherapy quite well. selected published protocols for the treatment of feline lymphoma are detailed in table 31 -8. most current combination protocols in north america are modifications of chop protocols initially designed for human oncologic use. in europe, cop (i.e., without the addition of doxorubicin) is used more often in cats with lymphoma, and one compilation reported results similar to those for chop. 133 some studies with relatively few case entries have reported limited activity for doxorubicin as a single agent in cats with lymphoma 19, 132 ; however, larger studies using combination protocols have more consistently reported that the addition of doxorubicin is necessary to attain more durable responses. 10, 129 in european and australian studies that reported less favorable doxorubicin responses, the patient populations consisted of a higher proportion of the mediastinal form in siamese cats, a population less frequently included in north american reports. 19, 133 the protocol the authors use in their practice is presented in table 31 -9. this protocol has been used in many cats with various forms of lymphoma and is well tolerated. currently, our canine lymphoma protocols (see section a) discontinue chemotherapy by week 25, and we have sufficient data exist to show that in dogs, this short, maintenance-free protocol is as good as if not superior to longer maintenance protocols. because long-term maintenance protocols have not proved superior to maintenance-free protocols in dogs and humans with lymphoma, the same likely holds true in the cat, although no data exist to document this. until such time as evidence to the contrary exists, we recommend discontinuation of chemotherapy at week 25 in cats that have attained complete remission. although doxorubicin appears to be the most effective agent for treating cats with lymphoma in north america, the species generally is less tolerant of doxorubicin than are dogs, and significant toxicity results when it is used at the dog dosage of 30 mg/m 2 given intravenously every 3 weeks. however, at lower dosages (e.g., 25 mg/m 2 or 1 mg/kg, given intravenously) doxorubicin can be used without significant toxicity. 10, 129 the major toxicities noted with doxorubicin are anorexia, myelosuppression, renal toxicity and, if perivascular leakage occurs, severe tissue damage. clinical doxorubicin-induced cardiac toxicity has not been documented in cats, but no information indicates that cats are resistant to myocardial damage. renal toxicity has been produced experimentally in rats and box 31-5 • single tumor (extranodal) with regional lymph node involvement • two or more nodal areas on the same side of the diaphragm • two single (extranodal) tumors with or without regional lymph node involvement on the same side of the diaphragm • resectable primary gastrointestinal tract tumor, usually in the ileocecal area, with or without involvement of associated mesenteric nodes only rabbits and has been reported in cats. [136] [137] [138] in our experience, the incidence is significant enough that renal function in cats should be monitored closely (i.e., serial creatinine and urine specific gravity analysis) before each therapy session. a stealth liposomal form of doxorubicin has been shown to increase the likelihood of renal toxicity and therefore is limited in its use in this species. 138 a small number of cats with lymphoma have been treated with single agent ccnu at dosages ranging from 30 to 60 mg/m 2 given orally every 3 to 6 weeks. 130,131 although activity was noted, only partial responses were reported. little is known about the treatment of solitary or regional nodes of the head and neck that closely resemble hodgkin's lymphoma histologically. 1, 54 one study described the clinical outcome in four cats with this condition; these researchers found that the course of disease was prolonged, but after surgical excision of the single affected node, only one cat showed recurrence 6 months after surgery. 54 a second surgery was performed in this cat, and a second recurrence was noted, again 6 months after excision. cats with granular cell lymphoma or globule leukocyte tumors tend to respond poorly to chemotherapy, although durable responses have been reported. 102 currently, too few cases have been treated with aggressive chemotherapy to allow an assessment of response to therapy for this disease. a distinct form of intestinal lymphoma in cats that is composed of small, mature lymphocytes has been referred to as lymphocytic lymphoma. this syndrome has been reported to respond well to oral prednisone (10 mg/cat/day) and chlorambucil (15 mg/m 2 given once daily for 4 consecutive days out of every 3 weeks). a complete response rate of 69% was reported, with median remission and survival durations of 16 and 17 months, respectively. 62 radiation therapy has been used effectively to treat localized lymphoma, such as epidural, mediastinal, and nasal lymphoma. total doses of 10 to 15 gy usually result in a complete remission. 77 in one study, 10 cats with localized lymphoma were treated with radiation, alone or with chemotherapy, at doses of 8 to 40 gy. 78 eight of the 10 cats achieved complete remission, with a median remission duration of just over 2 years. radiation therapy also has been used effectively to treat nasal lymphoma. 78 complete response is the norm (80% to 100%), and median remission durations exceeding 1.5 years can be expected in felv-negative cats with disease confined to the nasal and paranasal cavities. for nasal lymphoma, radiotherapy appears to be superior to chemotherapy, for which reported median remission durations are 151 to 380 days. 10 very little has been published about the treatment of cutaneous lymphoma or mycosis fungoides in cats; however, a report of a complete response to ccnu exists. 135 cats with a solitary mass should be treated with surgical excision, although clinical staging is necessary to rule out possible internal involvement. combination chemotherapy can be considered if multiple sites are involved. if the disease is localized to a small region, radiation therapy usually is also effective. mycosis fungoides may be treated effectively in dogs with retinoids, such as isotretinoin (accutane) at a dosage of 3 to 4 mg/kg given orally daily or etretinate (tegison) at a dosage of 1.25 mg/kg given orally daily; however, no providing precise treatment recommendations for the wide variety of clinical settings of cats with lymphoma is difficult. our current recommendation is to treat cats with lymphoma using the chop-based protocols (see table 31 -9). doxorubicin alone (25 mg/m 2 given iv every 3 weeks for five total treatments) or palliative prednisone therapy is offered if the client declines more aggressive therapy. nutritional support is especially important, particularly for cats with alimentary lymphoma. a feeding tube may need to be placed in cats undergoing chemotherapy for alimentary lymphoma, particularly if anorexia is a factor (see chapter 16, section b.) ultimately, most cats with lymphoma that are successfully treated with chemotherapy have a relapse of the disease. this often represents a recrudescence of the tumor in a more drug-resistant form. at the first recurrence, reinduction first should be attempted by using the induction protocol that was successful initially. in general, the likelihood of a response and the length of the response are half those for the initial therapy; however, a subset of animals enjoy long-term reinduction. if reinduction fails or if the cat does not respond to the initial induction, so-called rescue agents or rescue protocols can be attempted. these are drugs or drug combinations that typically are not found in the standard chop protocol and are withheld for use in drugresistant cases. a number of rescue protocols have been reported in the veterinary literature and were reviewed previously. 139,140 the most common rescue protocols advocated for cats with resistant relapse include single agent use or combination use of mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), ccnu, and mopp. in general, overall rescue response rates of 40% to 50% are reported, but these responses usually are not durable; median responses of 1.5 to 2 months are the norm. a small subset of animals enjoy longer rescue durations. in general, cats do not enjoy response rates as high or remission and survival durations as long as dogs with lymphoma. complete response rates range from 50% to 70% after combination chemotherapy, and overall median remission and survival durations are approximately 4 and 6 months, respectively. 1,5,6,10,129 however, a significant proportion of cats (25% to 30%) that achieve a complete response with combination chemotherapy enjoy more durable overall remission and survival times (i.e., 1 year or longer). the response rate and the length of response vary according to the presence or absence of several prognostic factors. the wide variation in frequency and the great heterogeneity of anatomic forms of lymphoma in cats make specific prognostications more difficult than in dogs with lymphoma. most studies have lumped anatomic groups together to produce remission and survival data, and individual group numbers generally have been too small to apply statistical analysis with meaningful power. that being said, the factors that appear to be most strongly associated with a more positive prognosis in cats are a complete response to therapy (figure 31-17) , which unfortunately cannot be determined before treatment; negative felv status ( figure 31-18) ; early clinical stage (figure 31-19) ; anatomic location; and addition of doxorubicin to the treatment protocol. 5,10,24,129 unlike in the dog, cd3 immunoreactivity has not been established as a negative prognostic factor in the cat. 10 early reports may contradict more recent studies partly because of the decline in felv-associated lymphoma, and patients reported in the early literature may not equate to more recent populations studied. in general, felv-negative cats that achieve a complete response on chop-based protocols have a high likelihood of long-term survival, proportion alive with approximately 30% alive at 1.5 years after diagnosis. for cats with alimentary lymphoma, median survival times of 7 to 10 months are expected with chemotherapy that includes doxorubicin. 10, 26, 129 in one study of 28 cats with alimentary lymphoma in which the treatment protocol did not include doxorubicin, the median survival time was only 50 days. 25 mediastinal lymphoma in felv-positive young cats is associated with the poorest prognosis, and survivals times of approximately 2 to 3 months are expected with chemotherapy. 6, 10, 19 in contrast, older, felv-negative siamese cats with mediastinal lymphoma appear to experience remission rates approaching 90%, and responses tend to be quite durable. overall, cats with nasal lymphoma have the best prognosis, because local radiotherapy (or chemotherapy if radiotherapy is not available) generally results in excellent control, with median survival times approaching 1.5 years. 10,78 however, cats with nasal lymphoma and concurrent felv infection have much shorter survival durations. renal lymphoma is associated with a shorter survival time, with published medians ranging from 3 to 6 months. 1,6,10,76 in a study by mooney and colleagues, 76 28 cats with primary renal lymphoma were treated with combination chemotherapy. a complete response (cr) was noted in 17 cats (61%), and nine (32%) had a partial response. the median survival times were 4 months for the cats that showed a complete response and 1 month for those that showed a partial response. the duration of response to chemotherapy did not correlate with the degree of renal insufficiency except in cats with a bun higher than 150 mg/dl. extension to the cns occurred in 40% of these treated cats. the investigators revised their chemotherapy protocol to include cytosine arabinoside, which can penetrate the blood barrier, theoretically to prevent or reduce cns metastasis; however, definitive improvement with this modification has not been documented. few studies have reported on the results of treatment of cats with spinal lymphoma. in one study of four cats treated with chemotherapy (l-asparaginase, vincristine, and prednisone) combined with spinal radiation (n = 3) or surgical cytoreduction (n = 1), most of the cats were euthanized by 5 months, although one cat survived 13 months. 23 in another study of nine cats treated with chemotherapy (vincristine, cyclophosphamide, and prednisone), three cats achieved a complete response with a duration of 14 weeks and three achieved a partial response with a duration of 6 weeks. 22 one cat treated with dorsal decompression laminectomy and chemotherapy survived 13 months. although the numbers are small overall, the prognosis for spinal lymphoma is poor. section c of this chapter presents a complete discussion of leukemias and mpds, including a general discussion of hematopoiesis, etiologies, lineage classification, and descriptions. the classification of leukemias in cats is difficult because of the similarity of clinical and pathologic features and the transition, overlap, or mixture of cell types involved. 110, [141] [142] [143] [144] [145] [146] [147] [148] leukemia is a neoplastic proliferation of hematopoietic cells that originate in the bone marrow. cell lineage includes myeloid cells, neutrophils, basophils, eosinophils, monocytes, lymphoid cells, megakaryocytes, and erythrocytes. box 31-6 presents a classification scheme for the leukemias identified in the cat. leukemias also must be classified according to their degree of differentiation. 110 well-differentiated leukemia is referred to as chronic leukemia, and poorly differentiated leukemia usually is called acute leukemia; this distinction is very important in the therapeutic management and prognosis of leukemias. cats with acute leukemia usually show signs of severe anemia (pale mucous membranes), splenomegaly, and febrile episodes. cats with chronic leukemia may have a longer duration of signs and mild anemia with or without splenomegaly. in cats suspected of having leukemia, bone marrow aspiration or biopsy usually is diagnostic. the preferred sites for bone marrow aspiration are the proximal humerus and the iliac crest. cats with acute leukemia are likely to have malignant cellular infiltrates in organs other than the bone marrow. 144 the bone marrow aspirate must contain more than 30% abnormal blast cells to support a diagnosis of an acute leukemia. in cats suspected of having cll, infiltration of the bone marrow with more than 20% mature lymphocytes helps confirm the diagnosis. all cats with leukemia should be tested for felv and fiv. determining the lineage of some leukemias can be challenging; most can be distinguished from one another by histologic appearance, histochemical stains, immunohistochemical analysis of cell surface antigens, and flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 141, 143, 149, 150 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. the french-american-british (fab) classification system (box 31-7) is considered useful in cats with myelodysplastic syndromes, and almost all these patients are felv antigenemic. 151,152 lymphoid leukemia is the most common leukemia in cats. approximately 25% of cats with lymphoma also have a leukemic blood profile. 153 all is the most common lymphoid leukemia. 154 all is characterized by the presence of poorly differentiated lymphoblasts and prolymphocytes in the blood and bone marrow. 155 most cats with all have normal to low wbc counts. a few cats have leucocytosis with circulating blasts. a moderate to marked anemia is common. bone marrow aspiration usually reveals extensive infiltration with lymphoblasts. approximately 60% to 80% of cats with all are felv positive, and most malignant cells have a t-cell phenotype. 156 cll, which is rarely reported in cats, is characterized by the presence of well-differentiated, small, mature lymphocytes in the peripheral blood and bone marrow. 155,156 although most of these cells are of the t-cell lineage (particularly t helper), b-cell cll has been reported. 150, 157, 158 most cats with cll have an elevated wbc count (i.e., over 50,000/µl), and most are felv negative. the total leukocyte count varies and may range from leukopenia to marked leukocytosis. chronic granulocytic leukemia (gl) must be distinguished from leukemoid reactions associated with infections. acute gl is characterized by a large percentage of myeloblasts and/or progranulocytes in the blood and bone marrow. myeloblasts can be difficult to distinguish from lymphoblasts, but the former have a finer chromatin pattern, a smaller nucleus-to-cytoplasm ratio, more prominent or multiple nucleoli, and sometimes cytoplasmic granules. it is not uncommon in cats with gl to have no recognizable neoplastic cells in the peripheral blood. the bone marrow is hypercellular as a result of granulocytic leukemia cells. 141,145,146,159 myelomonocytic leukemia (mml) results from malignant transformation of both neutrophils and monocytes. this form of leukemia is one of the most common forms reported. monocytic leukemia (ml) is a rarely reported leukemia. it generally is considered an acute leukemia regardless of the morphologic appearance of the cells. eosinophilic leukemia (el) is rarely diagnosed in cats and is considered a variant of chronic granulocytic or myeloid leukemia. 148,160 felv has induced el experimentally. 160 mature eosinophils outnumber immature stages, and anemia is uncommon in cats associated with el. cats usually have an eosinophil count of 15,000 cells/µl or higher with or without immature cells in the peripheral blood. the bone marrow shows hyperplasia of eosinophilic precursors, and the myeloid to erythroid ratio (m:e) is significantly increased. organ infiltration, such as in lymph nodes, the spleen, and the liver, can be seen. 161 in establishing a diagnosis of el, it is important to rule out eosinophilic enteritis, parasitism, eosinophilic granuloma complex, and allergic disorders. diagnosing el can be very difficult because of the hypereosinophilic syndrome (hes) seen with other disease conditions in cats. [161] [162] [163] hes is characterized by a marked increase in the eosinophil count, bone marrow hyperplasia of eosinophilic precursors, and multiple organ infiltration by mature eosinophils. most cats have signs related to gi involvement. basophilic leukemia (bl) is considered a variant of chronic granulocytic leukemia. only one confirmed case of basophilic leukemia has been reported in the cat. it is important to differentiate bl from systemic mastocytosis with mast cell leukemia. mast cells have numerous cytoplasmic granules and round nuclei. basophils have segmented nuclei and cytoplasmic granules that superimpose on the nucleus, giving it a moth-eaten appearance. erythremic myelosis [165] [166] [167] [168] erythremic myelosis (em) is an mpd characterized by excessive proliferation of erythroid elements, which results in an increase in nucleated erythrocytes (rubriblasts to metarubricytes). severe anemia is common, and the peripheral blood shows numerous nucleated erythrocytes, moderate to marked anisocytosis, and an increased erythrocyte mean cell volume. the bone marrow contains a preponderance of normal-appearing erythrocyte precursors. some cats undergo blast transformation to myeloblastic, granulocytic, or a poorly differentiated leukemia (previously called reticuloendotheliosis). the transition from erythremic myelosis to erythroleukemia to acute granulocytic leukemia is well recognized in humans. 169 110, 143, 146 erythroleukemia, or acute erythremic myelosis, can develop from blast transformation of erythremic myelosis. primitive erythroid precursors in the blood and bone marrow predominant. primitive cells resembling myeloblasts often are present in low numbers. [170] [171] [172] [173] primary erythrocytosis is rarely reported in cats, and the diagnosis is based on an elevated pcv (65% to 80%) with low to normal serum erythropoietin activity. most clinical signs are associated with an increased red blood cell (rbc) volume, which increases blood volume and viscosity, causing impaired blood flow, stasis, and tissue hypoxia. neurologic signs such as seizures, blindness, and mental depression are common. 172 the oral mucous membranes may appear brick red. it is important to differentiate this condition from secondary polycythemia caused by renal tumors, chronic hypoxia, and right-to-left cardiac shunts. [174] [175] [176] megakaryocytic leukemia is characterized by abnormal megakaryocytic hyperplasia in the bone marrow. the megakaryocytes are morphologically abnormal, and some are small (dwarf megakaryocytes) and have few or no nuclear lobulations. thrombocytopenia or thrombocytosis may be present. in humans this form of leukemia often is associated with extensive marrow fibrosis and an increase in reticulum or collagen. 169 primary thrombocythemia is a very rare, chronic mpd characterized by proliferation of megakaryocytes and elevated platelet counts exceeding 1 million. giant platelets and platelets with abnormal morphology may be seen in the peripheral blood. one case has been reported in the cat. [178] [179] [180] [181] [182] malignant histiocytosis, a rare condition in cats, is characterized by systemic proliferation of malignant macrophages (histiocytes) and their precursors. a distinguishing characteristic of this disease is erythrophagocytosis. hepatosplenomegaly with progressive anemia (sometimes coombs positive) and thrombocytopenia are characteristic. the erythrophagocytosis may be confused with a possible immune-mediated anemia. bone marrow biopsy, rather than aspiration, and splenic biopsy may be necessary to establish a diagnosis. special stains using acid phosphatase and nonspecific esterase with fluoride inhibition (naphthol butyrate substrate) may be necessary to indicate macrophage origin. metaplasia [183] [184] [185] [186] myelofibrosis and myeloid hyperplasia are characterized by abnormal growth and differentiation of erythroid, myeloid, and megakaryocytic cell types with varying proliferation of fibroblasts in the marrow. anemia, leukopenia, or thrombocytopenia or varying combinations are common. myelofibrosis has been diagnosed in felv-positive cats and is directly associated with the virus rather than a consequence of myeloproliferative disorders. myeloid metaplasia may terminate in acute leukemia and therefore may be considered a preleukemic event. the use of chemotherapy to treat all has been disappointing. a 27% complete response rate has been reported with a cop regimen. 1, 10, 138, 187 in 15 cats treated with cop for all, four achieved a cr and 6 cats had a partial response. 1 the median remission was 7 months (range, 1 to 24 months). one report described a short-term remission in a cat with lymphoid leukemia that was treated with a low dose of cytosine arabinoside (10 mg/m 2 given subcutaneously twice daily). 188 cll can be treated with chlorambucil (0.2 mg/kg given orally or 2 mg/cat given every other day) and prednisone at a dose of 1 mg/kg given orally daily. cats with cll have a better prognosis and survive 1 to 2 years when treated with chlorambucil. as in humans and dogs, treatment can be withheld if the patient has no significant clinical signs and no profound cytopenias. in one study, a cat with cll remained stable without chemotherapy for over a year. 158 the prognoses for acute nonlymphoblastic leukemias generally are very poor, although some exceptions exist. a treatment regimen consisting of a combination of cytosine arabinoside and cyclophosphamide and multiple blood transfusions was effective at inducing a response for 3 months in a cat with acute megakaryocytic leukemia. 176 hydroxyurea (hydrea) can be used to treat chronic myeloid leukemia and primary erythrocytosis. cats with primary erythrocytosis that go untreated are reported to survive 6 to 20+ weeks. 170, 172 phlebotomy alone every 2 to 3 months was used to treat one cat, which survived longer than 20 months. 171 hydroxyurea treatment for primary erythrocytosis was used in eight cats, and all survived longer than 1 year. 172 hydroxyurea is available in 500 mg capsules, and the dosage is 25 to 50 mg/kg given orally daily. some cats have been given 500 mg every 5 to 7 days, but methemoglobulinemia and hemolytic anemia with heinz bodies has been seen. 172 a better recommendation is to have the drug formulated into 125 mg capsules, which is a more appropriate dosage. however, care must be used in making these capsules because hydroxyurea is potentially carcinogenic. a recommended treatment schedule for hydroxyurea is 125 mg daily to every other day, depending on the type of leukemia under treatment. the drug is tolerated very well at this dosage. a case of acute ml in a cat undergoing treatment with cytarabine, doxorubicin, vincristine, and prednisolone has been reported; a partial remission was noted for approximately 2 months, and the cat survived for 3 months after diagnosis. 147 a retrospective study subcommittee to re-examine criteria for a classification system and to spearhead large multi-institutional studies to validate the criteria. mpds are uncommon or rare in the dog; they occur 10 times less frequently than lymphoproliferative disorders. 3 accurate information about the incidence and other epidemiologic information await consistent use of a uniform classification system (see later discussion). no known age, breed, or gender predisposition exists, although large breed dogs have been overrepresented in some retrospective studies. [4] [5] [6] [7] [8] [9] [10] [11] [12] in dogs, the etiology of spontaneously occurring leukemia is unknown. genetic and environmental factors (including exposure to radiation, drugs, or toxic chemicals) probably play a role. in humans, acquired chromosomal derangements lead to clonal overgrowth with arrested development. 13 chromosomal abnormalities have been reported in dogs with acute myeloid leukemia (aml), chronic myelogenous leukemia (cml), and lymphoid leukemia. 14, 15 however, because karyotyping is difficult to perform in dogs, owing to the large number and morphologic similarity of their chromosomes and their resistance to banding, definition of the genetic factors in canine mpds awaits application of molecular techniques and use of the canine genome map. 14, 16, 17 certain forms of leukemia in dogs have been produced experimentally by irradiation. [18] [19] [20] in contrast to mpds in cats, no causative viral agent has been demonstrated in dogs, although retrovirus-like budding particles were observed in the neoplastic cells of a dog with granulocytic leukemia. 21 a review of normal hematopoiesis can aid the clinician's understanding of the various manifestations of mpds. hematopoiesis is the process of proliferation, differentiation, and maturation of stem cells into terminally differentiated blood cells (a simplified schematic is presented in figure 31 -20) . pluripotent stem cells differentiate into either lymphopoietic or hematopoietic multipotent stem cells. 22 under the influence of specific regulatory and microenvironmental factors, multipotent stem cells in bone marrow differentiate into progenitor cells committed to a specific hematopoietic cell line, such as erythroid, granulocyticmonocytic, or megakaryocytic cells. maturation results in the production of terminally differentiated blood cells-erythrocytes, granulocytes, monocytes, and platelets-that are delivered to the circulation. in some cases, as in the maturation of reticulocytes to erythrocytes, final development may occur in the spleen. the proliferation and differentiation of hematopoietic cells are controlled by a group of regulatory growth factors. 22, 23 of these, erythropoietin is the best characterized; it regulates erythroid proliferation and differentiation and is produced in the kidneys, where changes in oxygen tension are detected. the myeloid compartment depends on a group of factors, collectively referred to as colony-stimulating factors (csfs). these factors act at the level of the committed progenitor cells but also influence the functional capabilities of mature cells. some of these factors have a broad spectrum of activity; others are more restricted in their target cells and actions. csfs are produced in vitro by a multitude of cell types, including monocytes, macrophages, lymphocytes, and endothelial cells, and these cells likely play a role in the production and regulation of these factors in vivo. the gene for thrombopoietin also has been cloned, and this hormone alone apparently can induce differentiation of megakaryocytes and platelet production. 24 recombinant forms of many of these hormones are becoming increasingly available. the clonal disorders of bone marrow include myeloaplasia (usually referred to as aplastic anemia), myelodysplasia, and myeloproliferation. a preleukemic syndrome, characterized by peripheral pancytopenia and bone marrow hyperplasia with maturation arrest, is more correctly called myelodysplasia because the syndrome does not always progress to overt leukemia. this syndrome has been described in cats, usually in association with felv infection, but it has only rarely been recognized in dogs. [25] [26] [27] [28] mpds may be manifested by abnormalities in any or all of the different cell lines, because hematopoietic cells share a common stem cell. in addition, transformation from one mpd to another may occur. 29 mpds are classified in several ways. the terms acute and chronic refer to the degree of cellular differentiation of the leukemic cells, but these terms also correlate with the biologic behavior of the neoplasm. 30 disorders resulting from uncontrolled proliferation of cells incapable of maturation lead to the accumulation of poorly differentiated, or blast, cells. these disorders have been called acute mpds or acute nonlymphocytic leukemias, but they now are included under the umbrella term acute myeloid leukemia. disorders resulting from unregulated proliferation of cells that exhibit progressive, albeit incomplete and defective, maturation lead to the accumulation of differentiated cells. these disorders are called chronic mpds. they include polycythemia vera, cml and its variants, essential thrombocythemia, and possibly primary myelofibrosis. mpds are further classified by the lineage of the dominant cell type or types, as defined by romanowsky stains, special cytochemical stains, ultrastructural features, and immunologic cell markers, and they recently have been classified into subtypes (see later discussion). acute leukemias have a more sudden onset and are more aggressive. in both acute and chronic disorders, however, abnormalities in proliferation, maturation, and functional characteristics can occur in any hematopoietic cell line. 1 in addition, normal hematopoiesis is adversely affected. animals with leukemia usually have decreased numbers of circulating normal cells. the pathogenesis of the cytopenias is complex and may result in part from production of inhibitory factors. eventually, neoplastic cells displace normal hematopoietic cells, a process called myelophthisis. anemia and thrombocytopenia are particularly common. neutropenia and thrombocytopenia result in infection and hemorrhage, which may be more deleterious to the animal than the primary disease process. despite the disseminated nature of the disease at the time of diagnosis, parenchymal organ dysfunction usually occurs only in very advanced cases of mpd. aml is rare and is characterized by aberrant proliferation of a clone of cells without maturation. this results in the accumulation of immature blast cells in the bone marrow and peripheral blood (figure 31-21) . the wbc count varies, ranging from leukopenia to counts greater than 150,000/ml. the spleen, liver, and lymph nodes commonly are involved, and other tissues may be infiltrated as well, including the tonsils, kidneys, heart, and cns. no characteristic age has been noted, and even very young dogs may be affected. 31 the clinical course of these disorders tends to be rapid. production of normal peripheral blood cells usually is diminished or absent, and anemia, neutropenia, and thrombocytopenia are common. infection and hemorrhage are common sequelae. occasionally, malignant blasts are present in the bone marrow but not the peripheral blood; this is called aleukemic leukemia. subleukemic leukemia suggests a normal or decreased wbc with some neoplastic cells in circulation. in 1985 the animal leukemia study group was formed under the auspices of the american society for veterinary clinical pathology to develop specific morphologic and cytochemical criteria for classifying acute nonlymphocytic leukemias in dogs and cats. recognition of specific subtypes of leukemia is necessary for the accumulation of accurate, useful information about prognosis and response to treatment and for comparison of studies from different sites. in 1991 this group proposed a classification system following adaptation of the french-american-british (fab) system and criteria established by the national cancer institute workshop. 2 the group members examined blood and bone marrow from 49 dogs and cats with mpds. romanowsky-stained specimens were examined first to identify blast cells and their percentages. lineage specificity then was determined using cytochemical markers. the percentage of blasts and the information about lineage specificity were used in combination to classify disorders as acute undifferentiated leukemia (aul), acute myeloid leukemia (aml, subtypes m1 to m5 and m7), and erythroleukemia with or without erythroid predominance (subtypes m6 and m6er) (see box 31-7). except for acute promyelocytic leukemia (subtype m3), all these subtypes have been described in dogs. however, because this modified fab system has only recently been adopted, the names given to these disorders in the literature vary considerably. in addition, in the absence of special cytochemical staining, immunophenotyping, and/or electron microscopy, the specific subtype of leukemia often has been uncertain, making retrospective analysis of epidemiologic information, prognosis, and response to therapy confusing at best. although defining specific subtypes may seem to be an academic exercise owing to the uniformly poor prognosis of acute leukemias, this information is critical to improving the management of these diseases. because of the low incidence of mpds, national and international cooperative efforts are required to accumulate information on the pathogenesis of specific subtypes and their response to different treatment modalities; use of a uniform classification system is an essential first step. different forms of aml are shown in figure 31 -21, a-e. the most frequently reported forms of aml in the dog are acute myeloblastic leukemia (m1 and m2) and acute myelomonocytic leukemia (m4). [3] [4] [5] [6] [7] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [32] [33] [34] [35] [36] [37] [38] [39] megakaryoblastic leukemia (m7) also is well recognized in dogs 8, [40] [41] [42] [43] [44] [45] [46] and may be associated with platelet dysfunction. 44 monocytic leukemias likely have included those with and without monocytic differentiation (m5a and m5b), 9, 47 but in some cases the diagnosis may have been chronic myelomonocytic or chronic monocytic leukemia (see later discussion). there are few reports in dogs of spontaneously occurring erythroleukemia (m6) in which the leukemic cells comprise myeloblasts, monoblasts, and erythroid elements. 48, 49 auls have uncertain lineages, because they are negative for all cytochemical markers. these leukemias should be distinguished from lymphoid leukemias by flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 50 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. chronic mpds are characterized by excessive production of differentiated bone marrow cells, which results in the accumulation of erythrocytes (polycythemia vera), granulocytes and/or monocytes (chronic myelogenous leukemia and its variants), or platelets (essential thrombocythemia). primary myelofibrosis as a clonal disorder of marrow stromal cells, characterized by proliferation of fibroblasts with accumulation of collagen in bone marrow, is not recognized in animals. myelofibrosis is considered a response to injury and may occur secondary to mpds. polycythemia vera (pv) is a clonal disorder of stem cells, although whether the defect is in the pluripotent stem cell or the hematopoietic multipotent stem cell is still unclear. in humans, progenitor cells have an increased sensitivity to insulin-like growth factor 1 (igf-1), which stimulates hematopoiesis. 51 whether this hypersensitivity is the primary defect or occurs secondary to another gene mutation is unknown. in any case, the result is overproduction of rbcs. the disease is rare and must be distinguished from more common causes of polycythemia, including relative and secondary absolute polycythemia (see later discussion). in pv, neoplastic proliferation of the erythroid series with terminal differentiation to rbcs occurs. the disease has been reported mostly in middle-aged dogs, and no breed or gender predilection has been noted. [52] [53] [54] [55] [56] [57] [58] [59] [60] pv is characterized by an increased rbc mass, evidenced by an elevated pcv, rbc count, and hemoglobin concentration. the pcv typically is in the range of 65% to 85%. the bone marrow is hyperplastic, although the m:e tends to be normal. in contrast to the disease in humans, other cell lines do not appear to be involved, and transformation to other mpds has not been reported. the disease in dogs may be more appropriately called primary erythrocytosis. cml is a neoplastic proliferation of the neutrophil series, although concurrent eosinophilic and basophilic differentiation can occur. cml can occur in dogs of any age. 31, [61] [62] [63] [64] [65] neutrophils and neutrophilic precursors accumulate in the bone marrow and peripheral blood and invade other organs. the peripheral wbc count usually, but not always, is higher than 100,000/ml. both immature and mature neutrophils are present (see figure 31 -21, f). mature forms usually are more numerous, but sometimes an "uneven" left shift is present. signs of dysplasia may be evident, including hypersegmentation, ringed nuclei, and giant forms. eosinophils and basophils may also be increased. the bone marrow is characterized by granulocytic hyperplasia, and morphologic abnormalities may not be present. erythroid and megakaryocytic lines may be affected, resulting in anemia, thrombocytopenia or, less commonly, thrombocytosis. cml must be distinguished from severe neutrophilic leukocytosis and "leukemoid reactions" caused by inflammation or immune-mediated diseases. leukemoid reactions also can occur as a paraneoplastic syndrome. in humans with cml, characteristic cytogenetic abnormalities are present in all bone marrow cells, signifying a lesion at the level of an early multipotent stem cell. typically these individuals have a chromosomal translocation, resulting in the philadelphia chromosome. 66 no consistent cytogenetic abnormalities have been demonstrated in spontaneously occurring cml in dogs. variants of cml are chronic myelomonocytic leukemia (cmml) and chronic monocytic leukemia (cmol). these diagnoses are made based on the percentage of monocytes in the leukemic cell population. besides accumulating in the bone marrow and peripheral blood, leukemic cells invade the red pulp of the spleen, the periportal and sinusoidal areas of the liver, and sometimes the lymph nodes. other organs (e.g., the kidneys, heart, and lungs) are less commonly affected. in addition, extramedullary hematopoiesis may be present in the liver and spleen. death usually results from complications of infection or hemorrhage secondary to neutrophil dysfunction and thrombocytopenia. in some cases, cml may terminate in "blast crisis," in which a transformation occurs from a predominance of well-differentiated granulocytes to excessive numbers of poorly differentiated blast cells in the peripheral blood and bone marrow; this phenomenon is well documented in the dog. 61, 62, 64 basophilic leukemia, although rare, has been reported in dogs and is characterized by an elevated wbc count with a high proportion of basophils in the peripheral blood and bone marrow. [67] [68] [69] hepatosplenomegaly, lymphadenopathy, and thrombocytosis may be present, and these dogs have all been anemic. basophilic leukemia should be distinguished from mast cell leukemia (mastocytosis). whether dogs develop eosinophilic leukemia remains in question. reported cases have had high blood eosinophil counts and eosinophilic infiltrates in organs. 70, 71 one dog responded well to treatment with corticosteroids. the distinction between neoplastic proliferation of eosinophils and idiopathic hypereosinophilic syndrome remains elusive. disorders associated with eosinophilia (e.g., parasitism, skin diseases, and diseases of the respiratory and gi tracts) should be considered first in an animal with eosinophilia. one distinguishing feature should be clonality, with reactive eosinophilia comprising polyclonal cells and the neoplastic condition arising from a single clone. as clonality assays become more available, this discrepancy may be resolved. in humans, essential thrombocythemia, or primary thrombocytosis, is characterized by platelet counts that are persistently higher than 600,000/ml. no blast cells are in circulation, and marked megakaryocytic hyperplasia of the bone marrow without myelofibrosis is present. thrombosis and bleeding are the most common sequelae, and most patients have splenomegaly. other mpds, especially pv, should be ruled out, and the patient should have no primary disorders associated with reactive thrombocytosis, 72 such as inflammation, hemolytic anemia, iron deficiency anemia, malignancies, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, or abscence of a spleen. in addition, certain drugs, such as vincristine, can induce thrombocytosis. essential thrombocythemia has been recognized in dogs. 29, [73] [74] [75] [76] in one dog, the platelet count exceeded 4 million/ml, and bizarre giant forms with abnormal granulation were present. the bone marrow contained increased numbers of megakaryocytes and megakaryoblasts, but circulating blast cells were not seen. other findings included splenomegaly, gi bleeding, and increased numbers of circulating basophils. causes of secondary or reactive thrombocytosis were ruled out. 73 basophilia also was reported in a more recent case. 75 in another dog, primary thrombocytosis was diagnosed and then progressed to cml. 29 in some cases reported in the literature as essential thrombocythemia, the dogs had microcytic hypochromic anemias. because iron deficiency anemia is associated with reactive or secondary thrombocytosis, care must be taken to rule out this disorder. however, spurious microcytosis may be reported if a dog has many giant platelets that are counted by an analyzer as small rbcs. 76 microscopic review of the blood film may be helpful in these cases. primary myelofibrosis with clonal proliferation of marrow fibroblasts has not been reported in dogs. 77 in humans, myelofibrosis is characterized by collagen deposition in the bone marrow and increased numbers of megakaryocytes, many of which have morphologic abnormalities. in fact, breakdown of intramedullary megakaryocytes and subsequent release of factors that promote fibroblast proliferation or inhibit collagen breakdown may be the underlying pathogenesis of the fibrosis. 78 focal osteosclerosis sometimes is present. anemia, thrombocytopenia, splenomegaly, and myeloid metaplasia (production of hematopoietic cells outside the bone marrow) are consistent features. the extramedullary hematopoiesis is ineffective at maintaining or restoring normal peripheral blood counts. in dogs, myelofibrosis occurs secondary to mpds, radiation damage, and congenital hemolytic anemias. [79] [80] [81] [82] in some cases the inciting cause is unknown (idiopathic myelofibrosis). concurrent marrow necrosis may occur in cases of ehrlichiosis, septicemia, or drug toxicity (estrogens, cephalosporins), and some speculate that fibroblasts proliferate in response to release of inflammatory mediators associated with the necrosis. 77 myeloid metaplasia has been reported to occur in the liver, spleen, and lungs. 82 extramedullary hematopoiesis is ineffective at preventing or correcting the pancytopenia that eventually develops. dysfunction of the hematopoietic system can be manifested by a variety of abnormalities that comprise myelodysplastic syndromes (mds). in dogs, in which the syndrome is rare, cytopenias usually are seen in two or three lines in the peripheral blood (anemia, neutropenia, and/or thrombocytopenia). other blood abnormalities can include macrocytic erythrocytes and metarubricytosis. the bone marrow typically is normocellular or hypercellular, and dysplastic changes are evident in several cell lines. if blast cells are present, they account for fewer than 30% of all nucleated cells, 2 although this threshold may be altered to fewer than 20%. 16 myelodysplasia sometimes is called preleukemia because in some cases it may progress to an acute leukemia. [25] [26] [27] in humans and in cats, mds are clonal disorders and are considered neoplastic. dogs with mpds have similar presentations regardless of the specific disease entity, although animals with acute mpds have a more acute onset of illness and a more rapid clinical course. a history of lethargy, inappetence, and weight loss is common. clinical signs include emaciation, persistent fever, pallor, petechiation, hepatosplenomegaly and, less commonly, lymphadenopathy and enlarged tonsils. shifting leg lameness, ocular lesions, and recurrent infections also are seen. vomiting, diarrhea, dyspnea, and neurologic signs are variable features. serum chemistries may be within reference intervals but can change if significant organ infiltration occurs. animals with msd may be lethargic and anorectic and may have pallor, fever, and hepatosplenomegaly. in pv, dogs often have erythema of mucous membranes because of the increased rbc mass. some dogs are polydipsic. in addition, neurologic signs may be present, such as disorientation, ataxia, or seizures, and these are thought to be the result of hyperviscosity or hypervolemia. 56 hepatosplenomegaly usually is absent. peripheral blood abnormalities are consistently found. in addition to the presence of neoplastic cells, other abnormalities may be present, including a decrease in the numbers of normal cells of any or all hematopoietic cell lines. occasional nucleated rbcs are present in the blood of about half of dogs with acute nonlymphocytic leukemia. 2 nonregenerative anemia and thrombocytopenia are present in most cases. the anemia usually is normocytic and normochromic, although macrocytic anemia sometimes is present. pathogenic mechanisms include the effects of inhibitory factors, leading to ineffective hematopoiesis, myelophthisis, immune-mediated anemia secondary to neoplasia, and hemorrhage secondary to thrombocytopenia, platelet dysfunction, or dic. anemia is most severe in acute mpds, although both anemia and thrombocytopenia may be milder in animals with the m5 subtype (acute monocytic leukemia). in myelofibrosis, the anemia is characterized by anisocytosis and poikilocytosis. in addition, pancytopenia and leukoerythroblastosis, in which immature erythroid and myeloid cells are in circulation may be present. these phenomena probably result from the replacement of marrow by fibrous tissue, with resultant shearing of red cells and escape of immature cells normally confined to bone marrow. in pv, the pcv is elevated, usually in the range of 65% to 85%. the bone marrow is hyperplastic, and the m:e usually is in the normal range. the neoplastic cells often are defective functionally. platelet dysfunction has been reported in a dog with acute megakaryoblastic leukemia (m7), 44 and in cml, the neutrophils have decreased phagocytic capacity and other abnormalities. one exception to this was a report of cml in a dog in which the neutrophils had enhanced phagocytic capacity and superoxide production. 83 the authors hypothesized that increased synthesis of granulocyte-macrophage colony-stimulating factor (gm-csf) resulted from a lactoferrin deficiency in the neoplastic neutrophils and mediated the enhanced function of these cells. in all cases of mpd, the diagnosis depends on the examination of peripheral blood and bone marrow. acute mpds are diagnosed on the basis of finding blast cells with clearly visible nucleoli in blood and bone marrow. most dogs with acute leukemia have circulating blasts. these cells may be present in low numbers in peripheral blood, and the smear should be searched carefully, especially at the feathered edge. even if blasts are not detected in circulation, indications of bone marrow disease, such as nonregenerative anemia or thrombocytopenia, usually are present. occasionally neoplastic cells can be found in csf in animals with invasion of the cns. smears of aspirates from tissues such as the lymph nodes, spleen, or liver may contain blasts but usually contribute little to the diagnostic workup. examination of blasts stained with standard romanowsky stains may give clues to the lineage of the cells (see figure 31 -21, a to c and e). in aml, in addition to myeloblasts, some progranulocytes with their characteristic azurophilic granules may be present. in myelomonocytic leukemia, the nuclei of the blasts usually are pleomorphic and have round to lobulated forms. in some cells the cytoplasm may contain large azurophilic granules or vacuoles. blasts in megakaryocytic leukemia may contain vacuoles and may have cytoplasmic blebs. in addition, bizarre macroplatelets may be present. although these distinguishing morphologic features may suggest a definitive diagnosis, cytochemical stains or immunophenotyping usually is required to define the lineage of the blasts. several investigators have reported modification of diagnoses after cytochemical staining. 84, 85 it is especially important to distinguish aml from lymphocytic leukemia to provide prognostic information to the owner. the animal leukemia group recommends the following diagnostic criteria (figure 31-22 ). 2 using well-prepared romanowsky-stained blood and bone marrow films, a minimum of 200 cells are counted to determine the leukocyte differential in blood and the percentage of blast cells in bone marrow or blood or both. in bone marrow, blast cells are calculated both as a percentage of all nucleated cells (anc) and nonerythroid cells (nec) and are further characterized using cytochemical markers. [84] [85] [86] neutrophil differentiation is identified by positive staining of blasts for peroxidase, sudan black b, and chloracetate esterase. nonspecific esterases (alpha-naphthyl acetate esterase or alphanaphthyl butyrate esterase), especially if inhibited by sodium fluoride, mark monocytes. canine monocytes may also contain a few peroxidase-positive granules. acetylcholinesterase is a marker for megakaryocytes in dogs and cats. in addition, positive immunostaining for von willebrand's factor (factor viii-related antigen) and platelet glycoproteins on the surface of blasts identifies them as megakaryocyte precursors. 8, [42] [43] [44] [45] [46] alkaline phosphatase (ap) only rarely marks normal cells in dogs and cats but is present in blast cells in acute myeloblastic and myelomonocytic leukemias. however, because of reports of ap activity in lymphoid leukemias in dogs, its specificity as a marker for myeloid cells is not certain. omega-exonuclease is a specific marker for basophils, which are also positive for chloracetate esterase activity. 69 blood and bone marrow differential counts and cytochemical staining should be performed and interpreted by experienced veterinary cytopathologists. if erythroid cells comprise less than 50% of anc and the blast cells account for more than 30%, a diagnosis of aml or aul is made. if erythroid cells are more than 50% of anc and the blast cells are more than 30%, a diagnosis of erythroleukemia (m6) is made. if rubriblasts comprise a significant proportion of the blast cells, a diagnosis of m6er, or erythroleukemia with erythroid predominance, can be made. (it should be noted that in the human aml classification system, the blast threshold has been lowered to 20%.) in some cases, electron microscopy is required to identify the lineage of the blast cells. for example, megakaryocyte precursors are positive for platelet peroxidase activity and contain demarcation membranes and alpha granules. 42, 46 both these features are detected at the ultrastructural level. immunophenotyping, used to identify cell lineages in human patients, awaits development of appropriate markers for animal species (see later discussion). hematopoietic cells from humans with leukemia often have abnormal chromosome patterns. cytogenetic abnormalities have been found in leukemic cells from a small number of dogs. 14,15 it is not clear whether chromosomal aberrations are primary (causative) or secondary (caused by the leukemia). if consistent karyotypic patterns can be identified and correlated with leukemic subtypes, cytogenetic analysis eventually may yield important diagnostic and prognostic information and become a valuable tool for evaluating remission and predicting relapse. although morphologic and cytochemical analyses have formed the mainstay of cell identification, newer technologies now are used routinely to classify leukemias by using monoclonal antibodies to detect antigens associated with certain cell types. cells can be immunophenotyped using flow cytometric analysis or immunocytochemistry. both lymphoid and nonlymphoid acute leukemias are positive for cd34. many lymphocyte markers, including cd3, cd4, cd8, cd18, bone marrow erythroid cells cd21, cd45, cd79, and igg, are available for dogs and can be used to rule out lymphoblastic leukemia in dogs with acute leukemias. 50, 87 other markers include myeloperoxidase (mpo) and cd11b for myeloid cells and cd41 for megakaryoblasts. some overlap in the expression of these cell antigens occurs. for example, canine (but not human) granulocytes express cd4. it is best to use a panel of antibodies (similar to using a battery of cytochemical stains), because antigens often are expressed on multiple lineages, and lineage infidelity can occur. these tests have become more valuable with the availability of canine reagents. currently, the myeloid neoplasm subcommittee of the acvp oncology committee recommends that the following immunophenotyping panel be done on bone marrow and/or blood smears to characterize animal leukemias: for b lymphocytes, cd79a; for t lymphocytes, cd3; for myeloid cells, mpo and cd11b; for megakaryoblasts, cd41; for dendritic cells, cd1c; and for acute leukemias, cd34. 16 owing to the degree of differentiation of cells in chronic mpds, these disorders must be distinguished from nonneoplastic causes of increases in these cell types. to allow a diagnosis of pv, tests first must establish that the polycythemia is absolute rather than relative. in relative polycythemias, plasma volume is decreased from hemoconcentration, dehydration, or hypovolemia, and the absolute red cell mass is not increased. splenic contraction also can result in relative polycythemia. absolute polycythemia, in which the rbc mass is increased, usually occurs secondary to tissue hypoxia, causing appropriate increased production of erythropoietin. in rare cases, erythropoietin may be produced inappropriately by a tumor (e.g., renal cell carcinoma) or in renal disease (pyelonephritis) or localized renal hypoxia. [88] [89] [90] these causes of polycythemia should be eliminated by appropriate laboratory work, thoracic radiographs, arterial blood gas analysis, and renal ultrasonography. in humans with pv, plasma erythropoietin (epo) levels are low. epo levels in dogs with pv tend to be low or low-normal, whereas in animals with secondary absolute polycythemia, the levels are high. 91, 92 samples for determination of epo concentrations should be taken before therapeutic phlebotomy to treat hyperviscosity and, owing to fluctuations in epo levels, should be repeated if results are incongruous with other information. cml in dogs has no pathognomonic features, and other common causes for marked leukocytosis with a left shift (leukemoid reaction) and granulocytic hyperplasia of bone marrow must be eliminated. these include infections, especially pyogenic ones; immunemediated diseases; and other malignant neoplasms. in cml, maturation sometimes appears disorderly, and variation in the size and shape of neutrophils may occur at the same level of maturation. in addition, neoplastic leukocytes may disintegrate more rapidly and appear vacuolated. 31 because of the invasive nature of cml, biopsy of the liver or spleen may also help distinguish true leukemia from a leukemoid reaction, assuming the animal can tolerate the procedure. if characteristic cytogenetic abnormalities can be found in dogs with cml, this analysis may be helpful. basophilic leukemia is diagnosed by finding excessive numbers of basophils in circulation and in bone marrow. basophilic leukemia must be differentiated from mastocytosis based on the morphology of the cell type present. basophils have a segmented nucleus and variably sized granules, whereas mast cells have a round to oval nucleus that may be partly or totally obscured by small, round, metachromatic-staining granules. this distinction usually is easy to make; however, in basophilic leukemia, changes in the morphology of the nucleus and granules make the distinction less clear. 68 essential thrombocythemia has been diagnosed based on finding persistent and excessive thrombocytosis (more than 600,000/ml) without circulating blast cells and in the absence of another mpd (e.g., polycythemia vera), myelofibrosis, or disorders known to cause secondary thrombocytosis. 72 these include iron deficiency anemia, chronic inflammatory diseases, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and absence of a spleen. thrombocytosis is transient in these disorders or abates with resolution of the primary disease. in essential thrombocythemia, platelet morphology may be abnormal, with bizarre giant forms and abnormal granulation. 73 in the bone marrow, megakaryocytic hyperplasia is a consistent feature, and dysplastic changes may be evident in megakaryocytes. 76 spurious hyperkalemia may be present in serum samples from dogs with thrombocytosis from any cause because of the release of potassium from platelets during clot formation. 93 measurement of the potassium in plasma is recommended in these cases and usually demonstrates a potassium concentration within reference interval. platelet aggregability has been variably reported as impaired 73 or enhanced. 76 in the one dog in which it was measured, the plasma thrombopoietin (tpo) concentration was normal. 75 whether tpo plays a role in essential thrombocythemia or is suppressed by the high platelet mass is unclear. elucidation of the pathogenesis of this disorder should be aided by the recent cloning of the genes for thrombopoietin and its receptor, the proto-oncogene mpl. 94 in mds, abnormalities in two or three cell lines usually are manifested in the peripheral blood as neutropenia with or without a left shift, nonregenerative anemia, or thrombocytopenia. other changes include macrocytosis and metarubricytosis. the bone marrow typically is normocellular or hypercellular with an increased m:e, and blasts cells, although increased, account for fewer than 30% of the nucleated cells. this blast threshold may be changed to fewer than 20%, and in a report of 13 dogs with primary or secondary mds, in all but one dog the blast cell percentage was less than 20%. 95 dysplastic changes can be detected in any cell line. dyserythropoiesis is characterized by asynchronous maturation of erythroid cells, typified by large hemoglobinized cells with immature nuclei (megaloblastic change). if the erythroid component is dominant, the mds is called mds-er (see box 31-7). 2, 28 in dysgranulopoiesis, giant neutrophil precursors and abnormalities in nuclear segmentation and cytoplasmic granulation can be seen. finally, dysthrombopoiesis is characterized by giant platelets and micromegakaryocytes. myelofibrosis should be suspected in animals with nonregenerative anemia or pancytopenia; abnormalities in erythrocyte morphology, especially shape; and leukoerythroblastosis. bone marrow aspiration usually is unsuccessful, resulting in a "dry tap"; this necessitates a bone marrow biopsy taken with a jamshidi needle. 96 the specimen is processed for routine histopathologic examination and, if necessary, special stains for fibrous tissue can be used. because myelofibrosis occurs secondary to other diseases of bone marrow (e.g., mpd), chronic hemolytic anemia, or bone marrow necrosis, the clinician should look for a primary disease process. because of the poor response of acute nonlymphocytic leukemias, treatment has been unrewarding to date. however, little information is available on the response of specific subtypes of leukemia to uniform chemotherapeutic protocols, partly because of the rarity of these disease processes and the paucity of cases in the literature. the clinician is advised to contact a veterinary oncologist for advice on new protocols and appropriate management of these cases. the therapeutic goal is to eradicate leukemic cells and re-establish normal hematopoiesis. currently, this is best accomplished by cytoreductive chemotherapy, and the agents most commonly used include a combination of cytosine arabinoside and an anthracycline (e.g., doxorubicin or cyclophosphamide), vincristine, and prednisone.* in humans, the introduction of cytosine arabinoside has been the single most important development in the therapy of acute nonlymphocytic leukemia. 99 in dogs, a regimen of 100 to 200 mg/m 2 of cytosine arabinoside, given by slow infusion (over 12 to 24 hours) daily for 3 days and repeated weekly, has been used. doxorubicin (30 mg/m 2 given intravenously every 2 to 3 weeks) can be administered at intervals alternating with cytosine arabinoside. if remission is achieved, as evidenced by normalization of the hemogram, the coap protocol (cyclophosphamide, vincristine, cytosine arabinoside, and prednisone), as described for canine lymphoma, could be used as maintenance therapy. 7, 97 another protocol that has been used to treat acute myeloblastic leukemia is presented in table 31-10. regardless of the chemotherapy protocol used, significant bone marrow suppression will develop, and intensive supportive care will be necessary. transfusions of whole blood or platelet-rich plasma may be required to treat anemia and thrombocytopenia, and infection should be managed with aggressive antibiotic therapy. because of the generally poor response, the major thrust of therapy may be to provide palliative supportive care. for pv, therapy is directed at reducing the red cell mass. the pcv should be reduced to 50% to 60% or by one sixth of its starting value. phlebotomies should be performed as needed, with administration of appropriate colloid and crystalloid solutions to replace lost electrolytes; 20 ml of whole blood per kilogram of body weight can be removed at regular intervals. 54 in humans, phlebotomy continues to be the therapeutic approach used most frequently. radiophosphorus ( 32 p) has been shown to provide long-term control but can be used only in specialized centers. 100 the chemotherapeutic drug of choice is hydroxyurea, an inhibitor of dna synthesis. this drug should be administered at an initial dosage of 30 mg/kg for 10 days; the dosage then is reduced to 15 mg/kg given orally daily. 56 the major goal of treatment is to maintain the pcv as close to normal as possible. cml is best managed with chemotherapy to control the proliferation of the abnormal cell line and improve the quality of life. hydroxyurea is the most effective agent for treating cml during the chronic phase. 61, 101 the initial dosage is 20 to 25 mg/kg given twice daily. treatment with hydroxyurea should continue until the leukocyte count falls to 15,000 to 20,000 cells/ml. 61, 65, 67 the dosage of hydroxyurea then can be reduced by 50% on a daily basis or to 50 mg/kg given biweeky or triweekly. in humans, the alkylating agent busulfan can be used as an alternative. 102 an effective dosage has not been established in the dog, but following human protocols, 0.1 mg/kg/day given orally is administered until the leukocyte count is reduced to 15,000 to 20,000 cells/ml. despite response to chemotherapy and control for many months, most dogs with cml eventually enter a terminal phase of disease. in one study of seven dogs with cml, four underwent terminal phase blast crisis. 61 in humans, blast crisis may be lymphoid or myeloid. 103 in dogs, the lineage of blast cells often has not been determined. these dogs have a poor prognosis, and the best treatment to consider, if any, would be that listed in table 31 -10. few cases have been reported, but one dog was treated successfully with a combination chemotherapy protocol that included vincristine, cytosine arabinoside, cyclophosphamide, and prednisone. 74 treatment is controversial in humans because of the lack of evidence that asymptomatic patients benefit from chemotherapy. patients with thrombosis or bleeding are given cytoreductive therapy. hydroxyurea is the drug of choice for initially controlling the thrombocytosis. 72 no standard therapeutic regimen exists for mds. often, humans receive no treatment if the cytopenias do not cause clinical signs. transfusions are given when necessary, and patients with fever are evaluated aggressively to detect infections. growth factors, such as erythropoietin, gm-csf, granulocyte colony-stimulating factor (g-csf), and il-3, are sometimes used in patients who require frequent transfusions to increase their blood cell counts and enhance neutrophil function. 104, 105 in one case report, human epo (100 u/kg given subcutaneously every 48 hours) was administered to a dog with msd because of profound anemia. the rationale for the erythropoietin was to promote terminal differentiation of dysplastic erythrocytes. the pcv increased from 12% to 34% by day 19 of epo treatment. this dog remained in remission for longer than 30 months. 28 other factors that induce differentiation of hematopoietic cells include retinoic acid analogs, 106 1,25 dihydroxyvitamin d 3 , 107 interferon-alpha, and conventional chemotherapeutic agents, such as 6-thioguanine and cytosine arabinoside. 108 the propensity of these factors to enhance progression to leukemia is not known in many cases, but the potential risk exists. in general, the prognosis for animals with chronic mpds is better than for dogs with acute mpds, for which the prognosis is grave. the prognosis for pv and chronic myelogenous leukemia is guarded, but significant remissions have been achieved with certain therapeutic regimens and careful monitoring. animals commonly survive a year or longer. 61, 67 the development of blast crisis portends a grave prognosis. the pathophysiology and therapy of nonlymphocytic leukemia in humans is being studied intensively. the mpds have been demonstrated to be clonal, with abnormalities evident in all hematopoietic cell lines. leukemogenesis likely is caused by mutation or amplification of proto-oncogenes in a two-step process that initially involves a single cell and is followed by additional chromosomal alterations that may involve oncogenes. 1, 13 these alterations are manifested as chromosomal abnormalities. environmental factors known to cause leukemia are exposure to high-dose radiation, benzene (chronic exposure), and alkylating agents. 109 new classification systems have incorporated genetic mutations, more accurately reflect prognoses, and facilitate use of consistent categorization among institutions. 110 therapeutic modalities under investigation or development include combination chemotherapy, immunotherapy, cytokine therapy, drug-resistance modulators, proapoptotic agents, antiangiogenic factors, signal transduction-active agents, and bone marrow transplantation. the prognosis for chronic mpds is better than for acute mpds. for acute nonlymphocytic leukemias, the prognosis is better for children than adults; only 10% of adults who receive chemotherapy maintain remissions for longer than 5 years. 109 the spontaneous canine diseases probably occur too infrequently to serve as useful models. mpds have been induced experimentally in the dog by irradiation and transplantation in an attempt to create models for study. many similarities between human and canine mpds exist, and veterinary medicine may benefit from any therapeutic advances made in the human field. plasma cell neoplasms arise when a cell of the b-lymphocyte plasma cell lineage proliferates to form a malignant population of similar cells. this population is believed in most instances to be monoclonal (i.e., derived from a single cell), because the cells typically produce homogenous immunoglobulin, although some examples of biclonal and polyclonal plasma cell neoplasms exist. a wide variety of clinical syndromes are represented by plasma cell neoplasms, including multiple myeloma, igm (waldenstrom's) macroglobulinemia, and solitary plasmacytoma (including solitary osseous plasmacytoma and extramedullary plasmacytoma). based on incidence and severity, multiple myeloma is the most clinically important plasma cell neoplasm. although multiple myeloma (mm) represents fewer than 1% of all malignant tumors in animals, it accounts for approximately 8% of all hematopoietic tumors and 3.6% of all primary and secondary tumors affecting bones in dogs. 1, 2 early studies indicated a male predisposition, 3 but subsequent reports do not suggest a gender predilection for the dog. 1, 4 older dogs are affected most often (average age, 8 to 9 years). 1, 3, 4 in one large case series, german shepherds were overrepresented based on the hospital population. 1 the true incidence of mm in the cat is unknown, although it is a much rarer diagnosis in that species than in the dog. mm accounted for only one of 395 and four of 3248 tumors in two large compilations of feline malignancies and 0.9% of all malignancies and 1.9% of hematologic malignancies in another report. [5] [6] [7] mm occurs in aged cats (median age, 12 to 14 years), most often in domestic shorthairs, and no gender predilection has been consistently reported, although a male preponderance may exist. 4,7,8 mm has not been associated with coronavirus, felv or fiv infection. the etiology of mm for the most part is unknown. genetic predispositions, molecular aberrations, viral infections, chronic immune stimulation, and exposure to carcinogen stimulation all have been suggested as contributing factors. 4, [8] [9] [10] [11] [12] [13] [14] support for a familial association in cats comes from cases reported among siblings. 8 evidence exists that molecular mechanisms of cellular control, including overexpression of cell cycle control components such as cyclin d1(see chapter 2) and receptor tyrosine kinase dysregulation, may be involved in canine myeloma and plasma cell tumors. 13, 14 in rodent models, chronic immune stimulation and exposure to implanted silicone gel has been associated with development of mm, 9, 10 as have chronic infections and prolonged hyposensitization therapy in humans. 11 viral aleutian disease of mink results in monoclonal gammopathies in a small percentage of cases. 12 working in the agricultural industry, petroleum products, and irradiation are known risk factors for the development of mm in humans. [15] [16] [17] progression of solitary plasma cell tumors to mm has been reported in both dogs and cats, and a single case of a b-cell lymphoma that progressed to mm has been reported in the dog. 18, 19 pathology and natural behavior mm is a systemic proliferation of malignant plasma cells or their precursors, which arise as a clone of a single cell that usually involves multiple bone marrow sites. malignant plasma cells can have a varied appearance on histologic sections and cytologic preparations. the degree of differentiation ranges from cells that resemble normal plasma cells in late stages of differentiation ( figure 31 -23) to very large, anaplastic round cells with a high mitotic index, representing early stages of differentiation. 3, 4, 7, 20 in cats with mm, most plasma cells (83% in one report) are immature and have marked atypia, including increased size, multiple nuclei, clefted nuclei, anisocytosis, anisokaryosis, variable nucleus-to-cytoplasm ratios, decreased chromatin density, and variable nucleoli. nearly one fourth of the cells have "flame cell" morphology, characterized by peripheral eosinophilic cytoplasmic processes. 7 malignant plasma cells typically produce an overabundance of a single type or component of immunoglobulin, referred to as the m component ( figure 31-24) . the m component may be represented by any class of the entire immunoglobulin or by only a portion of the molecule, such as the light chain (bence jones protein) or the heavy chain (heavy chain disease). in the dog, the m component usually is represented by either igg or iga immunoglobulin types in nearly equal incidence, whereas the ratio of igg to iga in cats is approximately 5:1.* if the m component is the igm type, the term macroglobulinemia (also waldenstrom's macroglobulinemia) often is applied. several cases of biclonal gammopathy in dogs and cats have been reported. 7, 8, [26] [27] [28] [29] [30] two cases of nonsecretory mm have been reported in dogs. 31 rarely, cryoglobulinemia has been reported in dogs with mm and igm macroglobulinemia, and it has been reported in a cat with igg myeloma. 4, [32] [33] [34] cryoglobulins are paraproteins that are insoluble at temperatures below 37°c. they require blood collection and clotting to be performed at 37°c before serum separation. if whole blood is allowed to clot at temperatures below this, the protein precipitates part iv • specific malignancies in the small animal patient in the clot and is lost. pure light-chain m component is rare but has been reported in dogs. 35 the pathology associated with mm is a result of high levels of circulating m component or of organ or bone infiltration with neoplastic cells, or both. associated pathologic conditions include bone disease, bleeding diathesis, hyperviscosity syndrome, renal disease, hypercalcemia, immunodeficiency (and subsequent susceptibility to infections), cytopenias secondary to myelophthisis, and cardiac failure. bone lesions can be isolated, discrete osteolytic lesions (including pathologic fractures [ figure 31 -25, a]) or diffuse osteopenias (figure 31-26 ). approximately one fourth to two thirds of dogs with mm have radiographic evidence of bony lysis or diffuse osteoporosis. 1, 3, 4, 36 skeletal lesions in cats with mm were rarely reported in the older literature, but more recent reports document lesions in 50% to 65% of cases. [6] [7] [8] 22 bones engaged in active hematopoiesis are more commonly affected, including the vertebrae, ribs, pelvis, skull, and proximal or distal long bones. 3 skeletal lesions are rare with igm (waldenstrom's) macrogammaglobulinemia, in which malignant cells often infiltrate the spleen, liver, and lymph tissue rather than bone. 4, 33, [37] [38] [39] bleeding diathesis can result from one or a combination of events. m components may interfere with coagulation by inhibiting platelet aggregation and the release of platelet factor 3; causing adsorption of minor clotting proteins; generating abnormal fibrin polymerization; and producing a functional decrease in calcium. 4, [40] [41] [42] approximately one third of dogs and one fourth of cats have clinical evidence of hemorrhage. 1, 7, 8 in dogs, nearly half have abnormal prothrombin (pt) and partial thromboplastin (ptt) times. thrombocytopenia may also play a role if bone marrow infiltration is significant (i.e., myelophthisis). hyperviscosity syndrome (hvs) represents one or a constellation of clinicopathologic abnormalities resulting from greatly increased serum viscosity. the magnitude of viscosity changes depends on the type, size, shape, and concentration of the m component in the blood. hvs is more common with igm macroglobulinemias because of the high molecular weight of this class of immunoglobulin. 37 iga myelomas, usually present as a dimer in the dog, may undergo polymerization, resulting in increased serum viscosity. 1, 4, 40 igg-associated hvs also can occur, albeit less frequently. high serum viscosity occurs in approximately 20% of dogs with mm and can result in bleeding diathesis, neurologic signs (e.g., dementia, depression, seizure activity, and coma), ophthalmic abnormalities (e.g., dilated and tortuous retinal vessels, retinal hemorrhage [ figure 31 -27], and retinal detachment), and increased cardiac workload with the potential for subsequent development of cardiomyopathy.* these consequences are thought to be a result of sludging of blood in small vessels, ineffective delivery of oxygen and nutrients, and coagulation abnormalities. hvs was reported less commonly in cats with mm in the older literature but has been reported in association with igg-, iga-, and igm-secreting tumors. 4, 6, 21, [23] [24] [25] in four of nine cats with mm, the relative serum viscosity was above control ranges. 8 renal disease is present in approximately one third to one half of dogs with mm, and azotemia was observed *references 1, 4, 37, 39, 40, and 43-45. in one third of cats in one report. 1, 3, 7 the pathogenesis of renal failure often is multifactorial. it can develop as a result of bence jones (light-chain) proteinuria, tumor infiltration into renal tissue, hypercalcemia, amyloidosis, diminished perfusion secondary to hyperviscosity syndrome, dehydration, or ascending urinary tract infection. 1, 4, [40] [41] [42] normally, heavy-chain and light-chain synthesis is well balanced in nonneoplastic immunoglobulin production. in mm, an unbalanced excess of light-chain products may result. 42 light chains have a low molecular weight and normally are filtered by the renal glomerulus; their presence can result in protein precipitates and subsequent renal tubular injury. the presence of light chains in urine without a concomitant monoclonal spike in serum, although rare, is indicative of pure light-chain disease. 35 tubules become obstructed by large laminated casts containing albumin, immunoglobulin, and light chains. 4, 35, [40] [41] [42] bence jones proteinuria occurs in approximately 25% to 40% of dogs with mm, and in two recent surveys, it occurred in approximately 50% of cases.* hypercalcemia is reported in 15% to 20 % of dogs with mm and is thought to result primarily from the production of osteoclast-activating factor by neoplastic cells. 1, 4, 46 other factors have been implicated in human mm, including elevated levels of various cytokines, tnf, il-1, and il-6. in two dogs with mm and hypercalcemia, serum elevations in circulating n-terminal pthrp were noted. 47 hypercalcemia may also be exacerbated by associated renal disease. initially thought to be a rare event in cats with mm, hypercalcemia was noted in 25% of recently reported cases. 7, 8, 48 susceptibility to infection and immunodeficiency long have been associated with mm and often are the ultimate cause of death in affected animals. 1, 4, 22 a b a, radiograph of a distal femur in a dog with severe osteolysis and a pathologic fracture secondary to a plasma cell tumor. b, radiograph of the same pathologic fracture after surgical repair with rush rods and bone cement. the local site was treated with adjuvant radiation. the dog was continued on chemotherapy for 2 more years and did well. infection rates in humans with mm are 15 times higher than normal and usually represent pneumonia or a urinary tract infection. 49 the response to vaccination also has been shown to be suppressed in humans with mm. 50 normal immunoglobulin levels often are severely depressed in affected animals. 4 in addition, leukopenias may develop secondary to myelophthisis. variable cytopenias may be observed in association with mm. approximately two thirds of dogs with mm have a normocytic, normochromic, nonregenerative anemia 1, 3, 4 ; this can result from marrow infiltration (myelophthisis), blood loss from coagulation disorders, anemia of chronic disease, or increased erythrocyte destruction secondary to high serum viscosity. in dogs with mm, similar factors lead to thrombocytopenia in nearly one third of the dogs and leukopenia in nearly one fourth. in cats, approximately two thirds are anemic, one half are thrombocytopenic, and one third are neutropenic. 7, 8 cardiac disease, if present, usually is a result of excessive cardiac workload and myocardial hypoxia secondary to hyperviscosity. 40, 41 myocardial infiltration with amyloid and anemia may be complicating factors. in one report nearly half of the cats with mm had a cardiac murmur, for which the etiology was not established. 7 clinical signs of mm may be present up to a year before diagnosis in dogs (median, 1 month). in one cat, m-component elevations were detected 9 years before clinical presentation. 1, 7 in the latter case, the m-component elevation was consistent with monoclonal gammopathy of unknown significance (mgus). mgus (i.e., "benign," "essential," or "idiopathic" monoclonal gammopathy) is a benign monoclonal gammopathy that is not associated with osteolysis, bone marrow infiltration, or bence jones proteinuria. mgus also has been reported in dogs. 51, 52 signs of mm can vary, depending on the wide range of possible pathologic effects. tables 31-11 multiple retinal hemorrhages on the fundus in a cat with hyperviscosity syndrome secondary to multiple myeloma. several reports. 1, 4, 7, 8, 22 bleeding diathesis usually is represented by epistaxis and gingival bleeding. funduscopic abnormalities may include retinal hemorrhage (see figure 31 -27), venous dilation with sacculation and tortuosity, retinal detachment and blindness. 1, 4, 40, [43] [44] [45] cns signs may include dementia, seizure activity, and deficiencies in midbrain or brainstem localizing reflexes secondary to hvs or extreme hypercalcemia. signs reflecting transverse myelopathies secondary to vertebral column infiltration, pathologic fracture, or extradural mass compression also can occur. 1, 4, 32, 36, 53 one case of ataxia and seizure activity in a dog with extramedullary plasmacytoma (emp) secondary to tumor-associated hypoglycemia has been reported. 54 in addition, paraneoplastic polyneuropathy has been reported in a dog with mm. 55 a history of chronic respiratory infections and persistent fever may also be present in cats. hepatosplenomegaly and renomegaly can occur as a result of organ infiltration. bleeding diathesis due to hvs is less common in the cat, but epistaxis, pleural and peritoneal hemorrhagic effusions, retinal hemorrhage, and central neurologic signs have been reported. 4, 6, polydipsia and polyuria can occur secondary to renal disease or hypercalcemia, and dehydration may develop. hind limb paresis secondary to osteolysis of lumbar vertebral bodies or extradural compression has been reported in cats. 8, 56 the diagnosis of mm usually follows the demonstration of bone marrow plasmacytosis (see figure 31 -23), the presence of osteolytic bone lesions (see figure 31 -26), and the demonstration of serum or urine myeloma proteins (m component; see figure 31 -24). in the absence of osteolytic bone lesions, a diagnosis can also be made if marrow plasmacytosis is associated with a progressive increase in the m component. in the cat, because the degree of bone marrow infiltration may not be as marked, some have suggested consideration of plasma cell morphology and visceral organ infiltration in cases with demonstrable m-component disease in the absence of marked marrow plasmacytosis (less than 20%). 7 all animals suspected of having plasma cell tumors should receive a minimal diagnostic evaluation, including a cbc, platelet count, serum biochemistry profile, and urinalysis. particular attention should be paid to renal function and serum calcium levels. serum electrophoresis and immunoelectrophoresis are performed to detect a monoclonal spike (see figure 31 -24) and to categorize the immunoglobulin class involved. heat precipitation with electrophoresis of urine is performed to detect bence jones proteinuria, because commercial urine dipstick methods are not capable of this determination. definitive diagnosis usually is done by bone marrow aspiration. a bone marrow core biopsy or multiple aspirates may be necessary because of the possibility of clustering of plasma cells in the bone marrow. normal marrow contains fewer than 5% plasma cells, whereas in myelomatoid marrow, this level often is greatly exceeded. current recommendations require the presence of marrow plasmacytosis greater than 20%; however, a 10% cutoff in cats recently has been recommended, with special attention to cellular atypia. 7 patel and colleagues 7 comment that even the 10% threshold is problematic, and cellular atypia and visceral organ involvement should be considered equally important. routine thoracic and abdominal radiographs are recommended. occasionally, bony lesions can be observed in skeletal areas on these standard films, and organomegaly (liver, spleen, and kidneys) is observed in most cats (figure 31-28) . 7 abdominal ultrasound scans are recommended in all cats suspected of having mm, because they reveal one or more abdominal tumors in nearly all such patients. 7 these include splenomegaly with or without nodules, diffuse hyperechoic hepatomegaly with or without nodules, renomegaly, and iliac lymph node enlargement. skeletal survey radiographs are recommended to detect and determine the extent of osteolytic lesions, which may have diagnostic, prognostic, and therapeutic implications. nuclear scintigraphy (bone scans) for clinical staging of dogs with mm has been performed; however, because of the predominant osteolytic activity with osteoblastic inactivity, these scans seldom give positive results and therefore are not useful for routine diagnosis. 57 in physician-based oncology, bone mineral density analysis (dexa scan) to document osteoporosis and mri scans of bone marrow are commonly used for staging; however, neither of these has been applied consistently in the veterinary literature. in rare cases, biopsy of osteolytic lesions (i.e., jamshidi core biopsy; see chapter 23) is necessary for diagnosis. in one case of mm in a dog, splenic aspirates were diagnostically helpful. 58 if clinical hemorrhage is present, a coagulation assessment (e.g., platelet count, pt, and ptt) and serum viscosity measurements should be done. all animals should undergo a careful funduscopic examination. table 31 -13 shows the overall frequency of clinical diagnostic abnormalities in dogs and cats with mm, as compiled from published series involving at least five cases each. a clinical staging system for canine mm has been suggested 1 ; however, currently no prognostic significance has been attributed to it. molecular diagnostic techniques for mm have had limited use thus far in veterinary oncology; however, pcr techniques have been used to determine the clonality of the immunoglobulin heavy-chain variable region gene in feline plasmacytomas and myelomas (see section a of this chapter and figure 31-8) . 59 the use of this technology in cases in which the diagnosis is not straightforward awaits further investigation. disease syndromes other than plasma cell tumors can be associated with monoclonal gammopathies and should be considered in any list of differentials. these include other lymphoreticular tumors (lymphoma, extramedullary plasmacytoma, chronic and acute lymphocytic leukemia), chronic infections (e.g., ehrlichiosis, leishmaniasis, fip), and mgus.* therapy for mm is directed at both the tumor cell mass and the secondary systemic effects. all diagnostic procedures should be completed before primary therapy is started to ensure a complete diagnosis and to procure baseline values for monitoring response. in most dogs with mm, chemotherapy is effective at reducing the myeloma cell burden, relieving bone pain, allowing for skeletal healing, and reducing levels of serum immunoglobulins. it also can greatly extend both the quality and duration of most patients' lives. 1, 4 complete elimination of neoplastic myeloma cells is rare, but the chemotherapeutic drugs currently available can make mm a gratifying disease to treat for both the clinician and the client. however, eventual relapse is to be expected. only one half of cats with mm respond to chemotherapy, and most responses are short-lived. however, several long-term responses (i.e., longer than 1 year) have been reported, and treatment should be attempted when educated clients decide on a therapeutic option. † melphalan, an alkylating agent, is the chemotherapeutic drug of choice for the treatment of mm. 1, 4 in the dog, an initial starting dosage of 0.1 mg/kg is given orally once daily for 10 days and then reduced to 0.05 mg/kg given orally once daily continuously. addition of prednisone therapy is thought to increase the efficacy of melphalan therapy. prednisone is started at a dosage of 0.5 mg/kg given orally once daily for 10 days and is then reduced to 0.5 mg/kg given orally every other day until the drug is discontinued after 60 days of therapy. melphalan, however, is continued at 0.05 mg/kg/day until clinical relapse occurs or myelosuppression necessitates a dose reduction. most dogs treated with this melphalan/prednisone combination tolerate the regimen well. the most clinically significant toxicity of melphalan is myelosuppression, particularly a delayed thrombocytopenia. a cbc, including a platelet count, should be performed biweekly for 2 months of therapy and monthly thereafter. if significant myelosuppression occurs (usually thrombocytopenia or neutropenia), the dosage or the treatment frequency may need to be reduced. the author has successfully used an alternative, pulse-dosing regimen for melphalan (7 mg/m 2 given orally daily for 5 consecutive days every 3 weeks) in a small number of cases in which myelosuppression was limiting the more conventional, continuous low-dose therapy. the author now uses this pulse-dose regimen as a first-line treatment with the caveat that long-term response data are lacking. melphalan/prednisone therapy also can be used in cats with mm; however, the protocol appears to be more myelosuppressive in cats than in dogs, and careful monitoring is required. in the cat, a dosing schedule similar to that for the dog has been reported 8, 22 ; 0.1 mg/kg (approximately 0.5 mg, or 1 ⁄4 of a 2 mg tablet) is given orally once daily for 10 to 14 days, then every other day until clinical improvement occurs or leukopenia develops. one report has advocated long-term continuous maintenance (0.1 mg/kg given orally once every 7 days). 8 cyclophosphamide has been used as an alternative alkylating agent or in combination with melphalan in dogs with mm. 1, 4, 64 no evidence indicates that it is superior to melphalan therapy. in the author's practice, cyclophosphamide is limited to patients with severe hypercalcemia or widespread systemic involvement in which a faster acting alkylating agent may alleviate systemic effects more quickly. cyclophosphamide is initiated at a dosage of 200 mg/m 2 given intravenously once, at the same time oral melphalan therapy is started. because cyclophosphamide is less likely to affect thrombocytes, it may be substituted in patients in which thrombocytopenia has developed secondary to long-term melphalan use. chlorambucil, another alkylating agent, has been used successfully for the treatment of igm macroglobulinemia in dogs at a dosage of 0.2 mg/kg given orally once daily. 4, 37 few or no clinical signs of toxicity result from this dosing schedule. ccnu, yet another alkylating agent, has been used in a limited number of cats with mm, and a partial response has been reported with a dosing schedule of 50 mg/m 2 given orally every 21 days. 65 evaluation of the response to therapy evaluation of the response to systemic therapy for mm is based on improvement in clinical signs and clinicopathologic parameters and radiographic improvement of skeletal lesions. 1, 4 subjective improvement in clinical signs of bone pain, lameness, lethargy, and anorexia should be evident within 3 to 4 weeks after initiation of therapy. objective laboratory improvement, including reduction in serum immunoglobulin or bence jones proteinuria, usually is noted within 3 to 6 weeks. radiographic improvement in osteolytic bone lesions may take months, and only partial resolution may occur. ophthalmic complications (including longstanding retinal detachment) and paraneoplastic neuropathies can be expected to resolve along with the tumor mass. 45, 55 in one report on cats that responded to melphalan and prednisone, clinical improvement was noted in 4 weeks and serum protein and radiographic bone abnormalities were greatly improved by 8 weeks. 8 as previously discussed, mm does not resolve completely, and a good response is defined as a reduction in measured m component (i.e., immunoglobulin or bence jones proteins) by at least 50% of pretreatment values. 4 reduction in the serum immunoglobulin levels may lag behind reduction in bence jones proteinuria, because the half-lives are 15 to 20 days and 8 to 12 hours, respectively. 66 for routine follow-up, quantification of the elevated serum immunoglobulin or urine bence jones protein is performed monthly until a good response is noted and then every 2 to 3 months. repeat bone marrow aspiration for evaluation of plasma cell infiltration occasionally may be necessary. this is particularly prudent when cytopenias develop during chemotherapy and drug toxicity must be differentiated from marrow recurrence. long-term control of complications such as hypercalcemia, hvs, bleeding diathesis, renal disease, immunosuppression, ophthalmic complications, and pathologic skeletal fractures is achieved by controlling the primary tumor mass. however, therapy directed more specifically at these complications may be indicated in the short term. if hypercalcemia is marked and significant clinical signs are present, standard therapies, including fluid diureses with or without pharmacologic agents (e.g., calcitonin), may be indicated (see chapter 5) . moderate hypercalcemia typically resolves within 2 to 3 days after initiation of melphalan/prednisone chemotherapy. hvs is best treated in the short term by plasmapheresis. 4, 40, 62, 67, 68 whole blood is collected from the patient and centrifuged to separate plasma from packed cells. packed red cells are resuspended in normal saline and reinfused into the patient. bleeding diathesis usually resolves along with hvs, but platelet-rich plasma transfusions may be necessary with thrombocytopenia. renal impairment may require aggressive fluid therapy in the short term and maintenance of adequate hydration in the long term. careful attention to secondary urinary tract infections and appropriate antimicrobial therapy are indicated. ensuring an adequate water intake at home is important, and in some cases owners must be taught home subcutaneous fluid administration. continued monitoring of renal function is recommended, along with follow-up directed at tumor response. patients with mm can be thought of as immunologic cripples. some have recommended prophylactic antibiotic therapy in dogs with mm, 4 but in humans, no benefit for this approach has been observed over diligent monitoring and aggressive antimicrobial management when indicated. 41 cidal antimicrobials are preferred over static drugs, and nephrotoxic antimicrobials should not be used. pathologic fractures of weight-bearing long bones and of vertebrae, resulting in spinal cord compression, may require immediate intervention in conjunction with systemic chemotherapy. orthopedic stabilization of fractures should be performed and may be followed with external beam radiotherapy (see figure 31-25, b) . recently, inhibition of osteoclast activity by bisphosphonate drugs has been shown to reduce the incidence and severity of skeletal complications of mm in humans. 57 this class of drugs may hold promise for use in dogs and cats with various skeletal tumors. 69 rescue therapy may be attempted when mm eventually relapses in dogs undergoing melphalan therapy or in the uncommon patient that initially is resistant to alkylating agents. the author has had success with the vad protocol, a combination of doxorubicin (30 mg/m 2 given intravenously every 21 days), vincristine (0.7 mg/m 2 given intravenously on days 8 and 15), and dexamethasone sodium phosphate (1 mg/kg given intravenously once a week on days 1, 8, and 15); this regimen is used in 21-day cycles. although most dogs initially respond to this rescue protocol, the duration of response tends to be short, lasting only a few months. high-dose cyclophosphamide (300 mg/m 2 given intravenously every 7 days) also been has used as a rescue agent, although with limited success. liposomal doxorubicin produced a long-term remission in a dog with mm that had been resistant to native doxorubicin. 70 because mm ultimately is a uniformly fatal disease in most species, including humans, significant effort is being put into investigational therapies for this disease. currently, bone marrow ablative therapy and marrow or stem cell rescue, thalidomide (and other antiangiogenic therapies), bortezomib (a proteasome inhibitor), arsenic trioxide, the bisphosphonates, and several molecular targeting therapies are under investigation; however, their use in veterinary species is limited or completely absent at present. 57 nonetheless, the promise of molecular-targeted therapies is foreshadowed by a case of a dog with mm that was resistant to melphalan, prednisone, and doxorubicin 14 ; this dog achieved a partial response to tyrosine kinase inhibitor therapy (su11654; see chapter 14, section b) that was maintained for 6 months. the prognosis for dogs with mm is good for initial control of the tumor and a return to good quality of life. in a group of 60 dogs with mm, approximately 43% achieved a complete remission (i.e., normalization of serum immunoglobulins), 49% achieved a partial remission (i.e., immunoglobulin levels less than 50% pretreatment values), and only 8% did not respond to melphalan/prednisone chemotherapy. 1 long-term survival is the norm, with a median of 540 days reported (figure 31-29) . hypercalcemia, bence jones proteinuria, and extensive bony lysis are known negative prognostic indices in the dog. 1 the long-term prognosis for dogs with mm is poor, because recurrence of the tumor mass and associated clinical signs is expected. eventually, the tumor no longer responds to available chemotherapeutic drugs, and death follows from renal failure, sepsis, or euthanasia for intractable bone or spinal pain. 1, 4 the prognosis for mm in the cat is not as favorable in the short term as it is in the dog. 4, 7, 8, 22 although most cats (approximately 60%) transiently respond to melphalan/prednisone-or cyclophosphamide-based protocols, most responses are partial and not durable. typically, cats with mm succumb to the disease within 4 months. [6] [7] [8] 22, 24, 68 however, long-term survivors (longer than 1 year) have been reported. 7, 8, 22, 30, 48 one investigator grouped mm in cats into two prognostic categories, based on criteria known to predict disease behavior in dogs (table 31-14) . 8 although no rigorous statistical analysis was performed on this small group of cats (nine), the median survival times were 5 days for cats with disease categorized as "aggressive" and 387 days for those with disease categorized as "nonaggressive." experience in dogs with igm macroglobulinemia is limited. 4, 35 response to chlorambucil is to be expected, and in nine treated dogs, 77% achieved remission, with a median survival time of 11 months. 4 solitary collections of monoclonal plasmacytic tumors can originate in soft tissues (extramedullary plasmacytoma) or bone (solitary osseous plasmacytoma [sop]). a number of large case compilations of cutaneous plasmacytoma have been reported in the dog. 13, [71] [72] [73] [74] [75] [76] [77] plasmacytomas represented 2.4% of all canine tumor submissions in one large compilation of cases. 78 in this series of 751 cases, the most common locations in the dog were cutaneous sites (86%) (figure 31-30) , the mucous membranes of the oral cavity and lips (9%) (figure 31-31) , and the rectum and colon (4%). the skin of the limbs and head (including the ears) is the most frequently reported cutaneous site. 71, 72, 77 all other sites accounted for the remaining 1% of cases; these sites may include the stomach, spleen, genitalia, eyes, uterus, and liver. the american cocker spaniel, english cocker spaniel, and west highland white terrier (and perhaps yorkshire terriers, boxers, german shepherds, and airedale terriers) have an increased risk of developing plasmacytomas, and the median age of affected dogs is 9 to 10 years. 77, 78 cutaneous and oral emp in dogs typically manifests as benign tumors that are highly amenable to local therapy. the natural behavior of noncutaneous/nonoral emp appears to be somewhat more aggressive in the dog. gastrointestinal emp has been reported in a number of sites in the veterinary literature, including the esophagus, 79 stomach ( figure 31-32) , 64,80 small intestine, 81 and large intestine. 78, 80, [81] [82] [83] metastasis to associated lymph nodes is more common in these cases; however, bone marrow involvement and monoclonal gammopathies are less commonly encountered. in nine cases of colorectal emp, only two dogs experienced recurrence after surgical excision, and two cases involved multiple lesions. 78 emp of the trachea, liver, and uterus also have been reported in a dog, and all had a benign course after local resection. [84] [85] [86] most cases of sop eventually progress to systemic mm; however, the time course from local tumor development to systemic mm may be many months to years. 31, 87 sops reported in the dog have involved the zygomatic arch and the ribs. 31 plasmacytomas are less common in cats, and fewer reports exist in the literature. 18, [88] [89] [90] [91] [92] [93] [94] they occur in older cats (mean age, 8.5 years) with no significant gender predilection. the skin is the most common site; other sites include the oral cavity, eye, gi tract, liver, subcutaneous tissues, and brain. two reports exist of cutaneous emp in cats that progressed to systemic disease; one cat developed lymph node and distant metastasis, the other progressed to mm. 18 cutaneous plasmacytoma on the limb of a dog. clinical signs associated with solitary plasmacytomas relate to the location of involvement; in the rare cases involving high levels of m component, hvs may occur. most cutaneous plasmacytomas are solitary, smooth, raised pink nodules that measure 1 to 2 cm in diameter (see figure 31 -30), although tumors as large as 10 cm have been reported. combination of data from large series shows that more than 95% of the tumors occur as solitary masses, and fewer than 1% occur as part of a systemic mm process. 13, [71] [72] [73] 77 the cutaneous and oral forms of emp usually have a benign course and no related clinical signs. gastrointestinal emp, however, typically produces relatively nonspecific signs that may suggest alimentary involvement. colorectal plasmacytomas usually cause rectal bleeding, hematochezia, tenesmus, and rectal prolapse. 78 one case of ataxia and seizure activity in a dog with emp secondary to tumor-associated hypoglycemia has been reported. 54 sop usually is associated with pain and lameness (if the appendicular skeleton is affected) or with neurologic signs (if vertebral bodies are involved). diagnosis of sops and emps usually requires tissue biopsy or fine-needle aspiration. the cells that make up a b solitary plasmacytic tumors in both cats and dogs have been classified histologically as mature, hyaline, cleaved, asynchronous, monomorphous blastic, and polymorphous blastic cell types. no prognostic significance has been observed with this classification system, although it has been suggested that the polymorphous blastic type may act more aggressively in the dog. 13, 77, 88 with poorly differentiated solitary plasmacytic tumors, immunohistochemical studies directed at detecting immunoglobulin, light and heavy chains, and thioflavine t may be helpful for differentiating the lesions from other round cell tumors. 31, 76, 77, [92] [93] [94] immunoreactivity has been demonstrated for canine igg f(ab) 2 and vimentin. 73 a variant characterized by an igg-reactive amyloid interspersed with the neoplastic cells also has been described. 74 in addition, pcr techniques can be used to determine the clonality of the immunoglobulin heavy-chain variable region gene in plasmacytomas and myelomas, which may be diagnostically useful in difficult cases. thorough staging is important in dogs and cats with plasmacytomas that are at higher risk for systemic spread. staging, which must be done before therapy is started, should include bone marrow aspiration, serum electrophoresis, and skeletal survey radiographs to ensure that the disease is confined to a local site. this is most important for sop and gastrointestinal emp because of these tumors' relatively high metastatic rate; it is less important for cutaneous and oral plasmacytomas because of their typically more benign behavior. in addition, endoscopic evaluation of the entire gi tract is recommended with gastrointestinal emp. cutaneous plasma cell tumors in the dog are almost always benign and have an excellent prognosis after conservative surgical excision. successful therapy with melphalan and prednisone has been rarely applied for a local recurrence or incomplete margins in dogs and cats. 53, 92 radiation therapy has been used infrequently for cases that are nonsurgical. surgery is recommended in combination with radiotherapy for sop when the lesion results in an unstable long bone fracture (see or the patient is nonambulatory because of neurologic compromise caused by a vertebral body sop. in the latter case, spinal cord decompression, mass excision, and possibly spinal stabilization may be necessary. 53 radiotherapy can be used without surgery, when fractures are stable, as a palliative measure for bone pain or, in the case of vertebral sop, if the patient is ambulatory and stable. good local control usually is achieved, but most patients eventually develop systemic mm. 31, 53, 87 sop of the axial skeleton can be managed by excision or radiotherapy alone. whether systemic chemotherapy should be initiated at the time of local therapy for sop when systemic involvement is not documented is the subject of debate. systemic spread may not occur for many months or even years beyond the primary sop diagnosis in humans and dogs, and studies in humans reveal no benefit from initiation of systemic chemotherapy before documentation of subsequent systemic spread. 42, 53 similarly, emp of the gi tract in humans most often is treated with surgical excision and thorough staging of disease. systemic therapy is not initiated unless systemic involvement is documented. systemic chemotherapy has been used following excision of a gastric emp in a cat, but the utility of adjuvant therapy in the species is unkown. 94 long-term follow-up of patients with a solitary plasmacytoma is indicated so that recurrence of disease and systemic spread can be recognized. in patients with sop, careful attention should be paid to the serum globulin levels, bone pain, and the radiographic appearance of bone healing. restaging of the disease, including bone marrow evaluation, is indicated if systemic spread is suspected. the prognosis for solitary plasma cell tumors generally is good. cutaneous and mucocutaneous plasmacytomas usually are cured by surgical excision. 13, 77 in large compilations of cases in dogs, the local recurrence rate was approximately 5%, and nodal or distant metastasis occurred in only seven of 349 cases (2%). 13, [71] [72] [73] 77 new cutaneous plasmacytomas at sites distant from the primary tumor developed in fewer than 2% of cases. one caveat: the author has encountered a handful of cases of aggressive multiple cutaneous plasmacytoma that eventual resulted in the death of the affected dogs. neither the tumor cell proliferation rate (as measured by ki-67 immunohistochemistry) in the dog nor histopathologic grading in dogs and cats was prognostic in large compilations of cases, although it has been suggested that the polymorphous blastic type may act more aggressively in the dog. 13, 77, 88 the presence of amyloid and overexpression of cyclin d1 (prognostic in human plasmacytomas) were not shown to be prognostic in dogs. 13 most dogs with emp of the alimentary tract and other abdominal organs (e.g., liver, uterus) that is treated by surgical excision alone or in combination with systemic chemotherapy (if metastasis is present) can enjoy long-term survival. 31, 64, [78] [79] [80] [82] [83] [84] [85] [86] in a compilation of nine dogs with colorectal plasmacytoma, two had local recurrence at 5 and 8 months after surgery; after surgery alone, the overall median survival time was 15 months. 78 dna ploidy and c-myc oncoprotein expression in biopsy samples were determined to be predictive for emps in dogs; however, tumors that were malignant all were from noncutaneous sites (i.e., lymph node, colon, and spleen), therefore location appears to be as predictive. 96 as previously discussed, lymphatic leukemia in dogs: an epizootiological clinical and haematological study survey of animal neoplasms in alameda and contra costa counties, california. ii. cancer morbidity in dogs and cats from alameda county immunohistochemical 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in two dogs macroglobulinemia in the dog, the canine analogue of gamma m monoclonal gammopathy macroglobulinemia with hyperviscosity syndrome in a dog serum hyperviscosity syndrome associated with iga multiple myeloma in two dogs plasma cell tumors plasma cell neoplasms ocular lesions in a dog with hyperviscosity secondary to an iga myeloma blindness in a dog with igaforming myeloma ophthalmic disease as the presenting complaint in five dogs with multiple myeloma bone destruction and hypercalcemia in plasma cell myeloma parathyroid hormone (pth)-related protein, pth, and 1,25-dihydroxyvitamin d in dogs with cancer associated hypercalcemia hypercalcemia in two cats with multiple myeloma infections complicating multiple myeloma and chronic lymphocytic leukemia infection, antibody response, and gamma globulin components in multiple myeloma and macroglobulinemia a benign hypergammaglobulinemia mimicking plasma cell myeloma idiopathic monoclonal (iga) gammopathy in a dog vertebral plasma cell tumors in 8 dogs hypoglycemia and polyclonal gammopathy in a dog with plasma cell dyscrasia multiple myeloma with associated polyneuropathy in a german shepherd dog plasma cell sarcoma in a cat plasma cell neoplasms fine-needle aspiration of the spleen as an aid in the diagnosis of splenomegaly characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of b-cell neoplasia use of plasmapheresis and chemotherapy for treatment of monoclonal gammopathy associated with ehrlichia canis infection in a dog monoclonal gammopathy associated with naturally occurring canine ehrlichiosis hyperviscosity syndrome associated with lymphocytic leukemia in three dogs monoclonal gammopathy in a dog with visceral leishmaniasis gastric extramedullary plasmacytoma in a dog hematological toxicity and therapeutic efficacy of lomustine in 20 tumor-bearing cats: critical assessment of a practical dosing regimen the treatment of multiple myeloma therapeutic plasmapheresis multiple myeloma in a cat response to liposome-encapsulated doxorubicin (tlc d-99) in a dog with myeloma extramedullary plasmacytomas in dogs: results of surgical excision in 131 cases mucocutaneous plasmacytomas in dogs: 75 cases cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study cutaneous plasmacytomas with amyloid in six dogs primary cutaneous plasmacytomas in the dog and cat an immunohistochemical study of canine extramedullary plasma cell tumours prognostic value of histopathological grading in canine extramedullary plasmacytomas colorectal plasmacytomas: a retrospective study of nine dogs esophageal plasmacytoma in a dog extramedullary plasmacytoma of the gastrointestinal tract in two dogs primary igg secreting plasma cell tumor in the gastrointestinal tract of a dog a solitary plasmacytoma in a dog with progression to a disseminated myeloma metastatic extramedullary plasmacytoma of the colon and rectum in a dog extramedullary plasmacytoma in the trachea of a dog uterine extramedullary plasmacytoma in a dog a primary hepatic plasma cell tumor in a dog part iv • specific malignancies in the small animal patient solitary plasmacytomas of bone and extramedullary plasmacytomas histopathologic and immunophenotypic characterization of extramedullary plasmacytomas in nine cats intracerebral plasma cell tumor in a cat: a case report and literature review intraocular extramedullary plasmacytoma in a cat extramedullary plasmacytoma and immunoglobulin-associated amyloidosis in a cat immunohistochemical staining of neoplastic and inflammatory plasma cell lesions in feline tissues immunoglobulin-producing tumours in dogs and cats identification of immunoglobulin light chains in canine extramedullary plasmacytomas by thioflavine t and immunohistochemistry gastric extramedullary plasmacytoma in a cat analysis of dna aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry most patients with sop eventually develop systemic disease, but long, disease-free periods usually precede the event.the prognosis in cats is less well defined, owing to the scarcity of reported cases. if disease is confined to a local site and/or regional nodes, surgical excision and chemotherapy can result in long-term control; however, early, widespread metastasis and progression to mm is also reported in cats.* key: cord-022561-rv5j1201 authors: boes, katie m.; durham, amy c. title: bone marrow, blood cells, and the lymphoid/lymphatic system date: 2017-02-17 journal: pathologic basis of veterinary disease doi: 10.1016/b978-0-323-35775-3.00013-8 sha: doc_id: 22561 cord_uid: rv5j1201 nan within the marrow spaces, a network of stromal cells and extracellular matrix provides metabolic and structural support to hematopoietic cells. these stromal cells consist of adipocytes and specialized fibroblasts, called reticular cells. the latter provides structural support by producing a fine network of a type of collagen, called reticulin, and by extending long cytoplasmic processes around other cells and structures. both reticulin and cytoplasmic processes are not normally visible with light microscopy but are visible with silver reticulin stains (e.g., gordon and sweet's and sometimes with periodic acid-schiff). bone marrow is highly vascularized but does not have lymphatic drainage. marrow of long bones receives part of its blood supply from the nutrient artery, which enters the bone via the nutrient canal at midshaft. the remaining arterial supply enters the marrow through an anastomosing array of vessels that arise from the periosteal arteries and penetrate the cortical bone. vessels from the nutrient and periosteal arteries converge and form an interweaving network of venous sinusoids that permeates the marrow. these sinusoids not only deliver nutrients and remove cellular waste but also act as the entry point for hematopoietic cells into blood circulation. sinusoidal endothelial cells function as a barrier and regulate traffic of chemicals and particles between the intravascular and extravascular spaces. venous drainage parallels that of the nutrient artery and its extensions. • bleeding time (template bleeding time, buccal mucosal bleeding time). this assay assesses primary hemostasis (platelet plug formation) by measuring the time interval between inflicting of standardized wound and cessation of bleeding. sedation may be required. in small animals the test is usually performed on the buccal mucosa; in large animals it may be performed on the distal limb. prolonged bleeding time may be because of a platelet function defect, von willebrand disease, or a vascular defect. the sensitivity of this test is low; reference intervals are species and site dependent (can perform test on a normal animal as a control). this test is contraindicated in cases of thrombocytopenia because significant thrombocytopenia can cause a prolonged bleeding time (invalidates interpretation of test results). • clot retraction test. this assay assesses retraction of a clot, in which platelets play an essential role. this is a crude test that is rarely performed. different protocols are described. significant thrombocytopenia invalidates interpretation of test results. • tests to characterize platelet function abnormalities more specifically are available through specialized laboratories. • aggregometry-to assess platelet aggregation in response to different physiologic agonists. • adhesion assays-to assess the ability of platelets to adhere to a substrate (e.g., collagen). • flow cytometry-to assay for expression of surface molecules. • pfa-100-an instrument that simulates a damaged blood vessel, by measuring time for a platelet plug to occlude an aperture; to date, this instrument has mainly been used in research applications. • thromboelastography (teg)-global assessment of hemostasis (platelets, coagulation, and fibrinolysis) based on viscoelastic analysis of whole blood. • tests for immune-mediated thrombocytopenia (imt). • flow cytometry-to detect immunoglobulin bound to the platelet surface, using a fluorescent-labeled antibody. • bone marrow immunofluorescent antibody (ifa) test-to detect bound immunoglobulin. sometimes referred to as the "antimegakaryocyte antibody test," this assay actually detects the presence of immunoglobulin nonspecifically: a smear of a bone marrow aspirate is incubated with a fluorescent-labeled antibody to species-specific immunoglobulin. other components of the marrow include myelinated and nonmyelinated nerves, as well as low numbers of resident macrophages, lymphocytes, and plasma cells. of note, the macrophages play an important role in iron storage and erythrocyte maturation. the following basic concepts provide a framework for understanding the mechanisms of injury and diseases presented later in the chapter. • hematopoietic tissue is highly proliferative. billions of cells per kilogram of body weight are produced each day. • pluripotent hematopoietic stem cells are a self-renewing population, giving rise to cells with committed differentiation programs, and are common ancestors of all blood cells. the process of hematopoietic differentiation is shown in fig. 13 -2. • hematopoietic cells undergo sequential divisions as they develop, so there are progressively higher numbers of cells as they mature. cells also continue to mature after they have stopped dividing. conceptually, it is helpful to consider cells in the bone marrow as belonging to mitotic and postmitotic compartments. examples of developing hematopoietic cells are shown in fig. 13 -3. • mature cells released into the blood circulation have different normal life spans, varying from hours (neutrophils), to days (platelets), to months (erythrocytes), and to years (some lymphocytes). • the hematopoietic system is under exquisite local and systemic control and responds rapidly and predictably to various stimuli. • production and turnover of blood cells are balanced so that numbers are maintained within normal ranges (steady-state kinetics) in healthy individuals. • normally the bone marrow releases mostly mature cell types (and very low numbers of cells that are almost fully mature) into the circulation. in response to certain physiologic or pathologic stimuli, however, the bone marrow releases immature cells that are further back in the supply "pipeline." the composition of the marrow changes with age. the general pattern is that hematopoietic tissue (red marrow) regresses and is replaced with nonhematopoietic tissue, mainly fat (yellow marrow). thus in newborns and very young animals the bone marrow consists largely of hematopoietically active tissue, with relatively little fat, whereas in geriatric individuals the marrow consists largely of fat. in adults, hematopoiesis occurs primarily in the pelvis, sternum, ribs, vertebrae, and the proximal ends of humeri and femora. even within these areas of active hematopoiesis, fat may constitute a significant proportion of the marrow volume. immature hematopoietic cells can be divided into three stages: stem cells, progenitor cells, and precursor cells. hematopoietic stem cells (hscs) have the capacity to self-renew, differentiate into mature cells, and repopulate the bone marrow after it is obliterated. progenitor cells and precursor cells cannot self-renew; with each cell division, they evolve into more differentiated cells. later-stage precursors cannot divide. stem cells and progenitor cells require immunochemical stains for identification, but precursor cells can be identified by their characteristic morphologic features (see fig. 13 -3). control of hematopoiesis is complex, with many redundancies, feedback mechanisms, and pathways that overlap with other physiologic and pathologic processes. many cytokines influence cells of different lineages and stages of differentiation. primary growth factors for primitive cells are interleukin (il) 3, produced by t lymphocytes, and stem cell factor, produced by monocytes, macrophages, fibroblasts, endothelial cells, and lymphocytes. interleukin 7 is an early lymphoid growth factor. lineage-specific growth factors are discussed in their corresponding sections. hematopoiesis occurs in the interstitium between the venous sinusoids in the so-called hematopoietic spaces. there is a complex functional interplay among hematopoietic cells with the supporting connective tissue cells, extracellular matrix, and soluble factors, which form the hematopoietic microenvironment. behavior of hematopoietic cells is influenced by direct cell-to-cell and cellmatrix interactions and by soluble mediators, such as cytokines and hormones that interact with cells and with matrix proteins. cells localize to specific niches within the hematopoietic microenvironment via adhesion molecules, such as integrins, immunoglobulins, lectins, and other receptors, which recognize ligands on other cells or matrix components. cells also express receptors for soluble molecules such as chemokines (chemoattractant cytokines) and hormones that influence cell trafficking and metabolism. iron is essential to hemoglobin synthesis and function. it is acquired through the diet and is transported to the bone marrow via the iron transport protein, transferrin. central macrophages either store iron as ferritin or hemosiderin, or transfer the iron to erythroid precursors for hemoglobin synthesis. hemosiderin is identifiable in routinely stained marrow preparations as an intracellular brown pigment. however, perls's prussian blue stain is more sensitive and specific for iron detection. the earliest erythroid precursor identifiable by routine light microscopy is the rubriblast, which undergoes maturational division to produce 8 to 32 progeny cells. late-stage erythroid precursors, known as metarubricytes, extrude their nuclei and become inhibiting apoptosis of developing erythroid cells. the stimulus for increased epo production is hypoxia. within the bone marrow, erythroid precursors surround a central macrophage in specialized niches, termed erythroblastic islands . the central macrophage, also known as a nurse cell, anchors the precursors within the island niche, regulates erythroid proliferation and differentiation, transfers iron to the erythroid progenitors for hemoglobin synthesis, and phagocytizes extruded metarubricyte nuclei. although erythroblastic islands occur throughout the marrow, those with more differentiated erythroid cells neighbor sinusoids, whereas nonadjacent islands contain mostly undifferentiated precursors. as erythroid cells mature from a rubriblast to a mature erythrocyte, their nuclei become smaller and more condensed. the nucleus is eventually extruded to form a polychromatophil. erythroid cells also become less basophilic and more eosinophilic as more hemoglobin is produced and as rna-rich organelles are lost during maturation. (hemoglobin stains eosinophilic, and rna stains basophilic with routine romanowsky's stains.) as granulocytes (e.g., neutrophils, eosinophils, and basophils) mature from a myeloblast to their mature forms, their nuclei become dense and segmented. granulocytes acquire their secondary or specific granules during the myelocyte stage and can be morphologically differentiated starting at this stage. neutrophils have neutral-staining secondary granules, eosinophil secondary granules have an affinity for acidic or eosin dyes, and basophil secondary granules have an affinity for basic dyes. monoblasts differentiate into promonocytes with ruffled nuclear boarders and then into monocytes. in most mammals, mature erythrocytes have a biconcave disk shape, called a discocyte. microscopically, these cells are round and eosinophilic with a central area of pallor. however, the central concavity may not be microscopically apparent in species other than the dog. camelids normally have oval erythrocytes, termed ovalocytes or elliptocytes, which facilitate better gas exchange at high altitudes. the erythrocytes of some animals are prone to in vitro shape change, including those of cervids, pigs, and some goat breeds (e.g., angora). erythrocyte size during health depends on the species, breed, and age of the animal. in dogs, some breeds have relatively smaller (e.g., akitas and shibas) or larger (e.g., some poodles) erythrocytes. akitas and shibas also have a high concentration of potassium, unlike erythrocytes in other dogs. juvenile animals may have larger erythrocytes because of the persistence of fetal erythrocytes, which is followed by a period of relatively smaller cells before reaching adult reference intervals. mature mammalian erythrocytes lack nuclei and organelles and are thus incapable of transcription, translation, and oxidative metabolism. however, they do require energy for various functions, including maintenance of shape and deformability, active transport, and prevention of oxidative damage. red blood cells generate this energy entirely through glycolysis (also known as the embden-meyerhof pathway). except in pigs, glucose enters erythrocytes from the plasma through an insulin-independent, integral membrane glucose transporter. within circulation the erythrocyte mean life span varies between species and is related to body weight and metabolic rate: approximately 150 days in horses and cattle, 100 days in dogs, and 70 days in cats. when erythrocytes reach the end of their life span, they are destroyed in a process termed hemolysis. hemolysis may occur within blood vessels (intravascular hemolysis) or by sinusoidal macrophages (extravascular hemolysis). during intravascular hemolysis, erythrocytes release their contents, mostly hemoglobin, directly into blood. however, during extravascular hemolysis, macrophages phagocytize entire erythrocytes, leaving little or no hemoglobin in the blood. normal turnover of erythrocytes occurs mainly by extravascular hemolysis within the spleen, and to a lesser extent in other organs such as the liver and bone marrow. the exact controls are not clear, but factors that likely play a role in physiologic hemolysis include the following: • exposure of membrane components normally sequestered on the inner leaflet of the erythrocyte membrane, particularly phosphatidylserine. reticulocytes, and subsequently mature erythrocytes. the normal transit time from rubriblast to mature erythrocyte is approximately 1 week. reticulocytes start maturing in the bone marrow but finish their maturation in the blood circulation and spleen. horses are an exception in that they do not release reticulocytes into circulation, even in situations of increased demand. unlike mature erythrocytes, which lack organelles, reticulocytes still contain ribosomes and mitochondria, mainly to support completion of hemoglobin synthesis. these remaining organelles impart a bluish-purple cast (polychromasia) to reticulocytes on routine blood smear examination. the resultant cells are termed polychromatophils. because older reticulocytes do not exhibit polychromasia, more sensitive laboratory techniques must be used for accurate reticulocyte quantification. when a blood sample is incubated with new methylene blue stain, the reticulocytes' ribosomal rna precipitates to form irregular, dark aggregates . cats also have a more mature form of reticulocyte, termed punctate reticulocyte, which is stippled when stained with new methylene blue. punctate reticulocytes indicate prior, not active, regeneration and do not appear polychromatophilic on routine blood smear evaluation. storage pool, which consists of a reserve of fully mature neutrophils. the size of the storage pool varies by species; it is large in the dog, but small in ruminants. in homeostasis mostly mature segmented granulocytes are released from the marrow into the blood. the first monocytic precursor identifiable by morphologic features is the monoblast, which develops into promonocytes and subsequently monocytes (see fig. 13 -3). unlike granulocytes, monocytes do not have a marrow storage pool; they immediately enter venous sinusoids upon maturation. after migrating into the tissues, monocytes undergo morphologic and immunophenotypic maturation into macrophages. within blood vessels there are two pools of leukocytes: the circulating pool and the marginating pool. circulating cells are free flowing in blood, whereas marginating cells are temporarily adhered to endothelial cells by selectins. in most healthy mammals there are typically equal numbers of neutrophils in the circulating and marginal pools. however, there are threefold more marginal neutrophils relative to circulating neutrophils in cats. only the circulating leukocyte pool is sampled during phlebotomy. the concentration of myeloid cells in blood depends on the rate of production and release from the bone marrow, the proportions of cells in the circulating and marginating pools, and the rate of migration from the vasculature into tissues. the fate of neutrophils after they leave the bloodstream in normal conditions (i.e., not in the context of inflammation) is poorly understood. they migrate into the gastrointestinal and respiratory tracts, liver, and spleen and may be lost through mucosal surfaces or undergo apoptosis and be phagocytized by macrophages. lymphopoiesis. lymphopoiesis-from lympha (latin, water)refers to the production of new lymphocytes, including b lymphocytes, t lymphocytes, and natural killer (nk) cells. b lymphocytes primarily produce immunoglobulins, also known as antibodies, and are key effectors of humoral immunity. they are distinguished by the presence of an immunoglobulin receptor complex, termed the b lymphocyte receptor. plasma cells are terminally differentiated b lymphocytes that produce abundant immunoglobulin. t lymphocytes, effectors of cell-mediated immunity, possess t lymphocyte receptors that bind antigens prepared by antigen-presenting cells. a component of innate immunity, nk cells kill a variety of infected and tumor cells in the absence of prior exposure or priming. main growth factors for b lymphocytes, t lymphocytes, and nk cells are il-4, il-2, and il-15, respectively. lymphocytes are derived from hscs within the bone marrow. b lymphocyte development occurs in two phases, first in an antigenindependent phase in the bone marrow and ileal peyer's patches (the site of b lymphocyte development in ruminants), then in an antigendependent phase in peripheral lymphoid tissues (such as spleen, lymph nodes, and mucosa-associated lymphoid tissue [malt] ). t lymphocyte progenitors migrate from the bone marrow to the thymus, where they undergo differentiation, selection, and maturation processes before migrating to the peripheral lymphoid tissue as effector cells. unlike granulocytes, which circulate only in blood vessels and migrate unidirectionally into target tissues, lymphocytes travel in both blood and lymphatic vessels and continually circulate between blood, tissues, and lymphatic vessels. also in contrast to nonlymphoid hematopoietic cells, blood lymphocyte concentrations in adult animals are primarily dependent upon extramedullary lymphocyte production and kinetics, and not lymphopoiesis by the marrow. in healthy nonruminant mammals, lymphocytes are the second most numerous blood leukocyte. according to conventional wisdom, • decreased erythrocyte deformability. • binding of immunoglobulin g (igg) and/or complement to erythrocyte membranes. complement binding may be secondary clustering of the membrane anion exchange protein, band 3. • oxidative damage to erythrocytes. macrophages degrade erythrocytes into reusable components, such as iron and amino acids, and the waste product bilirubin. bilirubin is then exported into circulation, where it is transported to the liver by albumin. the liver conjugates and subsequently excretes bilirubin into bile for elimination from the body. intravascular hemolysis normally occurs at only extremely low levels. hemoglobin is a tetramer that, when released from the erythrocyte into the blood, splits into dimers that bind to a plasma protein called haptoglobin. the hemoglobin-haptoglobin complex is taken up by hepatocytes and macrophages. this is the major pathway for handling free hemoglobin. however, free hemoglobin may also oxidize to form methemoglobin, which dissociates to form metheme and globin. metheme binds to a plasma protein called hemopexin, which is taken up by hepatocytes and macrophages in a similar manner to hemoglobin-haptoglobin complexes. free heme in the reduced form binds to albumin, from which it is taken up in the liver and converted into bilirubin. the concentration of circulating erythrocytes typically decreases postnatally and remains below normal adult levels during the period of rapid body growth. the age at which erythrocyte numbers begin to increase and the age at which adult levels are reached vary among species. in dogs, adult values are usually reached between 4 and 6 months of age; in horses, this occurs at approximately 1 year of age. granulopoiesis is the production of neutrophils, eosinophils, and basophils, whereas monocyte production is termed monocytopoiesis. granulocytic and monocytic cells are sometimes collectively referenced as myeloid cells. however, the term myeloid and the prefix myelo-can be confusing because they have other meanings; they may reference the bone marrow, all nonlymphoid hemic cells (erythrocytes, leukocytes, and megakaryocytes), only granulocytes, or the spinal cord. the main purpose of granulocytes and monocytes is to migrate to sites of tissue inflammation and function in host defense (see chapters 3 and 5 ). briefly, these cells have key immunologic functions, including phagocytosis and microbicidal activity (neutrophils and monocyte-derived macrophages), parasiticidal activity and participation in allergic reactions (eosinophils and basophils), antigen processing and presentation, and cytokine production (macrophages). neutrophils are the predominant leukocyte type in blood of most domestic species. primary stimulators of granulopoiesis and monocytopoiesis are granulocyte-macrophage colony-stimulating factor and il-1, il-3, and il-6 (granulocytes and monocytes), granulocyte colonystimulating factor (granulocytes), and macrophage colonystimulating factor (monocytes). in general, these cytokines are produced by various inflammatory cells, with or without contribution from stromal cells. the earliest granulocytic precursor identifiable by routine light microscopy is the myeloblast, which undergoes maturational division over 5 days to produce 16 to 32 progeny cells (see fig. 13 -3). these granulocytic precursors are conceptually divided into those stages that can divide, including myeloblasts, promyelocytes, and myelocytes (proliferation pool), and those that cannot, including metamyelocytes, and band and segmented forms (maturation pool). within the neutrophil maturation pool is a subpool, termed the platelet aggregation and adherence to subendothelial collagen. expansion of surface area and release of granule contents is aided by a network of membrane invaginations known as the open canalicular system. this system is not present in horses, cattle, and camelids. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. mechanisms of bone marrow disease are summarized in box 13-1. hematopoietic cells' response to injury is dependent upon whether the insult is on the marrow or within extramarrow tissues. in general, marrow-directed injury or disturbances result in production of abnormal hematopoietic cells (dysplasia), fewer hematopoietic cells (hypoplasia), or a failure of hematopoietic cell development (aplasia). dysplasia, hypoplasia, and aplasia may be specific for one cell line, such as pure red cell aplasia, or affect multiple lineages, as seen with aplastic anemia. accordingly, decreased blood concentrations of the involved cell types are expected with hypoplasia or aplasia. erythroid, myeloid, and megakaryocytic hypoplasia or aplasia causes nonregenerative anemia, neutropenia, and thrombocytopenia, respectively. bicytopenia is used to describe decreased blood concentrations of two cell lines, whereas pancytopenia indicates decreased blood concentrations of all three cell types. bicytopenia or pancytopenia may indicate generalized marrow disease, such as occurs with aplastic anemia or marrow malignancies (leukemia), necrosis, fibrosis (myelofibrosis), or inflammation (myelitis). replacement of hematopoietic tissue within the bone marrow by abnormal tissue, including neoplastic cells, fibrosis, or inflammatory cells, is termed myelophthisis. cattle normally have higher numbers of lymphocytes than neutrophils in circulation. however, recent studies suggest that is no longer the case, most likely due to changes in genetics and husbandry. in most species the majority of lymphocytes in blood circulation are t lymphocytes. the concentration of blood lymphocytes decreases with age. thrombopoiesis. thrombopoiesis-from thrombos (gr., clot)refers to the production of platelets, which are small (2 to 4 µm), round to ovoid, anucleate cells within blood vessels. platelets have a central role in primary hemostasis but also participate in secondary hemostasis (coagulation) and inflammatory pathways (see chapters 2 and 3). thrombopoietin (tpo) is the primary regulator of thrombopoiesis. the liver and renal tubular epithelial cells constantly produce tpo, which is then cleared and destroyed by platelets and their precursors. therefore plasma tpo concentration is inversely proportional to platelet and platelet precursor mass. if the platelet mass is decreased, less tpo is cleared, and there is subsequently more free plasma tpo to stimulate thrombopoiesis. the earliest morphologically identifiable platelet precursor is the megakaryoblast, which undergoes nuclear reduplications without cell division, termed endomitosis, to form a megakaryocyte with 8 to 64 nuclei. as the name suggests, megakaryocytes are very large cells, much larger than any other hematopoietic cell ; also see fig. 13 -1). megakaryocytes neighbor venous sinusoids, extend their cytoplasmic processes into vascular lumens, and shed membranebound cytoplasmic fragments (platelets) into blood circulation. orderly platelet shedding is partially facilitated by β 1 -tubulin microtubules within megakaryocytes. platelets circulate in a quiescent form and become activated by binding platelet agonists, including thrombin, adenosine diphosphate (adp), and thromboxane. platelet activation causes shape change, granule release, and relocation of procoagulant phospholipids and glycoproteins (gps) to the outer cell membrane. specific procoagulant actions include release of calcium, von willebrand factor (vwf), factor v, and fibrinogen, as well as providing phosphatidylserine-rich binding sites for the extrinsic tenase (factors iii, vii, and x), intrinsic tenase (factors ix, viii, and x), and prothrombinase (factors x, v, and ii) coagulation complexes. platelet gp surface receptors include those for binding vwf (gpib-ix-v), collagen (gpvi), and fibrinogen (gpiib-iiia), which facilitate increased destruction hemorrhage (especially erythrocytes) consumption (platelets) neoplasia altered distribution abnormal function bone marrow is not routinely sampled during postmortem examinations. however, indications for bone marrow evaluation include suspected leukemia, metastatic neoplasia within bone marrow, or infectious myelitis, as well as cytopenia(s) or hematopoietic dysplasia of unknown cause. multimodal evaluation is ideal, including a recent (<24 hours) complete blood count with bone marrow cytologic and histopathologic examination. however, antemortem blood analyses are not always available, and interpretation of hematopoietic cytomorphologic examination results becomes difficult to impossible shortly after death. postmortem bone marrow should be collected as soon as possible after death or euthanasia, preferably within 30 minutes. samples may be collected from the proximal femur, rib, sternum or vertebrae. when collecting from the femur, the femoral neck is removed with a bone saw, or a fragment of the shaft is removed with bone-cutting shears. cytologic samples are first collected using the paintbrush technique: gently sample the red marrow with a clean, dry, naturalbristle brush, and then carefully brush the material onto a clean glass microscope slide in two to four parallel wavy lines. the brush should be cleaned and dried before its use on a different animal. the slide is then air dried, stored away from formalin fumes, and then stained with a routine (romanowsky) stain. for histologic evaluation the entire femoral head or femoral shaft or rib fragment with exposed red marrow is submersed in 10% neutral buffered formalin. for cosmetic necropsies, samples may be obtained by antemortem techniques, such as needle biopsies for cytologic examination and core biopsies for histopathologic examination. the complete blood count (cbc) is the cornerstone for diagnosis of hematologic disturbances and is often part of a minimum database in sick patients. the cbc includes numeric data indicating the concentration of different cell types, as well as other estimations of red blood cell mass (hemoglobin concentration, packed-cell volume, and hematocrit), red blood cell volume (mean cell volume), and red blood cell hemoglobin content (mean cell hemoglobin and mean cell hemoglobin concentration). cell morphologic features and the presence or absence of hemic parasites are assessed upon microscopic review of a blood smear and are also included in a cbc report. (note: some parasites may infect blood cells, such as hepatozoon organisms within circulating neutrophils or monocytes or bartonella organisms within erythrocytes, but mainly cause disease in other body systems and are therefore not discussed in this chapter.) learning to evaluate blood smears is a valuable skill for any practicing veterinarian. the cbc also may include the plasma protein concentration, as measured with a refractometer. it is important to remember that changes in hydration status and in the distribution of body fluids between the vascular and extravascular compartments affect the concentration of both cells and proteins in the blood. other tests that may help with evaluation of the hematopoietic system include cell or tissue biopsies, the direct antiglobulin test, flow cytometry, immunophenotyping, and polymerase chain reaction (pcr). aspiration cytology and/or histopathology of organs other than the bone marrow can be pursued to assess for the presence of emh, increased destruction of erythrocytes, neoplasia, or infection. the coombs test, or direct antiglobulin test, detects excessive antibody or complement bound to red blood cells' surfaces and is the standard assay for immune-mediated hemolytic anemia. flow cytometry and immunofluorescent antibody tests may also be used to detect autoantibody bound to erythrocytes or other hematopoietic cells. immunophenotyping and pcr are further discussed in the section on hematopoietic neoplasia. structural or functional abnormalities of blood vessels, platelets, or coagulation factors may result in a tendency toward hypocoagulability (bleeding), hypercoagulability (inappropriate thrombosis), or both. in veterinary medicine there has been a great deal of work on specific mechanisms of hypocoagulability, whereas mechanisms of hypercoagulability are less fully characterized. disorders of primary hemostasis typically result in "small bleeds" (e.g., petechiation, mild ecchymosis, bleeding from mucous membranes, bleeding immediately after venipuncture), whereas disorders of secondary hemostasis typically result in "big bleeds" (e.g., hemorrhage into body cavities/ joints, marked ecchymosis, large hematomas, delayed bleeding after venipuncture). this chapter concentrates on primary disorders of hemostasis and also covers disseminated intravascular coagulation, which is the secondary condition. however, it is important to note that coagulation disorders can also result from other underlying disease processes. for example, advanced liver disease can lead to abnormal hemostasis through decreased or defective synthesis of coagulation factors or impaired clearance of fibrinolytic products that inhibit coagulation reactions and platelet function. vascular disorders may also result in a bleeding tendency because of abnormalities of endothelial function or collagen-platelet interactions. specific diseases involving abnormal structure or function of hematopoietic or hemostatic elements are discussed later in this chapter. the cbc provides basic information about platelets, including numeric values for platelet concentration and mean platelet volume (mpv), subjective assessment of platelet morphologic features (size, shape, and granularity), and a rough estimation of platelet numbers based on examination of a blood smear. some laboratories measure reticulated platelets (platelets recently released from the bone marrow), although this test is mostly used in the research setting at present. increased mpv and increased numbers of reticulated platelets tend to indicate increased thrombopoiesis. bone marrow examination is indicated with any unexplained cytopenia, including thrombocytopenia, to evaluate production. tests to evaluate the components of the hemostatic process are described and listed in e-appendix 13-1. secondary myelofibrosis is the enhanced deposition of collagen within the marrow by nonneoplastic fibroblasts and reticular cells. disease pathogenesis is unclear, but there are two leading theories. first, it may represent scar formation after marrow necrosis, as previously presented. and second, high concentrations of growth factors present during times of marrow injury or activation may stimulate fibroblast proliferation. in particular, stimulated megakaryocytes and macrophages produce fibrogenic cytokines, including plateletderived growth factor, transforming growth factor-β, and epidermal growth factor. early in disease there is reticulin deposition without reduction of hematopoietic elements. however, fibrous collagen replaces hematopoietic cells with disease progression. histologic identification of reticulin and collagen fibers can be aided with reticulin silver and masson's trichrome stains, respectively. in animals, secondary myelofibrosis occurs most commonly with leukemias, extramarrow malignancies, and chronic hemolytic anemias, but many cases are idiopathic. experimental whole-body gamma irradiation, dietary strontium-90 exposure, and certain drugs and toxins can also induce myelofibrosis. the responses of marrow adipocytes to systemic and localized disease are under current investigation, especially in relation to energy metabolism, inflammation, and bone trauma. during times of severe energy imbalance, such as cachexia, the marrow may undergo serous atrophy of fat, also known as gelatinous marrow transformation (efig. 13 -1). the pathogenesis of this phenomenon is unknown, but it is characterized by adipocyte atrophy, hematopoietic cell hypoplasia with subsequent cytopenias, and replacement of the marrow with extracellular hyaluronic acid-rich mucopolysaccharides. positive alcian blue staining identifies the extracellular material as mucin. marrow adipocytes secrete adipose-derived hormones, termed adipokines, including leptin and adiponectin. in general, leptin is proinflammatory, prothrombotic, and mitogenic for various cell types, including lymphocytes, hematopoietic progenitors, and leukemic cells. conversely, adiponectin has antiinflammatory and growth inhibitory properties. during times of inflammation and infection, leptin production is increased. in response to marrow trauma, such as orthopedic surgery, fat may enter the vasculature, embolize to various tissues, and cause tissue ischemia. the severity of tissue injury caused by fat embolism is dependent upon the quantity of fat entering circulation and the tissue's susceptibility to ischemia (see chapter 2). responses of circulating blood cells to injury include decreased survival (destruction, consumption, or loss), altered distribution, and altered structure or function (see box 13-1). these responses are not mutually exclusive-for example, altered erythrocyte structure may lead to decreased survival. often, but not always, these responses result in decreased concentrations of blood cells in circulation. abnormal concentrations of blood cells. the concentration of blood cells may be decreased, termed cytopenia (from kytos [gr., hollow vessel] and penia [gr., poverty]) or increased, designated cytosis (from osis [gr., condition]). a specific blood cell type is denoted as being decreased by using the suffix -penia (table 13-1) . a decreased concentration of erythrocytes is the exception and is termed anemia (from a [gr., without] and haima [gr., blood]). decreased concentrations of blood basophils are not recognized in domestic animals because the lower reference interval is typically zero. an increased blood cell type is denoted with the suffix -osis or -philia (see table 13 -1). postmortem quantification of blood cell insults to extramarrow tissues and cells tend to cause increased production of the involved cell types (hyperplasia) with or without dysplasia. loss of erythrocytes from blood vessels (hemorrhage), or premature destruction of erythrocytes (hemolysis) causes erythroid hyperplasia. tissue inflammation may cause neutrophilic, eosinophilic, basophilic, and/or monocytic hyperplasia, depending on the type of inflammation. megakaryocytic hyperplasia may occur with increased platelet use during hemorrhage or disseminated intravascular coagulation (dic) or with immune-mediated platelet destruction. exceptions to these generalizations, such as anemia of chronic disease, iron deficiency anemia, and anemia of renal failure, are discussed in more detail later. endothelial cell response to injury specifically within the marrow is poorly characterized, but it is likely similar to that of endothelial cells elsewhere, playing active roles in coagulation and inflammation (see chapters 2 and 3). however, one potential sign of marrow sinusoidal injury is the presence of circulating nucleated erythrocytes in the absence of erythrocyte regeneration, termed inappropriate metarubricytosis. it is proposed that injured marrow endothelial cells allow premature passage of metarubricytes into blood circulation during times of stress. however, a conflicting theory proposes that marrow stress causes decreased metarubricyte attachment to central macrophages, and subsequent release into circulation. specific causes of marrow injury-induced metarubricytosis include sepsis, hyperthermia, malignancies, hypoxia, and certain drugs and toxins. inappropriate metarubricytosis may also occur with erythroid dysplasia and splenic disorders. in addition to a suspected role in inappropriate metarubricytosis, marrow macrophages are integral to altered iron metabolism, including anemia of chronic disease and hemosiderosis. anemia of chronic disease is a mild to moderate nonregenerative anemia observed in animals with a variety of inflammatory and metabolic disorders. this anemia is discussed in more detail later, but briefly, it is primarily a result of iron sequestration within macrophages. hemosiderosis is the excessive accumulation of iron in tissues, typically macrophages. accumulation of iron in parenchymal organs, leading to organ toxicity, is termed hemochromatosis. in animals, iron overload due to blood transfusions or chronic hemolytic anemias may cause marrow hemosiderosis and hemochromatosis. myelitis can take different forms. granulomatous myelitis occurs with systemic fungal infections (e.g., histoplasmosis) or mycobacteriosis. acute or neutrophilic myelitis may occur with lower-order bacterial infections or those with an immune-mediated component. dogs and cats with nonregenerative immune-mediated hemolytic anemia (imha) often have myelitis, in addition to myelofibrosis and necrosis. the inflammation is evident as fibrin deposition, edema, and multifocal neutrophilic infiltrates; immune-mediated cytopenias may also concurrently occur with bone marrow lymphocytic and/or plasma cell hyperplasia. bone marrow necrosis is the necrosis of medullary hematopoietic cells, stromal cells, and stroma in large areas of bone marrow. potential causes include leukemias, extramarrow malignancies, infection (bovine viral diarrhea virus [bvdv] , ehrlichia canis, and feline leukemia virus [felv]), sepsis, drugs or toxins (carprofen, chemotherapeutic agents, estrogen, metronidazole, mitotane, and phenobarbital), and irradiation. direct hematopoietic or stromal cytotoxicity and altered marrow microvasculature (disseminated intravascular coagulation) are proposed pathogeneses. extensive marrow necrosis results in decreased hematopoiesis and subsequent blood cytopenias, including anemia, neutropenia, and thrombocytopenia. if the animal survives the initial insult, the marrow may recover and resume normal hematopoiesis, or it may undergo scar formation, termed myelofibrosis. concentrations is not possible due to perimortem coagulation. however, a complete blood count (cbc) with microscopic blood smear evaluation is the foundation for antemortem assessment of blood cells. anemia. anemia causes clinical signs referable to decreased red hemoglobin pigment (e.g., pale mucous membranes), decreased oxygen-carrying capacity (e.g., depression, lethargy, weakness, and exercise tolerance), and decreased blood viscosity (e.g., heart murmur). recumbency, seizures, syncope, or coma may occur with severe anemia. anemia is confirmed by identifying a decreased hemoglobin concentration or reduced erythrocyte mass, as measured by the packed-cell volume, hematocrit, or red blood cell concentration. the three general causes of anemia are blood loss (hemorrhage), red blood cell destruction or lysis (hemolysis), and decreased red blood cell production (erythroid hypoplasia). classifying anemia as regenerative or nonregenerative is clinically useful because it provides information about the mechanism of disease; regenerative anemia indicates hemorrhage or hemolysis, whereas erythroid hypoplasia or aplasia causes nonregenerative anemia (table 13 -2). the hallmark of regenerative anemias, except in horses, is reticulocytosis (i.e., increased numbers of circulating reticulocytes [immature erythrocytes]), which is evident as polychromasia on a routinely stained blood smear (see fig. 13 -5). reticulocytosis indicates increased bone marrow erythropoiesis ( fig. 13-7) and release of erythrocytes before they are fully mature. reticulocytosis is an appropriate marrow response to anemia and is often seen with hemorrhage or hemolysis. on a cbc a strong regenerative response may produce an increased mean cell volume (mcv) and decreased mean cell hemoglobin concentration (mchc) because reticulocytes are larger and have a lower hemoglobin concentration than mature erythrocytes. horses are an exception to this classification scheme because they do not release reticulocytes into circulation, even with erythroid hyperplasia. horses with a regenerative response may have an increased mcv and red cell distribution width (an index of variation in cell size). but definitive determination of regeneration in a horse requires demonstration of erythroid hyperplasia via bone marrow examination or an increasing red cell mass over sequential cbcs. in addition to reticulocytosis there may be increased numbers of nucleated red blood cells (nrbcs) in circulation with erythrocyte regeneration, termed appropriate metarubricytosis. when nrbcs are present as part of a regenerative response, they should be in low numbers relative to the numbers of reticulocytes. however, the presence of circulating nrbcs is not in itself definitive evidence of regeneration and may signify dyserythropoiesis (e.g., lead poisoning or bone marrow disease) or splenic dysfunction. these processes should be suspected when nrbcs are increased without reticulocysuch as into the peritoneal cavity, because iron is not lost from the body and can be reused for erythropoiesis. in hemolytic anemia, erythrocytes are destroyed at an increased rate. whether the mechanism is intravascular or extravascular, or a combination, depends on the specific disease process (specific diseases are discussed later in this chapter). some clinical indicators of hemolytic anemia and their pathogeneses are summarized in fig. 13 -9 and are further described in the following discussion. a classic sequela of hemolytic anemias in general is hyperbilirubinemia, which is an increase in the plasma bilirubin concentration. bilirubin is a yellow pigment, which explains why hyperbilirubinemia, if severe enough, causes icterus-the grossly visible yellowing of fluid or tissues ( fig. 13-10) . icterus, also known as jaundice, is usually detectable when the plasma bilirubin concentration exceeds 2 mg/dl. however, it is important to note that hyperbilirubinemia and icterus are not pathognomonic for hemolysis and may also occur with conditions of impaired bile flow (cholestasis), such as hepatopathy or cholangiopathy. in addition to icterus, hemolytic anemia often results in splenomegaly , which is secondary to extravascular hemolysis and macrophagic hyperplasia within the spleen, as well as splenic emh. splenomegaly may also occur in other conditions, as discussed elsewhere in this chapter. intravascular hemolysis is grossly evident as pink-tinged plasma or serum, termed hemolysis or hemoglobinemia. hemolysis is not apparent until the concentration of extracellular hemoglobin is greater than approximately 50 mg/dl. cell-free hemoglobin is scavenged by haptoglobin until haptoglobin becomes saturated with hemoglobin at a concentration of approximately 150 mg/dl. when haptoglobin is saturated, any remaining free hemoglobin has a low enough molecular weight to pass through the renal glomerular filter into the urine. this imparts a pink or red discoloration to the urine, called hemoglobinuria. thus extracellular hemoglobin can cause gross discoloration of the plasma, where it is bound to haptoglobin, before becoming grossly visible in urine. the half-life of haptoglobin is markedly decreased when bound to hemoglobin, so when large amounts of haptoglobin-hemoglobin complex are formed, the concentration of haptoglobin in the blood decreases and hemoglobin can pass through the glomerulus at even lower concentrations. hemoglobinuria is a contributing factor in the renal tubular necrosis (hemoglobinuric nephrosis) that often occurs in cases of acute intravascular hemolysis (see chapter 11). a similar lesion occurs in the kidneys of individuals with marked muscle damage and resulting myoglobinuria (see chapters 11 and 15). hemoglobinuria cannot be distinguished grossly from hematuria (erythrocytes in the urine) or myoglobinuria (myoglobin in the urine), and all three processes cause a positive reaction for "blood protein" on urine test strips. comparing the colors of the plasma and the urine may be informative. in contrast to hemoglobin, myoglobin causes gross discoloration of the urine before the plasma is discolored. this is because myoglobin is a low-molecular-weight monomer, freely filtered by the glomerulus, and does not bind plasma proteins to a significant degree. hematuria can be distinguished from hemoglobinuria on the basis of microscopic examination of urine sediment (i.e., erythrocytes are present in cases of hematuria). in addition to red plasma and urine, hemoglobinemia may also be identified by increased mch or mchc values on a cbc. this is because the hemoglobin concentration is measured by lysing all erythrocytes in the sample and then measuring the total hemoglobin via spectrophotometry. by this method, hemoglobin that originated within or outside of erythrocytes is measured together. however, calculations for mch and mchc, which include results for the hemoglobin and red blood cell concentrations, assume that all of tosis, or their numbers are high relative to the degree of reticulocytosis, termed inappropriate metarubricytosis. in ruminants, reticulocytosis is often accompanied by basophilic stippling (fig. 13-8) . however, like metarubricytosis, basophilic stippling without reticulocytosis is concerning for lead poisoning or other causes of dyserythropoiesis. recall that the stimulus for increased erythropoiesis is increased secretion of epo in response to tissue hypoxia. although the action of epo on erythropoiesis is rapid, evidence of a regenerative response is not immediately apparent in a blood sample. one of the main effects of epo is to expand the pool of early-stage erythroid precursors, and it takes time for these cells to differentiate to the point where they are released into circulation. in a case of acute hemorrhage or hemolysis, for example, it typically takes 3 to 4 days until reticulocytosis is evident on the cbc and several more days until the regenerative response peaks. the term preregenerative anemia is sometimes used to describe anemia with a regenerative response that is impending but not yet apparent on the cbc. confirming a regenerative response in such cases requires either evidence of erythroid hyperplasia in the bone marrow or emergence of a reticulocytosis on subsequent days. hemorrhage results in escape of erythrocytes and other blood components, such as protein, from the vasculature. as a result, a decreased plasma or serum protein concentration, termed hypoproteinemia, may be evident on a cbc or chemistry panel. if the hemorrhage is into the gastrointestinal lumen, some of the protein may be resorbed and converted to urea, resulting in an increased urea nitrogen concentration relative to creatinine in plasma. hemorrhage within the urinary tract may cause red urine with erythrocytes observed in the urine sediment. causes of hemorrhage include trauma, abnormal hemostasis, certain parasitisms, ulceration, and neoplasia. hemorrhage may be acute or chronic, or internal or external. during acute hemorrhage, there are ample iron stores within the body for hemoglobin synthesis and erythrocyte regeneration. however, with chronic external hemorrhage, continued loss of iron may deplete the body's iron stores. as iron stores diminish, so does erythrocyte regeneration, eventually leading to iron deficiency anemia. iron deficiency anemia is either poorly regenerative or nonregenerative and is discussed in more detail later in the chapter. iron deficiency anemia does not occur with chronic internal hemorrhage, spherocytes form when macrophages (mainly in the spleen) phagocytize part of an erythrocyte plasma membrane bound with autoantibody ( fig. 13-12) . the remaining portion of the erythrocyte assumes a spherical shape, thus preserving maximal volume. this change in shape results in decreased deformability of the cells. erythrocytes must be extremely pliable to traverse the splenic red pulp and sinusoidal walls; spherocytes therefore tend to be retained in the spleen in close association with macrophages with risk for further injury and eventual destruction. in the dog, spherocytes appear smaller than normal and have uniform staining ( fig. 13-13 , a), in contrast to normal erythrocytes, which have a region of central pallor imparted by their biconcave shape. this difference in staining between spherocytes and normal erythrocytes is not consistently discernible in many other domestic animals (including horses, the hemoglobin originated within erythrocytes. in the case of hemoglobinemia, the excess extracellular hemoglobin may cause an artifactual increase in the calculated mch and mchc. it is important to remember that similar artifactual increases may also occur with lipemia. once hemolytic anemia has been identified, the specific cause for hemolysis should be investigated based on signalment, clinical history, and microscopic blood smear evaluation. the most common causes of hemolytic anemia in domestic animals are immunemediated, infectious, oxidative, and mechanical fragmentation (i.e., microangiopathic) disorders (table 13-3) . spherocytosis and autoagglutination are hallmarks of immunemediated hemolytic anemia, either primary (also known as idiopathic) or secondary to infectious disease, drugs/toxins, or neoplasms. several initiating processes can cause intravascular hemolysis; formation of the complement membrane attack complex is pictured. with intravascular hemolysis, free hemoglobin is release directly into the plasma, where it is scavenged by haptoglobin and hemopexin. when haptoglobin and hemopexin are saturated, the cell-free hemoglobin causes red discoloration of the plasma (hemolysis) and is excreted in the urine (hemoglobinuria; dark red urine). the liver clears haptoglobinhemoglobin and hemopexin-methemoglobin complexes from plasma and converts hemoglobin to unconjugated bilirubin and then conjugated bilirubin. conjugated bilirubin is normally excreted in the bile and then converted to urobilinogen (yellow) and subsequently stercobilinogen (dark brown). however, excessive bilirubin will spill over into the plasma, resulting in hyperbilirubinemia, icteric plasma (if severe enough), and urinary excretion of bilirubin (bilirubinuria; icteric urine). extravascular hemolysis: during extravascular hemolysis, erythrocytes are phagocytized by macrophages, which digest erythrocytes, and convert hemoglobin to unconjugated bilirubin. excessive bilirubin in plasma causes hyperbilirubinemia with or without icteric plasma. unconjugated bilirubin is processed and excreted by the liver (as previously described) and in dogs, the kidney. kidney u-bilirubin cattle, and cats), whose erythrocytes differ from those of the dog in that they are smaller and have less pronounced biconcavity and therefore less pronounced central pallor. autoagglutination occurs because of cross-linking of antibodies bound to erythrocytes (see . autoagglutination is evident macroscopically as blood with a grainy consistency (see fig. 13-13, b) , and microscopically as clusters of erythrocytes (see fig. 13 -13, c). autoagglutination may also result in a falsely increased mcv and decreased red blood cell concentration when clustered cells are mistakenly counted as single cells by automated hematology analyzers. when autoagglutination is present, the packed-cell volume is the most reliable measurement of red blood cell mass. ghost cells are ruptured red blood cell membranes devoid of cytoplasmic contents (see a) . they indicate intravascular hemolysis and may be seen with a variety of hemolytic disorders, including those with immune-mediated, infectious, oxidative, or fragmentation causes. in the case of immune-mediated hemolytic anemia, antibody or complement binds to red blood cell membranes and activates the complement membrane attack complex (see fig. 13 -12). this causes pore formation in the red blood cell membrane and release of cytoplasmic contents into the plasma. ghost cells are eventually cleared from circulation by phagocytic macrophages, mainly within the spleen. oxidative damage to erythrocytes occurs when normal antioxidative pathways that generate reducing agents (such as reduced nicotinamide adenine dinucleotide [nadh] , reduced nicotinamide adenine dinucleotide phosphate [nadph] , and reduced glutathione [gsh]) are compromised or overwhelmed, resulting in hemolytic anemia, abnormal hemoglobin function, or both. hemolysis caused by oxidative damage may be extravascular or intravascular, or a combination. evidence of oxidative damage to erythrocytes may be apparent on blood smear examination as heinz bodies or eccentrocytes or on gross examination as methemoglobinemia. heinz bodies are foci of denatured globin that interact with the erythrocyte membrane. they are usually subtly evident on routine wright-stained blood smears as pale circular inclusions or blunt, rounded protrusions of the cell margin but are readily discernible on smears stained with new methylene blue . cats are particularly susceptible to heinz body formation and may have low numbers of heinz bodies normally. there is no unanimity of opinion, but some clinical pathologists believe that the presence of heinz bodies in up to 10% of all erythrocytes in cats is within normal limits. this predisposition is believed to reflect unique features of the feline erythrocyte, whose hemoglobin has more sulfhydryl groups (preferential sites for oxidative damage) than do erythrocytes of other species and may also have lower intrinsic reducing capacity. it is also possible that the feline spleen does not have as efficient a "pitting" function (splenic structure and function are discussed in more detail later in this chapter). eccentrocytes, evident as erythrocytes in which one side of the cell has increased pallor ( fig. 13-15, a) , are another manifestation of oxidative damage. they form because of cross-linking of total hemoglobin), methemoglobin imparts a grossly discernible chocolate color to the blood. by itself, mechanical fragmentation hemolysis tends to cause mild or no anemia. mechanical fragmentation results from trauma or shearing of erythrocytes within blood vessels. normal erythrocytes may be flowing through abnormal vasculature, such as with heart valve defects, intravascular fibrin deposition (e.g., disseminated intravascular coagulation), vasculitis, or hemangiosarcoma. alternatively, the red blood cells may be particularly fragile within normal blood vasculature, as occurs with iron deficiency. in either instance, microscopic evidence of mechanical fragmentation includes the presence of erythrocyte fragments (schistocytes [see fig. 13 -15, c]), erythrocytes with irregular cytoplasmic projections (acanthocytes), erythrocytes with blister-like projections (keratocytes), or ghost cells (see figs. 13-13, a, 13-15, d, and 13-15, e). membrane proteins, with adhesion of opposing areas of the cell's inner membrane leaflet, and displacement of most of the hemoglobin toward the other side. the fused membranes may fragment off of the eccentrocyte, leaving a slightly ruffled border; this cellular morphologic abnormality is called a pyknocyte (see fig. 13-15, b) . oxidative insult may also result in conversion of hemoglobin (iron in the fe 2+ state) to methemoglobin (iron in the fe 3+ state), which is incapable of binding oxygen. methemoglobin is produced normally in small amounts but reduced back to oxyhemoglobin by the enzyme cytochrome-b 5 reductase (also known as methemoglobin reductase). methemoglobinemia results when methemoglobin is produced in excessive amounts (because of oxidative insult) or when the normal pathways for maintaining hemoglobin in the fe 2+ state are impaired (as in cytochrome-b 5 reductase deficiency). when present in sufficiently high concentration (approximately 10% of degradation. antierythrocyte antibodies bind rbc surface antigens, resulting in rbc opsonization by immunoglobulins (mainly immunoglobulin g [igg] ) and complement (primarily c3b). immunoglobulin-or c3b-bound rbcs are phagocytized and digested by sinusoidal macrophages. 2, spherocytes. spherocytes form when the membrane of immunoglobulin-or c3b-bound rbcs are phagocytized by macrophages, without removing the entire rbc from circulation. compared to normal erythrocytes, spherocytes appear smaller, more eosinophilic, and lack central pallor. 3, rbc aggregation (agglutination). rbc aggregation occurs when antierythrocyte immunoglobulins (immunoglobulin m [igm] or high concentrations of igg) bind multiple erythrocytes simultaneously. 4, ghost cells. antierythrocyte antibodies bind rbc surface antigens, resulting in complement activation and formation of the membrane attack complex (mac). macs form membrane pores, resulting in rupture of rbcs, and the release of hemoglobin into the circulation. ghost cells are rbc membrane remnants that lack cytoplasm (hemoglobin normocytic, normochromic anemia). it has long been known that patients with inflammatory or other chronic disease often become anemic, and that this condition results in increased iron stores in the bone marrow. sequestration of iron may be a bacteriostatic evolutionary adaptation because many bacteria require iron as a cofactor for growth. in recent years, investigators have begun to elucidate the molecular mechanisms underlying anemia of inflammation. hepcidin, an acute phase protein and antimicrobial peptide synthesized in the liver, is a key mediator that limits iron availability. hepcidin expression increases with inflammation, infection, or iron overload and decreases with anemia or hypoxia. hepcidin exerts its effects by causing functional iron deficiency. it binds to and causes the degradation of the cell surface iron efflux molecule, ferroportin, thus inhibiting both absorption of dietary iron from the intestinal epithelium and export of iron from macrophages and hepatocytes into the plasma ( fig. 13-16 ). anemia of inflammation involves factors besides decreased iron availability. inflammatory cytokines are likely to inhibit erythropoiesis by oxidative damage to and triggering apoptosis of developing erythroid cells, by decreasing expression of epo and stem cell factor, and by decreasing expression of epo receptors. in addition, experimentally induced sterile inflammation in cats resulted in shortened erythrocyte survival, indicating that anemia of inflammation is likely also a function of increased erythrocyte destruction. other causes of decreased erythropoiesis are listed in table 13 -2. specific examples of diseases causing nonregenerative anemia by these mechanisms are discussed later in this chapter. neutropenia. neutropenia refers to a decrease in the concentration of neutrophils in circulating blood. neutropenia may be caused by decreased production, increased destruction, altered distribution, or a demand for neutrophils in tissues that exceeds the rate of granulopoiesis. decreased production is evident on bone marrow examination as granulocytic hypoplasia. this usually results from an insult that affects multiple hematopoietic lineages, such as chemical insult, radiation, neoplasia, infection, or fibrosis, but may also be caused by a process that preferentially targets granulopoiesis. in marked contrast to erythrocytes, neutrophils have a very short life span in circulation. once released from the bone marrow, a neutrophil is in the bloodstream only for hours before migrating into the tissues. when neutrophil production ceases, a reserve of mature neutrophils in the bone marrow storage pool may be adequate to maintain normal numbers of circulating neutrophils for a few days; however, after the bone marrow storage pool is depleted, neutropenia rapidly ensues. immune-mediated neutropenia is a rare but recognized condition in domestic animals. bone marrow findings range from granulocytic hypoplasia to hyperplasia, depending on where the cells under immune attack are in their differentiation programs. neutropenia with no evidence of decreased production and in which other causes of neutropenia have been excluded may be a result of destruction of neutrophils before they leave the bone marrow, a condition known as ineffective granulopoiesis. like other forms of ineffective hematopoiesis, this condition is often presumed to be immune mediated; in cats this condition may occur as a result of infection of hematopoietic cells with felv. as presented in the earlier section on granulopoiesis and monocytopoiesis (myelopoiesis), neutrophils within the blood vasculature are in two compartments: a circulating pool, consisting of those cells flowing freely in the blood, and a marginating pool, consisting of those cells transiently interacting with the endothelial surface. (in reality, neutrophils are constantly shifting between these two pools, but the proportion of cells in either pool normally remains fairly schistocytes are the only red blood cell morphologic abnormality specific for mechanical fragmentation because all other morphologic abnormalities can be seen with other disease processes. for example, ghost cells may be observed with other types of hemolysis. nonregenerative anemia is characterized by a lack of reticulocytosis on the cbc; however, reticulocytosis does not occur in horses even in the context of regeneration. most often this is a result of decreased production in the marrow (i.e., erythroid hypoplasia). erythrocytes circulate for a long time, so anemias caused by decreased production tend to develop slowly. the most common form of nonregenerative anemia is known as anemia of inflammation or anemia of chronic disease. in this form of anemia, erythrocytes are decreased in number but are typically normal in size and hemoglobin concentration (so-called c or because of one specifically depressing thrombopoiesis. in either case, decreased thrombopoiesis is evident as megakaryocytic hypoplasia upon bone marrow examination. general causes of decreased hematopoiesis outlined earlier in the sections on anemia and neutropenia also apply to thrombocytopenia. increased platelet destruction due to immune-mediated thrombocytopenia (imtp) is a fairly common disease in dogs and may also occur in other species. thrombocytopenia with immune-mediated thrombocytopenia is often severe (e.g., <10,000 platelets/µl), resulting in spontaneous multisystemic hemorrhage. increased use of platelets occurs with hemorrhage and disseminated intravascular coagulation. thrombocytopenia secondary to hemorrhage is often mild to moderate, whereas disseminated intravascular coagulation may cause mild to severe thrombocytopenia, often with evidence of mechanical fragmentation hemolysis (e.g., schistocytes). disseminated intravascular coagulation is a syndrome in which hypercoagulability leads to increased consumption of both platelets and coagulation factors in the plasma, with subsequent hypocoagulability and susceptibility to bleeding. risk factors for developing disseminated intravascular coagulation include severe inflammation, such as sepsis or pancreatitis, neoplasia, and organ failure. the spleen normally contains a significant proportion of total platelet mass (up to one-third in some species), and abnormalities involving the spleen may result in changes in the number of circulating platelets. for example, splenic congestion may result in platelet sequestration and thrombocytopenia, and splenic contraction may cause thrombocytosis. lymphopenia. lymphopenia refers to a decreased concentration of lymphocytes in blood. it is a common hematologic finding in sick animals. usually the precise mechanism of lymphopenia is not clear but is often presumed secondary to endogenous glucocorticoid excess that occurs with stress. excess glucocorticoids, either endogenous or exogenous, cause an altered distribution of lymphocytes; there is increased trafficking of lymphocytes from blood to lymphoid tissue, and decreased egress of lymphocytes from lymphoid tissue to blood. at higher concentrations of glucocorticoids, lymphocytes are destroyed. other causes of lymphotoxicity include chemotherapeutic agents, radiation therapy, and some infectious agents. lymphopenia may occur with various mechanisms, including loss of lymphocyte-rich lymphatic fluid (e.g., gastrointestinal disease, repeated drainage of chylous effusions), and disruption of the normal lymphoid tissue architecture because constant in any given species.) circulating neutrophils are part of the blood sample collected during routine venipuncture and are thus counted in the cbc, whereas marginating neutrophils are not. pseudoneutropenia refers to the situation in which there is an increased proportion of neutrophils in the marginating pool. this may occur because of decreased blood flow or in response to stimuli, such as endotoxemia, that increase expression of molecules promoting interaction between neutrophils and endothelial cells. this mechanism of neutropenia is rarely observed in clinical practice. neutropenia may also result from increased demand for neutrophils in the tissue. how rapidly such a situation develops depends not only on the magnitude of the inflammatory stimulus but also on the reserve of postmitotic neutrophils in the bone marrow. the size of this reserve, or storage pool, is species dependent. in dogs this pool contains the equivalent of 5 days' normal production of neutrophils. cattle represent the other extreme in that they have a small storage pool and thus are predisposed to becoming neutropenic during times of acute inflammation. horses and cats are somewhere between the two extremes, closer to cattle and dogs, respectively. it stands to reason that the clinical significance of neutropenia because of a supply and demand imbalance is also species dependent. in dogs, neutropenia as a result of inflammation is an alarming finding because it is evidence of a massive tissue demand for neutrophils that has exhausted the patient's storage pool and is exceeding the rate of granulopoiesis in the bone marrow. however in cows, neutropenia is commonly noted in a wide range of conditions involving acute inflammation and does not necessarily indicate an overwhelming demand. eosinopenia/basopenia. eosinopenia and basopenia are decreased concentrations of blood eosinophils and basophils, respectively. in many laboratories, cbc reference values for eosinophils and basophils are as low as zero cells per microliter, precluding detection of eosinopenia or basopenia. when detectable, eosinopenia is often a result of stress (i.e., glucocorticoid mediated). monocytopenia. monocytopenia denotes a decreased concentration of monocytes in blood; it is of little to no pathologic significance by itself. thrombocytopenia. thrombocytopenia refers to a decrease in the concentration of circulating platelets. mechanisms of thrombocytopenia include decreased production, increased destruction, increased consumption, and altered distribution. decreased production may occur because of a condition affecting cells of multiple hematopoietic lineages, including megakaryocytes, is often used interchangeably with erythrocytosis, but technically and for the purposes of this chapter, polycythemia refers to a specific type of leukemia called primary erythrocytosis or polycythemia vera. causes of erythrocytosis are either relative or absolute. relative erythrocytosis results from a fluid deficit or an altered distribution of erythrocytes within the body (i.e., the body's total erythrocyte mass of generalized lymphadenopathy (e.g., lymphoma, blastomycosis). some hereditary immunodeficiencies, such as severe combined immunodeficiency or thymic aplasia, can cause lymphopenia due to lymphoid aplasia. erythrocytosis. an increase in the measured red cell mass above the normal range is known as erythrocytosis. the term polycythemia rarely, an epo-secreting tumor may cause inappropriately elevated levels of epo in the absence of hypoxia. absolute erythrocytosis, whether primary or secondary, causes increased viscosity of the blood, resulting in impaired blood flow and microvasculature distention. affected individuals are at increased risk for tissue hypoxia, thrombosis, and hemorrhage. clinical signs of hyperviscosity syndrome may include erythematous mucous membranes ( fig. 13-17) , prolonged capillary refill time, prominent scleral vessels, evidence of thrombosis or hemorrhage, and secondary signs related to specific organ systems affected (e.g., neurologic and cardiovascular signs). neutrophilia. neutrophilia, an increased blood concentration of neutrophils, occurs in response to a number of different stimuli, which are not mutually exclusive. major mechanisms of neutrophilia are shown in fig. 13-18 . understanding the cbc findings characteristic of these responses is an important part of clinical veterinary medicine. inflammation can result in neutropenia, as discussed earlier, or neutrophilia, as discussed next. however, before moving on to a discussion of inflammatory neutrophilia and the so-called left shift, it is important to mention two other common is not increased). it occurs most frequently with dehydration, when the decreased proportion of water in the blood results in hemoconcentration. it is observed less frequently with epinephrine-mediated splenic contraction, wherein erythrocytes move from the spleen into peripheral circulation. erythrocytosis from splenic contraction occurs to the most pronounced degree in horses and cats, especially in young, healthy animals. absolute erythrocytosis is a true increase in red blood cell mass due to erythroid neoplasia or hyperplasia and includes causes of primary and secondary erythrocytosis. primary erythrocytosis, or polycythemia vera, is a neoplastic proliferation of erythroid cells with a predominance of mature erythrocytes. diagnosis is based on a marked increase in red cell mass (hematocrit in normally hydrated dogs ranges from 65% to >80%), an absence of hypoxemia, an absence of other tumors, and a normal or decreased plasma epo concentration. secondary erythrocytosis refers to epo-mediated erythroid hyperplasia causing an increased red blood cell mass. the erythroid hyperplasia may be an appropriate response to chronic hypoxia, such as occurs with right-to-left cardiac shunts or chronic pulmonary inflammatory mediators, including interleukin-1 (il-1), interleukin-6 (il-6), interferon (inf), and tumor necrosis factor (tnf), cause anemia of inflammatory disease due to oxidative hemolysis, iron sequestration within enterocytes and macrophages, and impaired erythroid responsiveness to erythropoietin (epo). during homeostasis the membrane transport molecule, ferroportin, transports iron from the cytosol to the extracellular space. the iron is then used for various physiologic processes, including hemoglobin production within bone marrow erythroid precursors. during times of inflammation the liver increases production of hepcidin, which binds ferroportin and causes its internalization and lysosomal degradation. with fewer membrane ferroportin molecules, less iron is absorbed from the diet and mobilized from macrophages. rbc, red blood cell. ( of course, neutrophilia may also indicate inflammation, and inflammatory stimuli of varying magnitude and duration produce different patterns of neutrophilia. a classic hematologic finding in patients with increased demand for neutrophils is the presence of immature forms in the blood, known as a left shift. not all inflammatory responses have a left shift, but the presence of a left shift almost always signifies active demand for neutrophils in the tissue. the magnitude of a left shift is assessed by the number of immature cells and their degree of immaturity. the mildest form is characterized by increased numbers of band neutrophils, the immediate predecessor to the segmented neutrophil normally found in circulation. progressively immature predecessors are seen with increasingly severe inflammation. a left shift is considered orderly if the number of immature neutrophils in circulation decreases as they become progressively immature. the term degenerative left shift is sometimes used to describe cases in which the number of immature forms exceeds the number of segmented neutrophils. as with glucocorticoidmediated neutrophilia, the typical magnitude of neutrophilia caused by inflammation varies by species, with dogs having the most pronounced response. it might be useful to think of neutrophil kinetics in terms of a producer-consumer model in which the bone marrow is the factory, and the tissues (where the neutrophils eventually go) are the customers. the bone marrow storage pool is the factory inventory, and the neutrophils in the bloodstream are in delivery to the customer. within the blood vessels, circulating neutrophils are on the highway, and marginating neutrophils are temporarily pulled off to the side of the road. during health, there is an even flow of neutrophils from the factory to the customer. thus the system is in steady state, and neutrophil numbers remain relatively constant and within the normal range. however, disease states may perturb this system at multiple levels. decreased granulopoiesis is analogous to a factory working below normal production level. ineffective granulopoiesis is analogous to goods that are produced at a normal to increased rate but are damaged during manufacturing and never leave the factory. a left shift is analogous to the factory meeting increased customer demand by shipping out unfinished goods. cases of persistent, established inflammation are characterized by bone marrow granulocytic hyperplasia and mature neutrophilia, analogous to a factory that has had time to adjust to increased demand and is meeting it more efficiently by increasing its output. eosinophilia/basophilia. eosinophilia and basophilia are increased concentrations of blood eosinophils and basophils, respectively. they may occur with parasitism, hypersensitivity reactions, paraneoplastic responses (e.g., lymphoma, mast cell neoplasia, or leukemia), and nonparasitic infectious disease. eosinophilia has also been documented with hypoadrenocorticism and rare idiopathic conditions (e.g., hypereosinophilic syndrome). most cases of eosinophilia and basophilia are due to eosinophilic and basophilic hyperplasia within the bone marrow in response to inflammatory growth factors. however, cortisol deficiency is thought to cause eosinophilia in dogs with hypoadrenocorticism. monocytosis. monocytosis is an increased concentration of monocytes in blood. it most commonly occurs with excessive glucocorticoids or inflammation and uncommonly to rarely with monocytic leukemia, immune-mediated neutropenia, and cyclic hematopoiesis. with excessive endogenous or exogenous glucocorticoids, monocytes shift from the marginating pool to the circulating pool. this stress monocytosis is most common in dogs, less frequent in cats, and rare in horses and cattle. inflammatory diseases cause monocytosis by cytokine-mediated monocytic hyperplasia in the bone marrow. causes of neutrophilia: glucocorticoid excess and epinephrine excess. less common causes of neutrophilia, such as leukocyte adhesion deficiency and neoplasia, are discussed later in the chapter. glucocorticoid excess, either because of endogenous production or exogenous administration, results in a cbc pattern known as the stress leukogram, characterized by mature neutrophilia (i.e., increased concentration of segmented neutrophils without immature neutrophils) and lymphopenia, with or without monocytosis and eosinopenia. mechanisms contributing to glucocorticoid-mediated neutrophilia include the following: • increased release of mature neutrophils from the bone marrow storage pool • decreased margination of neutrophils within the vasculature, with a resulting increase in the circulating pool • decreased migration of neutrophils from the bloodstream into tissues the magnitude of neutrophilia tends to be species dependent, with dogs having the most pronounced response (up to 35,000 cells/µl) and in decreasing order of responsiveness, cats (30,000 cells/µl), horses (20,000 cells/µl), and cattle (15,000 cells/µl) having less marked responses. with long-term glucocorticoid excess, neutrophil numbers tend to normalize, whereas lymphopenia persists. epinephrine release results in a different pattern, known as physiologic leukocytosis or excitement leukocytosis, characterized by mature neutrophilia (like the glucocorticoid response) and lymphocytosis (unlike the glucocorticoid response). this phenomenon is short lived (i.e., <1 hour). neutrophilia occurs primarily because of a shift of cells from the marginating to the circulating pool. physiologic leukocytosis is common in cats (especially when they are highly stressed during blood collection) and horses, less common in cattle, and uncommon in dogs. of a regenerative response in patients recovering from thrombocytopenia, as a result of redistribution after splenic contraction, or within the several weeks after splenectomy. in these cases, thrombocytosis is transient. in the case of splenectomy, thrombocytosis may be marked but normalizes after several weeks. because the body's total platelet mass regulates thrombopoiesis, and a significant portion of the platelet mass is normally in the spleen, it makes sense that splenectomized animals develop thrombocytosis. however, the reason that the number of circulating platelets normalizes in these individuals in the weeks after splenectomy is not thrombocytosis. thrombocytosis, or an increased concentration of platelets in the blood, is a relatively common, nonspecific finding in veterinary patients. in the vast majority of cases, thrombocytosis is reactive-a response to another, often apparently unrelated, disease process. examples of conditions having reactive thrombocytosis include inflammatory and infectious diseases, iron deficiency, hemorrhage, endocrinopathies, and neoplasia. factors that may contribute to reactive thrombocytosis include increased plasma concentration of thrombopoietin, inflammatory cytokines (e.g., il-6), or catecholamines. thrombocytosis may also occur as part 1, neutrophils and their precursors are distributed in five pools: a bone marrow precursor pool, which includes mitotically active and inactive immature cells; a bone marrow storage pool, consisting of mitotically inactive mature neutrophils; a peripheral blood marginating pool; a peripheral blood circulating pool; and a tissue pool. the relative size of each pool is represented by the size of its corresponding wedge. the peripheral blood neutrophil count measures only neutrophils within the circulating peripheral blood pool, which can be enlarged by (2) increased demargination, (3) diminished extravasation into tissue, (4) increased release of cells from the marrow storage pool, and (5) cells. the preceding section focused on abnormalities in the number of blood cells. there are also various acquired and congenital conditions involving abnormal structure or function of blood cells. this section briefly discusses abnormal blood cell structure or function occurring secondary to other underlying disease. primary disorders of blood cells are discussed later in the chapter in the section on specific diseases. morphologic abnormalities detected on routine microscopic examination of blood smears may provide important clues about underlying disease processes. poikilocytosis is a broad term referring to the presence of abnormally shaped erythrocytes in circulation. etable 13 -1 lists conditions with and mechanisms involved in the formation of a number of specific types of erythrocyte morphologic abnormalities, and fig. 13-15 shows some examples. the acquired neutrophil morphologic abnormality known as toxic change (fig. 13 -20) reflects accelerated production of neutrophils as part of the inflammatory response. features of toxic change include increased cytoplasmic basophilia, the presence of small bluegray cytoplasmic inclusions known as döhle bodies (often noted incidentally in cats), and in more severe cases, cytoplasmic vacuolation. although not causing impaired neutrophil function, toxic change occurs during granulopoiesis and thus is technically a form of dysplasia (e.g., döhle bodies are foci of aggregated endoplasmic reticulum). toxic change may accompany any inflammatory response, but in general the more marked the toxic change, the higher the index of suspicion for infection or endotoxemia. other secondary changes to neutrophils may not be evident morphologically. for example, studies in human beings and dogs have shown that individuals with cancer have abnormal neutrophil function (including phagocytic activity, killing capacity, and oxidative burst activity) before initiation of therapy. the clinical significance of this finding is not clear. platelet function disorders, also known as thrombopathies or thrombopathias, may be primary or secondary. many conditions are known or suspected to cause secondary platelet dysfunction (hypofunction or hyperfunction) by altering platelet adhesion or aggregation or by mechanisms that are not fully understood. box 13-2 shows underlying conditions having secondary platelet dysfunction. clear. there is also a rare form of megakaryocytic leukemia known as essential thrombocythemia, which is characterized by marked thrombocytosis. lymphocytosis. lymphocytosis refers to an increase in the concentration of lymphocytes in blood circulation. there are several causes of lymphocytosis, including age, excessive epinephrine, chronic inflammation, hypoadrenocorticism, and lymphoid neoplasia; lymphoid neoplasms are presented later in the chapter. young animals normally have higher concentrations of lymphocytes than older animals, and normal healthy young animals may have counts that exceed adult reference values. because this is not pathologic lymphocytosis, but normal physiologic variation, it is often termed pseudolymphocytosis of young animals. as discussed earlier in the section on neutrophilia, lymphocytosis is also a feature of epinephrine-mediated physiologic leukocytosis, resulting from redistribution of lymphocytes from the blood marginating pool into the blood circulating pool. epinephrine-mediated lymphocytosis may be more marked than neutrophilia, particularly in cats (lymphocyte counts of > 20,000/µl are not uncommon). antigenic stimulation may result in lymphocytosis, which may be marked in rare cases (up to approximately 30,000/µl in dogs and 40,000/µl in cats); however, this is not usually the case, even when there is clear evidence of increased immunologic activity in lymphoid tissues. in cases of antigenic stimulation, it is common for a minority of lymphocytes to have "reactive" morphologic features-larger lymphocytes with more abundant, deeply basophilic cytoplasm and more open chromatin ( fig. 13-19 ). just as glucocorticoid excess can cause lymphopenia, glucocorticoid deficiency (hypoadrenocorticism) can cause lymphocytosis, or lack of lymphopenia during conditions of stress that typically result in glucocorticoid-mediated lymphopenia. a condition known as persistent lymphocytosis (pl) occurs in approximately 30% of cattle infected with the bovine leukemia virus (blv). the condition is defined as an increase in the blood concentration of lymphocytes above the reference interval for at least 3 months. this form of lymphocytosis is a nonneoplastic proliferation (i.e., hyperplasia) of b lymphocytes. in the absence of other disease, cattle with persistent lymphocytosis are asymptomatic. however, cattle infected with blv, especially those animals with persistent lymphocytosis, are at increased risk for developing b lymphocyte lymphoma. aplastic anemia (aplastic pancytopenia) aplastic anemia, or more accurately aplastic pancytopenia, is a rare condition characterized by aplasia or severe hypoplasia of all hematopoietic lineages in the bone marrow with resulting cytopenias. the term aplastic anemia is a misnomer because affected cells are not limited to the erythroid lineage. many of the conditions reported to cause aplastic anemia do so only rarely or idiosyncratically; more frequently, they cause other hematologic or nonhematologic abnormalities. a partial list of reported causes of aplastic anemia in domestic animals includes the following: most of these causes, especially the chemical agents, are directly cytotoxic to hscs or progenitor cells, resulting in their destruction. however, another proposed mechanism is disruption of normal stem cell function because of mutation or perturbation of hematopoietic cells and/or their microenvironment. this pathogenesis is mostly recognized in retroviral infections. aplastic anemia occurs in both acute and chronic forms. most of the chemical causes result in acute disease. grossly, affected animals may show signs of multisystemic infection and hemorrhage due to severe neutropenia and thrombocytopenia, respectively. severe neutropenia typically develops within 1 week of an acute insult to the bone marrow, and severe thrombocytopenia occurs in the second week. this sequence is a result of the circulating life spans of each cell type; in health, neutrophils have a blood half-life of 5 to 10 hours, whereas platelets circulate for 5 to 10 days. the development of signs of anemia, such as pale mucous membranes, is more variable. the presence and severity of anemia depends on how rapidly the marrow recovers from the insult and the erythrocyte life span of the particular species. microscopically, bone marrow is hypocellular with markedly reduced hematopoietic cells. hematopoietic cells are replaced with adipose tissue, and there is a variable inflammatory infiltrate of lymphocytes, plasma cells, and macrophages. in addition, there may be necrosis, hematopoietic cell apoptosis, and an increase in phagocytic macrophages. fig. 13 -21 shows bone marrow aspirates from a dog with pancytopenia from acute 5-fluorouracil toxicosis, before and during recovery. many inherited or presumably inherited disorders of blood cells have been recognized in domestic animals, including rare or sporadic cases and conditions that are of questionable clinical relevance. this section and the later sections covering species-specific disorders are invading cells or microorganism gain access to the bone marrow or blood circulation either hematogenously or by trauma. trauma may be as obvious as a gaping wound or as subtle as the bite of an insect. portals of entry for the bone marrow are summarized in box 13-3. diseases that arise from the bone marrow, such as leukemia, typically spread to other tissues hematogenously. the bone marrow is encased by a protective shell of cortical bone, and blood supply to the marrow provides access to systemic humoral and cellular defenses. of course, leukocytes themselves function as an essential part of inflammation and immune function, as discussed briefly in the section on granulopoiesis and monocytopoiesis (myelopoiesis) and in greater detail in chapters 3 and 5. biochemical steps in the glycolytic pathway or linked to it generate antioxidant molecules that enable erythrocytes to withstand oxidative insults throughout their many days in circulation. in addition to producing energy in the form of adenosine triphosphate (atp), glycolysis generates nadh, which helps convert the oxidized, nonfunctional form of hemoglobin, known as methemoglobin, back to its active, reduced state. another antioxidant erythrocyte metabolic pathway, the pentose shunt or hexose uremia antiplatelet antibodies (also cause immune-mediated thrombocytopenia) infection (bvdv, felv) hyperglobulinemia increased fibrinolytic products hypoammonemia snake envenomation platelet inhibitors nsaids-irreversible (aspirin) or reversible inhibition of cyclooxygenase colloidal plasma expanders (e.g., hydroxyethyl starch) other drugs and exogenous agents (many) hematogenously direct penetration (trauma) including hypercellular glomeruli, thickened glomerular and tubular basement membranes, and tubular epithelial lipidosis, degeneration, and necrosis. other porphyrias have been diagnosed in cattle, pigs, and cats but are not known to cause hemolytic anemia. pyruvate kinase deficiency. pyruvate kinase (pk) deficiency is an inherited autosomal recessive condition due to a defective r-type pk isoenzyme that is normally present in high concentrations in mature erythrocytes. to compensate for this deficiency, there is persistence of the m2-type pk isoenzyme, which is less stable than the r-type isoenzyme. the disease is reported in many dog breeds and fewer cat breeds (e.g., abyssinian, somali, and domestic shorthair). erythrocyte pk deficiency results in decreased atp production and shortened erythrocyte life spans. in dogs the hemolytic anemia is typically chronic, moderate to severe, extravascular, and strongly regenerative. with chronicity, hemolytic anemia causes enhanced intestinal absorption of iron and subsequent hemosiderosis, especially of the liver and bone marrow. dogs typically die at 1 to 5 years of age of hemochromatosis-induced liver and bone marrow failure. however, cats with pk deficiency typically show no clinical signs, have milder anemia, and do not develop organ failure. grossly, affected animals have lesions attributed to hemolytic anemia, including splenomegaly, pale mucous membranes, and rarely icterus. dogs with end-stage disease have cirrhosis, myelofibrosis, and osteosclerosis. dogs with pk deficiency do not necessarily have the same genetic defect, so mutation-specific dna-based assays are required. in contrast, a single dna-based test is available to detect the common mutation affecting abyssinian, somali, and domestic shorthair cats. cytochrome-b 5 reductase deficiency. deficiency of cytochrome-b 5 reductase (cb5r, also known as methemoglobin reductase), the enzyme that catalyzes the reduction of methemoglobin (fe 3+ ) to hemoglobin (fe 2+ ), has been recognized in many dog breeds and in domestic shorthair cats. it is probably an autosomal recessive trait. affected animals may have cyanotic mucous membranes or exercise intolerance but usually lack anemia and clinical signs of disease. life expectancies are normal. glucose-6-phosphate dehydrogenase deficiency. deficiency of glucose-6-phosphate dehydrogenase (g6pd), the ratecontrolling enzyme of the pentose phosphate pathway (ppp), has been reported in an american saddlebred colt, its dam, and one male dog. the ppp is an antioxidative pathway that generates nadph, which maintains glutathione in its reduced form (gsh). therefore in animals with g6pd deficiency, oxidants are not scavenged, and erythrocyte oxidative injury occurs. the colt with g6pd deficiency had severe oxidative hemolytic anemia with eccentrocytes on blood smear evaluation. however, the colt's dam only had eccentrocytes, and showed no hematologic signs of disease. leukocyte adhesion deficiency. leukocyte adhesion deficiency (lad) is a fatal autosomal recessive defect of leukocyte integrins, in particular the β 2 chain (also known as cluster of differentiation [cd] 18 [cd18]). disease has been recognized in holstein cattle (known as bovine leukocyte adhesion deficiency [blad] ) and irish setter dogs (known as canine leukocyte adhesion deficiency [clad]) (see chapter 3). without normal expression of this adhesion molecule, leukocytes have severely impaired abilities to migrate from the blood into tissues. as a result, animals with leukocyte adhesion deficiency have marked neutrophilia with nonsuppurative multisystemic infections. blood neutrophils often have nuclei with greater than five nuclear segments, termed not comprehensive but instead focus on the more common, wellcharacterized, or recently reported conditions. erythropoietic porphyrias. porphyrias are a group of hereditary disorders in which porphyrins accumulate in the body because of defective heme synthesis. inherited enzyme defects in hemoglobin synthesis have been identified in holstein cattle, siamese cats, and other cattle and cat breeds, resulting in bovine congenital erythropoietic porphyria and feline erythropoietic porphyria, respectively. accumulation of toxic porphyrins in erythrocytes causes hemolytic anemia, whereas accumulation of porphyrins in tissues and fluids produces discoloration, including red-brown teeth, bones, and urine (see fig. 1 -59). because of the circulation of the photodynamic porphyrins in blood, these animals have lesions of photosensitization of the nonpigmented skin. all affected tissues, including erythrocytes, exhibit fluorescence with ultraviolet light. histologically, animals may exhibit perivascular dermatitis, as well as multisystemic porphyrin deposition, hemosiderosis, emh, and marrow erythroid hyperplasia. cats may show evidence of renal disease, a b syndrome exhibit oculocutaneous albinism (due to altered distribution of melanin granules) and are prone to infection and bleeding. blood smear evaluation reveals granulocytes with large cytoplasmic granules. glanzmann thrombasthenia. glanzmann thrombasthenia (gt) is an inherited platelet function defect caused by a mutated α iib subunit of the integrin α iib β 3 (also known as glycoprotein iib-iiia [gpiib-iiia]). the disorder has been recognized in great pyrenees and otterhound dogs and several horse breeds, including a quarter horse, a standardbred, a thoroughbred-cross, a peruvian paso mare, and an oldenburg filly. the α iib β 3 molecule has multiple functions but is best known as a fibrinogen receptor that is essential for normal platelet aggregation. bleeding tendencies vary widely between affected individuals but mainly occur on mucosal surfaces. the condition is characterized by an in vitro lack of response to all platelet agonists and severely impaired clot retraction (i.e., whole blood samples without anticoagulant often fail to clot). molecular testing is available to detect diseased or carrier states in dogs and horses. thrombopathia. calcium diacylglycerol guanine nucleotide exchange factor i (caldag-gefi) is a molecule within the signaling pathway that results in platelet activation in response to platelet agonists. mutated caldag-gefi has been documented in basset hound, eskimo spitz, and landseer dogs, and simmental cattle. all reported mutations have a bleeding tendency. in vitro platelet aggregation responses to platelet agonists, such as adp, collagen, and thrombin, are absent or impaired. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. oxidative agents. a variety of oxidative toxins cause hemolytic anemia and/or methemoglobinemia in domestic species. more common or well-characterized oxidants are listed here: • horses-acer rubrum (red maple) • ruminants-brassica spp. (cabbage, kale, and rape), copper hypersegmented neutrophils, due to neutrophil aging within blood vessels ( fig. 13-22 ). these animals are highly susceptible to infections and usually die at a young age. pelger-huët anomaly. pelger-huët anomaly (pha) is a condition of hyposegmented granulocytes due to a lamin b receptor mutation. it has been described in dogs, cats, horses, and rabbits, especially in certain breeds. in australian shepherd dogs the mode of inheritance is autosomal dominant with incomplete penetrance. most cases of pelger-huët anomaly are the heterozygous form and of no clinical significance. however, skeletal abnormalities, stillbirths, and/or early mortality may accompany pelger-huët anomaly in rabbits and cats, especially homozygotes. in pelger-huët anomaly the nuclei of neutrophils, eosinophils, and basophils fail to segment, resulting in band-shaped, bean-shaped, or round nuclei. although the nuclear shape is similar to that of an inflammatory left shift, healthy animals with pelger-huët anomaly do not have clinical signs or other laboratory findings indicating inflammation. for example, neutrophils in healthy animals with pelger-huët anomaly have mature (clumped) chromatin and do not show signs of toxicity ( fig. 13-23 ). an acquired, reversible condition mimicking pelger-huët anomaly, known as pseudo-pelger-huët anomaly, is occasionally noted in animals with infectious disease, neoplasia, or drug administration. chédiak-higashi syndrome. chédiak-higashi syndrome (chs) is a rare autosomal recessive defect in the lysosomal trafficking regulator (lyst) protein. the syndrome has been identified in hereford, brangus, and japanese black cattle, persian cats, and several nondomestic species. the defective lyst protein results in granule fusion in multiple cell types, including granulocytes, platelets, and melanocytes, as well as abnormal cell function. individuals with chédiak-higashi syndrome have severely impaired cellular innate immunity because of neutropenia, impaired leukocyte chemotaxis, and impaired killing by granulocytes and cytotoxic lymphocytes. platelets lack the dense granules that normally contain key bioactive molecules involved in hemostasis, including platelet agonists, such as adp and serotonin. in vitro platelet aggregation is severely impaired. as a result, animals with chédiak-higashi 746.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system von willebrand disease (vwd) is the most common canine hereditary bleeding disorder and has also been described in many other domestic species. the disease actually refers to a group of inherited conditions characterized by a quantitative or qualitative deficiency of vwf. this factor is a multimeric glycoprotein that is stored in platelet α-granules and endothelial cells and circulates as a complex with coagulation factor viii. its primary functions are to stabilize factor viii and mediate platelet binding to other platelets and subendothelial collagen. although not technically a platelet disorder, von willebrand disease is often classified as such because it results in a loss of normal platelet function. different types of von willebrand disease vary in terms of mode of inheritance and severity of clinical disease. type i von willebrand disease is characterized by low plasma vwf concentration but normal multimeric proportions and a mild to moderate clinical bleeding tendency; it has been reported in many dog breeds. type ii von willebrand disease is characterized by low vwf concentration, absence of large multimers, and a moderate to severe bleeding tendency; it has been reported in german short-haired pointer and german wirehaired pointer dogs. type iii von willebrand disease is characterized by absence of vwf and a severe bleeding tendency; familial and sporadic cases have been reported in numerous dog breeds. the buccal mucosal bleeding time is prolonged with von willebrand disease, often with adequate concentrations of platelets and normal prothrombin time and partial thromboplastin time (ptt). however, ptt may be mildly prolonged because vwf stabilizes factor viii, and deficiency of vwf results in enhanced factor viii degradation. grossly, affected animals exhibit bleeding tendencies, especially in the form of inherited coagulation factor deficiencies have been documented in most domestic species, including deficiencies of prekallikrein and factors i, ii, vii, viii, ix, x, xi, and xii. of these disorders, hereditary coagulation factor viii (hemophilia a) and factor ix (hemophilia b) deficiencies are most common. hemophilia a has been recognized in horses, cattle, dogs, and cats, and hemophilia b occurs in dogs and cats. both disorders have an x-linked recessive mode of inheritance, meaning that clinical disease is more common in males. affected males have variable tendencies to bleed, depending on the severity of the deficiency, exposure to trauma, and size and activity level of the affected individual. carrier females are usually asymptomatic. laboratory tests often reveal adequate platelets, normal prothrombin times, and prolonged partial thromboplastin times. hereditary defects in γ-glutamyl carboxylase, the enzyme required for normal carboxylation of vitamin k-dependent coagulation factors, have been recognized in a flock of rambouillet sheep and two devon rex cats from the same litter. the genetic defect is not known in cats, but in sheep it is an autosomal recessive trait that results in a premature stop codon and truncated γ-glutamyl carboxylase. in sheep there is increased lamb mortality with excessive bleeding during parturition, especially through the umbilicus or into subcutaneous tissues. gingival bleeding, epistaxis, and hematuria or at sites of injections, venipuncture, or surgery. of loss is through the gastrointestinal tract (e.g., neoplasia in older animals or hookworm infection in puppies). chronic blood loss may also be caused by marked ectoparasitism (e.g., pediculosis in cattle or massive flea burden in kittens and puppies), neoplasia in locations other than the gastrointestinal tract (e.g., cutaneous hemangiosarcoma), coagulation disorders, and repeated phlebotomy of blood donor animals. rapidly growing nursing animals may be iron deficient when compared with adults because milk is an iron-poor diet. in most cases this has little clinical significance (and in fact is normal). an important exception is piglets with no access to iron, which may cause anemia, failure to thrive, and increased mortality. neonatal piglets are routinely given parenteral iron for this reason. copper deficiency can cause iron deficiency in ruminants and may occur because of copper-deficient forage or impaired usage of copper by high dietary molybdenum or sulfate. it is believed that copper deficiency impairs production of ceruloplasmin, a copper-containing enzyme involved in gastrointestinal iron absorption. iron deficiency causes anemia by impaired hemoglobin synthesis. iron is an essential component of hemoglobin, and when it is absent, hemoglobin synthesis is depressed. because erythrocyte maturation is dependent upon obtaining a critical hemoglobin concentration, maturing erythroid precursors undergo additional cell divisions during iron-deficient states. these additional cell divisions result in small erythrocytes, termed microcytes (see fig. 13 -15, g). however, erythrocytes with low hemoglobin concentrations are produced when microcyte formation can no longer compensate for iron deficiency. the classic hematologic picture with iron deficiency anemia is microcytic (i.e., decreased mcv), hypochromic (i.e., decreased mchc) anemia. microcytes and hypochromasia (see fig. 13 -15, g) may also be discernible on blood smear examination as erythrocytes that are abnormally small and paler-staining, respectively. early iron deficiency anemia is poorly regenerative, whereas continued hemorrhage and iron loss cause nonregenerative anemia. additional hematologic changes may include evidence of erythrocyte mechanical fragmentation (e.g., schistocytes) and reactive thrombocytosis. hypophosphatemic hemolytic anemia. marked hypophosphatemia is recognized as a cause of intravascular hemolytic anemia in postparturient dairy cows and diabetic animals receiving insulin therapy. in postparturient cows, hypophosphatemia results from increased loss of phosphorus in their milk. insulin therapy may cause hypophosphatemia by shifting phosphorus from the extracellular space to the intracellular space. in either case, marked hypophosphatemia (e.g., 1 mg/dl in cows, or ≤ 1.5 mg/dl in cats) is thought to decrease erythrocyte production of atp, leading to inadequate energy required for maintenance of membrane and cytoskeletal integrity. an accompanying decrease in reducing capacity and increase in methemoglobin concentration have also been noted in experimental studies of hypophosphatemic hemolytic anemia in dairy cattle, suggesting that oxidative mechanisms may also contribute to anemia. affected animals are anemic and hemoglobinuric. gross postmortem findings include pallor, decreased viscosity of the blood, and lesions arising from the underlying metabolic derangement (e.g., discolored pale yellow and swollen liver due to hepatic lipidosis). renal tubular necrosis and hemoglobin pigment within the tubules is evident microscopically. this section covers infectious agents within the same genus that are recognized to cause disease in multiple species. other infectious agents with more limited host specificity (e.g., cytauxzoonosis in cats, feline and equine retroviruses) are covered in later sections on species-specific diseases. throughout both sections, diseases are • dogs-acetaminophen, propofol, zinc • cats-acetaminophen, propofol, propylene glycol • all species-allium spp. (chives, garlic, and onions) in horses, red maple leaves and bark are toxic, especially wilted or dried leaves. the toxic principle is believed to be gallic acid. plants that contain high concentrations of nitrates, such as cabbage, kale, and rape, may cause oxidative injury to erythrocytes; cattle are more susceptible than sheep and goats. however, sheep are more prone to copper toxicosis relative to other ruminants. the condition occurs in animals that have chronically accumulated large amounts of copper in the liver through the diet. the copper is then acutely released during conditions of stress, such as shipping or starvation. continuous rate infusions of the anesthetic propofol may cause oxidative hemolytic anemia in dogs and cats, but single or multiple single doses are not expected to cause clinical hemolysis. zinc toxicosis has been identified in a wide range of animals; however, it is most common in dogs due to their indiscriminate eating habits. common sources include pennies, batteries, paints, creams, automotive parts, screws, nuts, and coating on galvanized metals. propylene glycol is an odorless, slightly sweet solvent and moistening agent in many foods, drugs, and tobacco products. although it is "generally recognized as safe" for animal foods other than for cats by the food and drug administration, it has been banned from cat food since 1996. grossly and microscopically, animals show varying signs of oxidative hemolysis and/or methemoglobinemia, as previously presented in the section discussing anemias (see bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in sheep with copper toxicosis, hemoglobinuric nephrosis, frequently described as gunmetal-colored kidneys with port wine-colored urine, is a classic postmortem lesion. snake envenomation. hemolytic anemia from snake envenomation has been reported in horses, dogs, and cats. it is most commonly reported with viper and pit viper envenomations, including those from rattlesnakes. hemolysins within viper venom directly injure erythrocytes, causing intravascular hemolysis. other mechanisms of hemolysis include the action of phospholipase a 2 on erythrocyte membranes and erythrocyte mechanical fragmentation due to intravascular coagulation and vasculitis. nonhemolytic lesions depend on the venom's additional components and may include hemorrhage, paralysis, and/or tissue edema, inflammation, and necrosis. on blood smear evaluation, animals with snake envenomation may have ghost cells, spherocytes, and/or echinocytes (see figs. 13-13 and 13-15). information on this topic is available at www.expertconsult.com. severe malnutrition is probably a cause of nonregenerative anemia in all species attributable to combined deficiencies of molecular building blocks, energy, and essential cofactors. by far the most commonly recognized specific deficiency that results in anemia is iron deficiency. other specific nutritional deficiencies causing anemia in animals are uncommon or rare. acquired cobalamin (vitamin b 12 ) and folate deficiencies are recognized as causes of anemia in human beings but are rare in animals. iron deficiency anemia. iron deficiency is usually not a primary nutritional deficiency but rather occurs secondary to depletion of iron stores via chronic blood loss. the most common route 747.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system antagonism of vitamin k leads to production of a nonfunctional form of some coagulation factors and resulting coagulopathy; a similar condition results from vitamin k deficiency. conditions with avitaminosis k include poisoning with coumarin-related molecules, fat malabsorption (vitamin k is a fat-soluble vitamin) caused by primary intestinal disease or impaired biliary outflow (uncommon), dietary deficiency (rare), and antibiotics that interfere with vitamin k absorption or usage. a number of coagulation factors-factors ii, vii, ix, and x (collectively known as the vitamin k-dependent factors), as well as the regulatory molecules protein c and protein s-must undergo carboxylation to be functional. this posttranslational modification is catalyzed by the enzyme γ-glutamyl carboxylase, and requires vitamin k as a cofactor. vitamin k is oxidized during the carboxylation reaction and is converted back into its active reduced form by the enzyme vitamin k epoxide reductase. coumarin-related rodenticides, such as warfarin, act by inhibiting vitamin k epoxide reductase, resulting in an absence of vitamin k in its active reduced form (efig. 13 -2). this inhibition lasts until the rodenticide is metabolized and cleared. how long this takes depends on the half-life of the rodenticide and dose, but it may take many weeks. secondgeneration rodenticides, such as bromadoline and brodifacoum, are more potent than warfarin, with longer half-lives. spoiled sweet clover contains dicumarol, which causes coagulopathy by the same mechanism. efigure 13 -2 mechanism of anticoagulant rodenticide toxicity. anticoagulant rodenticides inhibit the enzyme that converts vitamin k back to its active reduced form. active factors (carboxylated) inactive factors (ii, vii, ix, x) = inactivation of vitamin k epoxide reductase, the enzyme that maintains vitamin k in the active form vitamin k epoxide (inactive) x x laboratory findings include prolonged coagulation times (prothrombin time [pt] , ptt, and activated clotting time [act] ). early in the course of rodenticide and related toxicoses, pt may be the only one of these tests that is prolonged because factor vii has the shortest half-life of the vitamin k-dependent factors. however, the other tests become prolonged as nonfunctional forms of the other factors accumulate. in uncomplicated cases, patients are not thrombocytopenic. a wide range of hemorrhagic lesions may occur in affected individuals, including ecchymoses, epistaxis, gingival bleeding, hematomas, hemoptysis, melena or hematochezia, hematuria, and other forms of hemorrhage. there are also lesions with regenerative anemia, such as pale mucous membranes and splenomegaly. histologically, there is hemorrhage, emh, and marrow erythroid hyperplasia. the treatment of cases of rodenticide and related toxicoses is regular administration of exogenous vitamin k 1 until the toxin is cleared (determined by repeat coagulation testing after withholding treatment). babesiosis may cause intravascular and extravascular hemolytic anemia via direct red blood cell injury, the innocent bystander effect, and secondary immune-mediated hemolytic anemia. infection with highly virulent strains may cause severe multisystemic disease. in these cases, massive immunostimulation and cytokine release cause circulatory disturbances, which may result in shock, induction of the systemic inflammatory response, and multiple organ dysfunction syndromes. babesia organisms can usually be detected on a routine blood smear in animals with acute disease. infected erythrocytes may be more prevalent in capillary blood, so blood smears made from samples taken from the pinna of the ear or the nail bed may increase the likelihood of detecting organisms microscopically. buffy coat smears also have an enriched population of infected erythrocytes. pcr-based tests are the most sensitive assay for detecting infection in animals with very low levels of parasitemia. at necropsy, gross lesions are mainly related to hemolysis and include pale mucous membranes, icterus, splenomegaly, dark red or black kidneys, and reddish-brown urine. the cut surface of the congested spleen oozes blood. the gallbladder is usually distended with thick bile. less common lesions include pulmonary edema, ascites, and congestion, petechiae, and ecchymoses of organs, including the heart and brain. parasitized erythrocytes are best visualized on impression smears of the kidney, brain, and skeletal muscle. microscopic findings in the liver and kidney are typical of a hemolytic crisis and include anemia-induced degeneration, necrosis of periacinar hepatocytes and cholestasis, and hemoglobinuric nephrosis with degeneration of tubular epithelium. erythroid hyperplasia is present in the bone marrow. in animals that survive the acute disease, there is hemosiderin accumulation in the liver, kidney, spleen, and bone marrow. in chronic cases there is hyperplasia of macrophages in the red pulp of the spleen. theileriosis (piroplasmosis). theileria spp. are tick-borne protozoal organisms that infect many domestic and wild animals worldwide. numerous theileria spp. have been documented, but only the more economically or regionally important species are mentioned here. diseases with the greatest economic impact in ruminants are east coast fever (theileria parva infection) and tropical theileriosis (theileria annulata infection). • horses-theileria equi (formerly babesia equi) • cattle-theileria annulata, theileria buffeli, t. parva • sheep and goats-theileria lestoquardi (formerly theileria hirci) like babesiosis, theileriosis is generally restricted to tropical and subtropical regions, including parts of africa, asia, the middle east, and europe. except for t. buffeli, all previously listed species are exotic to the united states. infection is characterized by schizonts within lymphocytes or monocytes, and pleomorphic intraerythrocytic piroplasms (merozoites and trophozoites). within host leukocytes the parasite induces leukocyte cellular division, which expands the parasitized cell population. infected cells disseminate throughout the lymphoid system via the lymphatic and blood vessels. the infected leukocyte may block capillaries, causing tissue ischemia. later in infection some schizonts cause leukocyte lysis and release of merozoites. merozoites then invade and parasitize erythrocytes, causing hemolytic anemia. possible mechanisms of anemia in theileriosis include invasion of erythroid precursors by merozoite stages and associated erythroid hypoplasia (as occurs with t. parva infection), immune-mediated hemolysis, mechanical fragmentation because of vasculitis or microthrombi, enzymatic destruction by proteases, and oxidative damage. gross and microscopic lesions are similar to those of babesiosis, except that cattle with east coast fever tend not to develop organized by taxonomy (protozoal, bacterial and rickettsial, and viral). babesiosis (piroplasmosis). babesia spp. and theileria spp., presented in the next section, are members of the order piroplasmida, and are generally referenced as piroplasms. these organisms are morphologically similar but have different life cycles; babesia spp. are primarily erythrocytic parasites, whereas theileria spp. sequentially parasitize leukocytes and then erythrocytes. both are protozoan parasites spread by ticks, but other modes of transmission are possible (e.g., biting flies, transplacental, and blood transfusions). evidence is accumulating that dog fighting also transmits babesia gibsoni infection. babesia organisms are typically classified as large (2 to 4 µm) or small (<2 µm) with routine light microscopy ( fig. 13-24 ). over 100 babesia species have been identified, some of which are listed here, along with their relative microscopic size in parentheses: geographic distributions vary with the species, but most have higher prevalences in tropical and subtropical regions. for example, equine and bovine babesiosis are endemic in parts of africa, the middle east, asia, central and south america, the caribbean, and europe. both were eradicated from the united states and are now considered exotic diseases in that country. of the previously mentioned species, only agents of canine babesiosis are thought to be endemic in the united states. fever. anaplasmosis, ehrlichiosis, heartwater, and tick-borne fever are tick-borne diseases caused by small, pleomorphic, gramnegative, obligate intracellular bacteria within the order rickettsiaceae, also colloquially known as rickettsias. as a group, rickettsias primarily infect hematopoietic cells and endothelial cells. rickettsias that predominantly infect endothelial cells (e.g., rickettsia rickettsii [rocky mountain spotted fever]), or cause gastrointestinal disease (e.g., neorickettsia helminthoeca [salmon poisoning disease] and neorickettsia risticii [potomac horse fever]) are discussed elsewhere (see chapters 4 and 7). less commonly, transmission may occur via blood transfusions or blood-contaminated medical supplies. rickettsias that infect erythrocytes include the following species (the disease name follows in parentheses): • cattle-anaplasma marginale, anaplasma centrale (bovine anaplasmosis) • sheep and goats-anaplasma ovis (ovine and caprine anaplasmosis, respectively) anaplasma marginale and a. ovis have worldwide distributions, but a. centrale is mostly restricted to south america, africa, and the middle east. hemolytic anemia. in acute east coast fever, lymph nodes are enlarged, edematous, and hemorrhagic. but with chronic cases they may be shrunken. there is often splenomegaly, hepatomegaly, and hemorrhagic enteritis with white foci of lymphoid infiltrates (pseudoinfarcts) in the liver and kidney. microscopically, infected leukocytes may block capillaries. african trypanosomiasis. trypanosomes are flagellated protozoa that can infect all domesticated animals. the most important species that cause disease are trypanosoma congolense, trypanosoma vivax, and trypanosoma brucei ssp. brucei. disease is most common in parts of africa where the biologic vector, the tsetse fly, exists. however, t. vivax has spread to central and south america and the caribbean, where other biting flies transmit the parasite mechanically. in africa, cattle are mainly affected due to the feeding preferences of the tsetse fly. african trypanosomiasis must be distinguished from nonpathogenic trypanosomiasis, such as trypanosoma theileri infection in cattle. animals become infected when feeding tsetse flies inoculate metacyclic trypanosomes into the skin of animals. the trypanosomes grow for a few days, causing a localized chancre sore, and then sequentially enter the lymph nodes and bloodstream. trypanosomal organisms do not infect erythrocytes but rather exist as free trypomastigotes (i.e., flagellated protozoa with a characteristic undulating membrane) in the blood ( fig. 13 -25, a) or as amastigotes in tissue. the mechanism of anemia is believed to be immune mediated. cattle with acute trypanosomiasis have significant anemia, which initially is regenerative, but less so with time. the extent of parasitemia is readily apparent with t. vivax and t. theileri infections because the organisms are present in large numbers in the blood. this is in contrast to t. congolense, which localizes within the vasculature of the brain and skeletal muscle. chronically infected animals often die secondary to poor body condition, immunosuppression, and concurrent infections. gross examination of animals with acute disease often reveals generalized lymphadenomegaly, splenomegaly, and petechiae on serosal membranes. an acute hemorrhagic syndrome may occur in cattle, resulting in lesions of severe anemia (e.g., pale mucous membranes) and widespread mucosal and visceral hemorrhages. main lesions of chronic infections include signs of anemia, lymphadenopathy (e.g., enlarged or atrophied lymph nodes), emaciation, subcutaneous edema, pulmonary edema, increased fluid in body cavities, and serous atrophy of fat. trypanosoma cruzi is the flagellated protozoal agent of american trypanosomiasis. infections have been reported in more than 100 mammal species in south america, central america, and the southern united states, but dogs and cats are among the more common domestic hosts. infected triatomine insects, or "kissing bugs," defecate as they feed on their mammalian host, releasing infective t. cruzi organisms. the parasite then enters the body through mucous membranes or breaks in the skin. like the other trypanosomes described previously, t. cruzi lives in the blood as extracellular trypomastigotes (see fig. 13 -25, b) and in the tissues as intracellular amastigotes. trypanosoma cruzi primarily causes heart disease. lesions of acute disease include a pale myocardium, subendocardial and subepicardial hemorrhages, and yellowish-white spots and streaks. there may also be secondary lesions, such as pulmonary edema, ascites, and congestion of the liver, spleen, and kidneys. in chronic disease the heart may be enlarged and flaccid with thin walls. microscopically, there is often myocarditis and amastigotes within cardiomyocytes. most of these rickettsias have worldwide distributions. however, e. ewingii has been reported only in the united states, and e. ruminantium is endemic only in parts of africa and the caribbean. although a. phagocytophilum has a wide geographic distribution, strain variants are regionally restricted. for example, a. phagocytophilum causes disease in ruminants in europe, but it has not been documented in ruminants in the united states. reservoirs of disease vary, depending upon the rickettsial species. cattle are the reservoir host for e. ruminantium, canids are the reservoir host for a. platys and e. canis, and the other rickettsias have wildlife reservoirs. pathogenesis of disease involves endothelial cell, platelet, and leukocyte dysfunction. those agents that infect endothelial cells cause vasculitis and increased vascular permeability of small blood vessels. if only plasma is lost, then there is hypotension and tissue edema. however, more severe vasculitis causes microvascular hemorrhage with the potential for platelet consumption thrombocytopenia, disseminated intravascular coagulation, and hypotension. infection of platelets may cause thrombocytopenia by direct platelet lysis, immune-mediated mechanisms, or platelet sequestration within the spleen. pathogenesis of leukocyte dysfunction is unclear, but may involve sepsis, inhibited leukocyte function, endothelial cell activation, and platelet consumption. chronic e. canis infection may cause aplastic anemia with pancytopenia by an unknown mechanism. some studies indicate that german shepherd dogs with ehrlichiosis are predisposed to have particularly severe clinical disease. some breeds of cattle (bos taurus), sheep (merino), and goats (angora and saanen) are more susceptible to heartwater. upon blood smear evaluation, thrombocytopenia is the most common hematologic abnormality; anemia and neutropenia occur less frequently. in early stages of infection, blood cells may contain morulae, which are clusters of rickettsial organisms within cytoplasmic, membrane-bound vacuoles ( fig. 13-27 ). examination of buffy coat smears increases the probability of detecting the organism. chronic infection may cause lymphocytosis, particularly of granular lymphocytes. anaplasma platys causes recurrent marked thrombocytopenia. in general, more common gross lesions are splenomegaly, lymphadenomegaly, and pulmonary edema and hemorrhage. more severe cases may also exhibit multisystemic petechiae, ecchymoses, and bovine anaplasmosis causes anemia mainly by immune-mediated extravascular hemolysis. the severity of disease in infected animals varies with age. infected calves under 1 year of age rarely develop clinical disease, whereas cattle 3 years of age or older are more likely to develop severe, potentially fatal, illness. the reason for this discrepancy is not clear. indian cattle (bos indicus) are more resistant to disease than european cattle (bos taurus). surviving cattle become chronic carriers (and thus reservoirs for infection of other animals) and develop cyclic bacteremia, which is typically not detectable on blood smears. splenectomy of carrier animals results in marked bacteremia and acute hemolysis. pcr testing is the most sensitive means of identifying animals with low levels of bacteremia. grossly, acute disease causes lesions of acute hemolytic anemia, including pale mucous membranes, low blood viscosity, icterus, splenomegaly, hepatomegaly, and a distended gallbladder. in animals with acute disease it is usually easy to detect a. marginale organisms on routine blood smear evaluation ( fig. 13-26 ) or impression smears from cut sections of the spleen. however, in recovering animals, the organisms may be difficult to find. rickettsias that infect leukocytes are broadly divided into those that preferentially infect granulocytes ( in addition to anemia, common findings in animals with leptospirosis-induced hemolysis include hemoglobinuria and icterus. on necropsy, renal tubular necrosis, which occurs in part because of hemoglobinuria (hemoglobinuric nephrosis), may also be present. hemotropic mycoplasmosis (hemoplasmosis). the term hemotropic mycoplasmas, or hemoplasmas, encompasses a group of bacteria, formerly known as haemobartonella or eperythrozoon spp., that infect erythrocytes of many domestic, laboratory, and wild animals. hemotropic mycoplasmas affecting common domestic species are as follows: • cattle-mycoplasma wenyonii • camelids-"candidatus mycoplasma haemolamae" • sheep and goats-mycoplasma ovis • pigs-mycoplasma suis (efig. 13-4) • dogs-mycoplasma haemocanis, "candidatus mycoplasma haematoparvum" • cats-mycoplasma haemofelis, "candidatus mycoplasma haemominutum," "candidatus mycoplasma turicensus" like other mycoplasmas, hemoplasmas are small (0.3 to 3 µm in diameter) and lack a cell wall. they are epicellular parasites, residing in indentations and invaginations of red blood cell surfaces. the mode of transmission is poorly understood, but blood-sucking arthropods are believed to play a role; transmission in utero, through biting or fighting, and transfusion of infected blood products are also suspected. effects of infection vary from subclinical to fatal anemia, depending on the specific organism, dose, and host susceptibility. most hemoplasmas are more likely to cause acute illness in individuals that are immunocompromised or have concurrent disease. however, m. haemofelis is an exception and tends to cause acute hemolytic anemia in immunocompetent cats. anemia occurs mainly because of extravascular hemolysis, but intravascular hemolysis also occurs. although the pathogenic mechanisms are not completely understood, an immune-mediated component is highly probable, as well as direct red blood cell injury by the bacteria and the innocent bystander effect. hemotropic mycoplasmas induce cold agglutinins in infected individuals, although it is not clear whether these particular antibodies are important in the development of hemolytic anemia. when detected on routine blood smear evaluation, the organisms are variably shaped (cocci, small rods, or ring forms) and sometimes arranged in short, branching chains ( fig. 13-28) . the organisms may also be noted extracellularly, in the background of the blood smear, especially if the smear is made after prolonged storage of the blood in an anticoagulant tube. in animals dying of acute hemoplasma infection, the gross findings are typical of extravascular hemolysis, with pallor, icterus, splenomegaly, and distended gallbladder ( fig. 13-29 ). additional lesions documented in cattle include scrotal and hind limb edema and swelling of the teats. microscopic lesions in the red pulp of the spleen include congestion, erythrophagocytosis, macrophage hyperplasia, emh, and increased numbers of plasma cells. bone marrow has varying degrees of erythroid hyperplasia, depending on the duration of hemolysis. immune-mediated hemolytic anemia. immune-mediated hemolytic anemia is a condition characterized by increased destruction of erythrocytes because of binding of immunoglobulin to red blood cell surface antigens. it is a common, life-threatening condition in dogs but also has been described in horses, cattle, and cats. immune-mediated hemolytic anemia may be idiopathic (also called edema, cavitary effusions, and effusive polyarthropathy. hydropericardium gives heartwater its name but is more consistently found in small ruminants than in cattle. chronically infected dogs are emaciated. the bone marrow is hyperplastic and red in the acute disease but becomes hypoplastic and pale in dogs with chronic e. canis infection. equine anaplasmosis is often mild but may cause edema and hemorrhages. disease in cats is rare and poorly documented. histologic findings include generalized perivascular plasma cell infiltration, which is most pronounced in animals with chronic disease. multifocal, nonsuppurative meningoencephalitis, interstitial pneumonia, and glomerulonephritis are present in most dogs with the disease. rickettsial organisms are difficult to detect histologically; examination of wright-giemsa-stained impression smears of lung, liver, lymph nodes, and spleen is a more effective method for detecting the morulae within leukocytes. heartwater is often diagnosed by observing morulae in endothelial cells of giemsastained squash preparations of brain. rickettsial diseases are often diagnosed on the basis of serologic testing, but pcr testing is more sensitive. clostridial diseases. certain clostridium spp. may cause potentially fatal hemolytic anemias in animals; nonhemolytic lesions are presented elsewhere (see chapters 4, 7, 8, and 19) . clostridium haemolyticum and clostridium novyi type d cause the disease in cattle known as bacillary hemoglobinuria. (the phrase "red water" has also been used for this disease and for hemolytic anemias in cattle caused by babesia spp.) similar naturally occurring disease has been reported in sheep. in cattle the disease is caused by liver fluke (fasciola hepatica) migration in susceptible animals. ingested clostridial spores may live in kupffer cells for a long time without causing disease. however, when migrating flukes cause hepatic necrosis, the resulting anaerobic environment stimulates the clostridial organisms to proliferate and elaborate their hemolytic toxins, causing additional hepatic necrosis. the mechanism of hemolysis involves a bacterial β-toxin (phospholipase c or lecithinase), which enzymatically degrades cell membranes, causing acute intravascular hemolysis. bacillary hemoglobinuria also occurs with liver biopsies in calves. clostridium perfringens type a causes intravascular hemolytic anemia in lambs and calves-a condition known as yellow lamb disease, yellows, or enterotoxemic jaundice because of the characteristic icterus. the organism is a normal inhabitant of the gastrointestinal tract in these animals but may proliferate abnormally in response to some diets. c. perfringens causes intravascular hemolytic anemia in horses with clostridial abscesses, and clostridial mastitis in ewes. c. perfringens type a produces hemolytic α-toxin, which also has phospholipase c activity. leptospirosis. leptospirosis is recognized as a cause of hemolytic anemia in calves, lambs, and pigs. specific leptospiral organisms that cause hemolytic disease include leptospira interrogans serovars pomona and ictohaemorrhagiae. leptospira organisms are ubiquitous in the environment. infection occurs percutaneously and via mucosal surfaces and is followed by leptospiremia; organisms then localize preferentially in certain tissues (e.g., kidney, liver, and pregnant uterus). proposed mechanisms of hemolytic disease include immune-mediated (immunoglobulin m [igm] cold agglutinin) extravascular hemolysis and enzymatic (phospholipase produced by the organism) intravascular hemolysis. leptospirosis can also cause many disease manifestations besides hemolysis (e.g., renal failure, liver failure, abortion, and other conditions) that are not discussed here. primary immune-mediated hemolytic anemia or autoimmune hemolytic anemia) or secondary to a known initiator, termed secondary immune-mediated hemolytic anemia. although the cause of idiopathic immune-mediated hemolytic anemia is unknown, certain dog breeds (e.g., cocker spaniels) are predisposed to developing disease, suggesting the possibility of a genetic component. causes of secondary immune-mediated hemolytic anemia include certain infections (e.g., hemoplasmosis, babesiosis, and theileriosis), drugs (e.g., cephalosporins, penicillin, and sulfonamides), vaccines, and envenomations (e.g., bee stings). immune-mediated hemolysis directed at nonself antigens, such as in neonatal isoerythrolysis, is presented later. in most cases of idiopathic immune-mediated hemolytic anemia, the reactive antibody is igg, and the hemolysis is extravascular (i.e., erythrocytes with surface-bound antibody are phagocytized by macrophages, mainly in the spleen). igm and/or complement proteins may also contribute to idiopathic immune-mediated hemolytic anemia. complement factor c3b usually acts as an opsonin that promotes phagocytosis and extravascular hemolysis. however, formation of the complement membrane attack complex on red blood cell surfaces causes intravascular hemolysis; this mechanism more commonly occurs with igm autoantibodies. most immunoglobulins implicated in immune-mediated hemolytic anemia are reactive at body temperature (warm hemagglutinins). a smaller portion, usually igm, are more reactive at lower temperatures, young (hours to days old) with typical gross and microscopic changes of immune-mediated hemolytic anemia. pure red cell aplasia. pure red cell aplasia (prca) is a rare bone marrow disorder characterized by absence of erythropoiesis and severe nonregenerative anemia. primary and secondary forms of pure red cell aplasia have been described in dogs and cats. primary pure red cell aplasia is apparently caused by immune-mediated destruction of early erythroid progenitor cells, a presumption supported by the response of some patients to immunosuppressive therapy and by the detection of antibodies inhibiting erythroid colony formation in vitro in some dogs. administration of recombinant human erythropoietin (rhepo) has been identified as a cause of secondary pure red cell aplasia in dogs, cats, and horses, presumably caused by induction of antibodies against rhepo that cross-react with endogenous epo. experimentation with the use of speciesspecific recombinant epo has produced mixed results. dogs treated with recombinant canine epo have not developed pure red cell aplasia. however, in experiments reported thus far involving cats treated with recombinant feline epo, at least some animals have developed pure red cell aplasia. parvoviral infection has been suggested as a possible cause of secondary pure red cell aplasia in dogs. infection with felv subgroup c causes secondary erythroid aplasia in cats, probably because of infection of early-stage erythroid precursors. grossly, animals with pure red cell aplasia have pale mucous membranes without indicators of hemolysis (e.g., icterus). microscopic examination of the bone marrow shows an absence or near absence of erythroid precursors with or without lymphocytosis, plasmacytosis, and myelofibrosis; production of other cell lines (e.g., neutrophils and platelets) is normal or hyperplastic. immune-mediated neutropenia. immune-mediated neutropenia is a rare condition that has been reported in horses, dogs, and cats. this disease is characterized by severe neutropenia from immune-mediated destruction of neutrophils or their precursors. the range of causes is presumably similar to that of other immunemediated cytopenias (e.g., immune-mediated hemolytic anemia, pure red cell aplasia, and immune-mediated thrombocytopenia). affected animals may have infections, such as dermatitis, conjunctivitis, or vaginitis, which are secondary to marked neutropenia and a compromised innate immune system. microscopically, there may be neutrophil hyperplasia, maturation arrest, or aplasia in the bone marrow, depending on which neutrophil maturation stage is targeted causing a condition known as cold hemagglutinin disease. this results in ischemic necrosis at anatomic extremities (e.g., tips of the ears), where cooling of the circulation causes autoagglutination of erythrocytes and occlusion of the microvasculature. typically immunemediated hemolytic anemia targets mature erythrocytes, causing a marked regenerative response. however, as discussed earlier in the chapter, immune-mediated destruction of immature erythroid cells in the bone marrow may also occur, resulting in nonregenerative anemia. pathogenesis of secondary immune-mediated hemolytic anemia is dependent upon the cause. erythrocytic parasites may cause immune-mediated hemolysis by altering the red blood cell surface and exposing "hidden antigens" that are not recognized as selfantigens by the host's immune system. alternatively, the immune attack may be directed at the infectious agent, but erythrocytes are nonspecifically destroyed because of their close proximity-this is called the innocent bystander mechanism. certain drugs, such as penicillin, may cause immune-mediated hemolytic anemia by binding to erythrocyte membranes and forming drug-autoantigen complexes that induce antibody formation, termed hapten-dependent antibodies. other proposed mechanisms include binding of drugantibody immune complexes to the erythrocyte membrane, or induction of a true autoantibody directed against an erythrocyte antigen. hematologic, gross, and histopathologic abnormalities are typical of those of hemolytic anemia, as presented in the earlier section on bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in brief, there may be spherocytes and autoagglutination on blood smear evaluation, icterus and splenomegaly on gross examination, and emh, erythrophagocytic macrophages, and hypoxia-induced or thromboemboli-induced tissue necrosis on histopathologic examination. dogs with immune-mediated hemolytic anemia also frequently develop an inflammatory leukocytosis and coagulation abnormalities (prolonged coagulation times, decreased plasma antithrombin concentration, increased plasma concentration of fibrin degradation products, thrombocytopenia, and disseminated intravascular coagulation). intravascular hemolysis plays a relatively insignificant role in most cases of immune-mediated hemolytic anemia, but evidence of intravascular hemolysis (e.g., ghost cells, red plasma and urine, dark red kidneys) is noted occasionally, presumably in those cases in which igm and complement are major mediators of hemolysis. neonatal isoerythrolysis. neonatal isoerythrolysis (ni) is a form of immune-mediated hemolytic anemia in which colostrumderived maternal antibodies react against the newborn's erythrocytes. it is common in horses ( fig. 13-30 ) and has been reported in cattle, cats, and some other domestic and wildlife species. in horses, neonatal isoerythrolysis occurs as a result of immunosensitization of the dam from exposure to an incompatible blood type inherited from the stallion (e.g., transplacental exposure to fetal blood during pregnancy or mixing of maternal and fetal blood during parturition). a previously mismatched blood transfusion produces the same results. some equine blood groups are more antigenic than others; in particular, types aa and qa are very immunogenic in mares. in cattle, neonatal isoerythrolysis has been caused by vaccination with whole blood products or products containing erythrocyte membrane fragments. neonatal isoerythrolysis has been produced experimentally in dogs, but there are no reports of naturally occurring disease. in cats the recognized form of neonatal isoerythrolysis does not depend on prior maternal immunosensitization but on naturally occurring anti-a antibodies in queens with type b blood. affected animals are s l rather a secondary complication of many types of underlying disease, including severe inflammation, organ failure, and neoplasia. it is included in the section on inflammatory disorders because the coagulation cascade is closely linked to inflammatory pathways. information on this topic, including efig. 13 -5, is available at www.expertconsult.com. as well as in chapter 2. the term hematopoietic neoplasia encompasses a large and diverse group of clonal proliferative disorders of hematopoietic cells. historically, numerous systems have been used to classify hematopoietic neoplasms in human medicine, some of which have been applied inconsistently to veterinary species (examples include the kiel classification and national cancer institute working formulation). the world health organization (who) classification of hematopoietic neoplasia was first published in 2001 (updated in 2008) and is based on the principles defined in the revised european-american classification of lymphoid neoplasms (real) from the international lymphoma study group. the who classification system is considered the first true worldwide consensus on the classification of hematopoietic malignancies and integrates information on tumor topography, cell morphology, immunophenotype, genetic features, and clinical presentation and course. a veterinary reference of the who classification system, published in 2002, was later validated in 2011 using the canine model of lymphoma. this project, modeled after the study to validate the system in human beings, yielded an overall accuracy (i.e., agreement on a diagnosis) among pathologists of 83%. currently this classification system is accepted as the method of choice in both human and veterinary medicine. the who classification broadly categorizes neoplasms primarily according to cell lineage: myeloid, lymphoid, and histiocytic. this distinction is based on the fact that the earliest commitment of a pluripotent hsc is to either a lymphoid or nonlymphoid lineage. many pathologists and clinicians distinguish leukemias from other hematopoietic neoplasms. leukemia refers to a group of hematopoietic neoplasms that arise from the bone marrow and are present within the blood. leukemia may be difficult to differentiate from other forms of hematopoietic neoplasms that originate outside of the bone marrow but infiltrate the bone marrow and blood. for simplicity, cases of secondary bone marrow or blood involvement may not be considered leukemia but rather the "leukemic phase" of another primary neoplasm. it is now recognized that certain lymphomas and leukemias are different manifestations of the same disease (e.g., chronic lymphocytic leukemia and small lymphocytic lymphoma), and the designation of lymphoma or leukemia is placed on the tissue with the largest tumor burden. based on their degree of differentiation, leukemias are classified as acute or chronic. acute leukemias are poorly differentiated or undifferentiated, meaning that there are high percentages of early progenitor and precursor cells, including lymphoblasts, myeloblasts, monoblasts, erythroblasts, and/or megakaryoblasts. in contrast, well-differentiated cells predominate in chronic leukemias. because well-differentiated cells also predominate with nonneoplastic proliferations, chronic leukemias must be differentiated from reactive processes, such as those cells that occur in chronic and/or granulomatous inflammation. diagnosis of chronic leukemia is often made by excluding all other causes for the proliferating cell type. for example, causes of relative and secondary erythrocytosis are excluded to be able to diagnose polycythemia vera. furthermore, the designation of acute or chronic also refers to the disease's clinical course. acute leukemias tend to have an acute onset of severe and rapidly progressive clinical signs, whereas animals with chronic leukemia for destruction. marrow lymphocytosis and plasmacytosis may be marked (e.g., >60% of nucleated cells). the diagnosis may be supported by flow cytometric detection of immunoglobulin bound to neutrophils but is most often made on the basis of exclusion of other causes of neutropenia and response to immunosuppressive therapy. immune-mediated thrombocytopenia. immune-mediated thrombocytopenia (imtp) is a condition characterized by immunemediated destruction of platelets. it is a fairly common condition in dogs and is less frequent in horses and cats. the disease is usually idiopathic but may be secondary to infection (e.g., equine infectious anemia and ehrlichiosis), drug administration (e.g., cephalosporins and sulfonamides), neoplasia, and other immunemediated diseases. when immune-mediated thrombocytopenia occurs together with immune-mediated hemolytic anemia, the condition is called evans's syndrome. the thrombocytopenia is often severe (e.g., < 20,000 platelets/µl), resulting in varying degrees of bleeding tendencies, mainly in skin and mucous membranes. microscopically, there are multifocal perivascular hemorrhages in multiple tissues, and the bone marrow exhibits megakaryocytic and erythroid hyperplasia. rarely, immune-mediated destruction of megakaryocytes may cause megakaryocytic hypoplasia, termed amegakaryocytic thrombocytopenia. neonatal alloimmune thrombocytopenia. a form of immune-mediated thrombocytopenia, known as neonatal alloimmune thrombocytopenia, is recognized in neonatal pigs and foals. the pathogenesis of this disease is virtually identical to that of neonatal isoerythrolysis as a cause of anemia: a neonate inheriting paternal platelet antigens absorbs maternal antibodies against these antigens through the colostrum. in principle, a similar situation may occur after platelet-incompatible transfusion of blood or blood products containing platelets. gross and microscopic changes are similar to those of immune-mediated thrombocytopenia except that the animal is young (e.g., 1 to 3 days). hemophagocytic syndrome. hemophagocytic syndrome is a term used to describe the proliferation of nonneoplastic (i.e., polyclonal), well-differentiated but highly erythrophagic macrophages. the condition is rare but has been recognized in dogs and cats. unlike hemophagocytic histiocytic sarcoma, which is a neoplastic proliferation of phagocytic macrophages, hemophagocytic syndrome is secondary to an underlying disease, such as neoplasia, infection, or an immune-mediated disorder. the primary disease process causes increased production of stimulatory cytokines, which results in macrophage proliferation and hyperactivation. these activated macrophages phagocytize mature hematopoietic cells and hematopoietic precursors at an enhanced rate, resulting in one or more cytopenias. affected animals usually have lesions of the primary disease, as well as signs of the anemia (e.g., pale mucous membranes), neutropenia (e.g., bacterial infections), and thrombocytopenia (e.g., petechiae and ecchymoses). microscopically, phagocytic macrophages are found in high numbers in the bone marrow and commonly in other tissues, including lymph nodes, spleen, and liver. additional bone marrow findings reported in animals with hemophagocytic syndrome vary widely, ranging from hypoplasia to hyperplasia of cell lines with peripheral cytopenias. disseminated intravascular coagulation. disseminated intravascular coagulation is a syndrome characterized by continuous activation of both coagulation and fibrinolytic pathways and is also known as consumptive coagulopathy. it is not a primary disease, but 754.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system disseminated intravascular coagulation is a consumptive coagulopathy resulting from activation of both coagulation and fibrinolytic pathways. it is a secondary complication of many types of underlying disease, including many infectious diseases, trauma, burns, heat stroke, immune-mediated disease, hemolysis, shock, neoplasia, organ failure, obstetric complications, and noninfectious inflammatory disease, such as pancreatitis. it is common in critically ill domestic animals. disseminated intravascular coagulation involves an initial hypercoagulable phase, resulting in thrombosis and ischemic tissue damage, and a subsequent hypocoagulable phase as a result of consumption of coagulation factors and platelets, resulting in hemorrhage (efig. 13-5) . the pathogenesis of disseminated intravascular coagulation typically involves the release of tissue factor (thromboplastin) and subsequent activation of coagulation pathways and platelets but may also involve defective normal inhibition of coagulation or defective fibrinolysis. classically diagnosis of disseminated intravascular coagulation is based on clinical evidence of hemorrhage and/or thromboembolic disease and a triad of laboratory findings: thrombocytopenia, usually moderate (below the lower reference value but above 50,000/µl); prolonged coagulation times (prothrombin time and/or partial thromboplastin time); and decreased fibrinogen or increased concentration of plasma fibrin degradation products or d-dimer. milder forms of disseminated intravascular coagulation that do not meet all of the diagnostic criteria also occur. decreased plasma antithrombin (antithrombin iii) concentration and schistocytosis are other laboratory abnormalities often found in patients with disseminated intravascular coagulation. dysplasia of myeloid cells, and fewer than 20% myeloblasts and "blast equivalents." 3 acute myeloid leukemia. acute myeloid leukemia (aml) is uncommon in domestic animals but most frequently occurs in dogs and cats. in veterinary species, acute myeloid leukemia is most commonly of neutrophil, monocyte, and/or erythroid origin, with rare reports of eosinophil, basophil, or megakaryocytic lineages. it is caused by felv infection in cats. evaluations of blood smears show many early myeloid precursors, including myeloblasts and blast equivalents ( fig. 13-31, a) . in dogs the total leukocyte concentration averages approximately 70,000/µl; anemia, neutropenia, and thrombocytopenia commonly occur. grossly, animals show lesions attributed to anemia, neutropenia, and thrombocytopenia, such as pale mucous membranes, secondary infections, and multisystemic bleeding, respectively. neoplastic cells often infiltrate tissues, resulting in splenomegaly, hepatomegaly, and lymphadenomegaly. microscopically, myeloid cells efface (replace) the bone marrow and infiltrate extramedullary tissues, especially lymphoid tissue. chronic myeloid leukemia. chronic myeloid leukemia (cml), also called chronic myelogenous leukemia or myeloproliferative neoplasia, is rare in animals. most reported cases occur in dogs and cats. there are various subclassifications of chronic myeloid leukemia, including excessive production of erythrocytes (polycythemia vera), platelets (essential thrombocythemia), neutrophils (chronic neutrophilic leukemia), monocytes (chronic monocytic leukemia), neutrophils and monocytes (chronic myelomonocytic leukemia), eosinophils (chronic eosinophilic leukemia), or basophils (chronic basophilic leukemia). complete peripheral blood count analysis often reveals very high concentrations of the neoplastic cells, such as greater than 50,000 to 100,000 leukocytes/µl (see fig. 13-31, b) or 2,000,000 platelets/µl. cellular morphologic features are often normal, but slight dysplasia may be observed. later in the disease there may be cytopenias of nonneoplastic cell types. animals with polycythemia vera often have red mucous membranes and lesions of hyperviscosity syndrome, such as bleeding and dilated, tortuous retinal vessels. essential thrombocythemia results in multisystemic bleeding due to dysfunctional platelets, or multisystemic infarcts from hyperaggregability and excessive platelets. chronic myeloid leukemias of leukocytes often result in splenomegaly, hepatomegaly, and lymphadenomegaly because of infiltration by the neoplastic cells. histologically, the bone marrow shows proliferation of the neoplastic cell type characterized by dysplasia and low numbers (e.g., <20%) of myeloblasts and blast equivalents. mast cell neoplasia. mast cell tumors (mcts) of the skin and other sites are common in animals (see chapters 6, 7, and 17), but mast cell leukemia is rare. in cats, mcts are the most common neoplasm in the spleen (efig. 13-6) . mast cells normally are not present in the blood vascular system, but the finding of mast cells in the blood (mastocytemia) is highly suggestive of disseminated mast cell neoplasia (systemic mastocytosis) in cats. however, mastocytemia does not necessarily indicate myeloid neoplasia in dogs. in fact, one study found that the severity of mastocytemia in dogs was frequently higher in animals without mcts than those with mcts and that random detection of mast cells in blood smears usually is not the result of underlying mct. granulocytic sarcoma. granulocytic sarcoma is a poorly characterized extramedullary proliferation of myeloid precursors, most typically have indolent, slowly progressive disease. this classification scheme is summarized in table 13 -4. subcategories exist within each of these groups, as discussed further later. information on this topic is available at www.expertconsult.com. this section discusses examples of myeloid neoplasms, including myelodysplastic syndrome, myeloid leukemias, and mast cell neoplasms (technically a form of myeloid neoplasia), and lymphoid neoplasms, including lymphoid leukemias and multiple myeloma. other lymphoid neoplasms, such as the numerous subtypes of lymphoma and extramedullary plasmacytomas (emps), as well as histiocytic disorders are described in the section on lymphoid/lymphatic system, disorders of domestic animals, neoplasia. additional discussion of hematopoietic neoplasia occurs in the species-specific sections at the end of this chapter. myelodysplastic syndrome. myelodysplastic syndrome (mds) most commonly occurs in dogs and cats and may be caused by felv infection in cats. the disease refers to a group of clonal myeloid proliferative disorders with ineffective hematopoiesis in the bone marrow, resulting in cytopenias of more than one cell line. hematopoietic proliferation in bone marrow with concurrent peripheral blood cytopenias is likely a result of increased apoptosis of neoplastic cells within the bone marrow, before their release into circulation. clinical illness and death often result from secondary manifestations, such as secondary infections or cachexia, attributable to the effects of cytopenias and/or transformation of the neoplasm into acute myeloid leukemia. gross lesions are dependent upon the type and severity of the cytopenias. however, essential microscopic findings within the bone marrow are normal or increased cellularity, 3 "blast equivalents" include other stages of immature myeloid cells, such as abnormal promyelocytes, monoblasts, promonocytes, erythroblasts, and megakaryoblasts. before the discussion of specific diseases, it is worthwhile to describe the diagnostic techniques required to classify hematopoietic neoplasms that are becoming increasingly available for routine use in veterinary medicine. immunophenotyping refers to the use of antibodies recognizing specific molecules expressed on different cell types to determine the identity of a cell population of interest. immunophenotyping on the basis of these lineage-specific or lineage-associated markers can be performed on histologic sections (immunohistochemistry [see fig. 13 -86]), air-dried cytologic examination smears (immunocytochemistry), or by laser analysis of cells in suspension in blood or buffer solutions (flow cytometry). in cases of lymphoid neoplasia, immunophenotyping most routinely refers to determination of b or t lymphocyte origin. clonality assays, pcr for antigen receptor rearrangement (parr), can help identify neoplastic lymphoid proliferations on the basis of clonal rearrangements of genes encoding lymphocyte antigen receptors. in terms of practical application the parr assay is most useful in helping to distinguish lymphoid neoplasms from those nonneoplastic lymphoid proliferations mimicking neoplasia. cytogenetic testing has not been routinely used in veterinary medicine, though several genetic mutations have been identified in dogs. for example, breakpoint cluster region-abelson (bcr-abl) translocations have been identified in some canine leukemias, including acute myeloblastic leukemia and chronic monocytic leukemia. dogs with burkitt-like lymphoma have a translocation leading to constitutive c-myc expression. a b thrombocytopenia commonly occur. gross and microscopic lesions also are similar to those that occur in cases of acute myeloid leukemia, except that neoplastic cells may differentiate into morphologically identifiable lymphoid cells. chronic lymphocytic leukemia. chronic lymphocytic leukemia (cll) is uncommon in veterinary medicine. it is predominantly a disease of middle-aged to older dogs but is also documented in horses, cattle, and cats. most canine chronic lymphocytic leukemia cases are of t lymphocyte origin, typically cytotoxic t lymphocytes expressing cd8. in cats the majority of chronic lymphocytic leukemia cases have a t helper lymphocyte immunophenotype. a cbc often shows very high numbers of small lymphocytes with clumped chromatin and scant cytoplasm. proliferating cytotoxic t lymphocytes frequently contain a few pink cytoplasmic granules when stained with most methanol-based romanowsky stains (e.g., wright-giemsa). however, these granules may not be appreciated with some aqueous-based romanowsky stains (e.g., diff-quik). although the number of total blood lymphocytes is often greater than 100,000/µl, relatively mild lymphocytosis (e.g., 15,000/µl) has been reported. seventy-five percent of affected dogs also have often of eosinophilic or neutrophilic cell lines. although rare, there are reports of granulocytic sarcoma in dogs, cats, cattle, and pigs, and it may arise in a number of sites, such as lung, intestine, lymph nodes, liver, kidney, skin, and muscle. acute lymphoblastic leukemia. acute lymphoblastic leukemia (all) is uncommon in dogs and cats, and rare in horses and cattle. in a recent immunophenotype study of 51 cases of acute lymphoblastic leukemia in dogs, 47 arose from b lymphocytes and 4 arose from double-negative t lymphocytes that were immunonegative for cd4 and cd8 markers. in the blood of animals with acute lymphoblastic leukemia, there are typically many medium to large lymphoid cells with deeply basophilic cytoplasm, reticular to coarse chromatin, and prominent, multiple nucleoli (see fig. 13-31, c) . in affected dogs the mean blood lymphoid concentration is approximately 70,000/µl, but cats with acute lymphoblastic leukemia often have low numbers of neoplastic cells in the circulation. as with animals with acute myeloid leukemia, anemia, neutropenia, and c d m the light chains deposit as nonamyloid granules, it is termed light chain deposition disease. light chains are low-molecular-weight proteins that pass through the glomerular filter into the urine, wherein they are also known as bence jones proteins. they tend to not react with urine dipstick protein indicators and are most specifically detected by electrophoresis and immunoprecipitation. in addition to aiding in the diagnosis of multiple myeloma, paraproteins have an important role in pathogenesis of disease. these proteins may inhibit platelet function, increase blood viscosity, deposit in glomerular basement membranes (see chapter 11; see figs. 11-27 and 11-28, or precipitate at cool temperatures, which results in bleeding tendencies, hyperviscosity syndrome, glomerulopathies, and cryoglobulinemia, respectively. hyperviscosity syndrome refers to the clinical sequelae of pathologically increased blood viscosity, which are slowed blood flow and loss of laminar flow. clinical signs include mucosal hemorrhages, visual impairment due to retinopathy, and neurologic signs, such as tremors and abnormal aggressive behavior. cryoglobulinemia is the condition in which proteins, typically igm, precipitate at temperatures below normal body temperature (cold agglutinins). precipitation often occurs in blood vessels of the skin and extremities, such as the ears and digits, and results in ischemic necrosis. in multiple myeloma the neoplastic proliferation of plasma cells results in osteolysis. work with human cell cultures has shown that anemia, and 15% have thrombocytopenia. autopsy findings depend on the stage of disease. in advanced cases with marked infiltration of organs with neoplastic cells, there is often uniform splenomegaly, hepatomegaly, and lymphadenomegaly, and the bone marrow is highly cellular (efig. 13-7 ; see fig. 13-31, d) . other lesions depend on whether there are concurrent cytopenias, such as anemia, neutropenia, and thrombocytopenia, and if the neoplastic cells produce excessive immunoglobulin. lesions caused by excessive immunoglobulin are further discussed in the section on multiple myeloma. histologically, the bone marrow is densely cellular with welldifferentiated lymphocytes. small lymphocytes infiltrate and often efface in the architecture of the lymph nodes and spleen. the liver may have dense accumulations of neoplastic cells in the connective tissue around the portal triad. plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone; the latter is discussed in the section on lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia. multiple myeloma. multiple myeloma (mm) is a rare, malignant tumor of plasma cells that arises in the bone marrow and usually secretes large amounts of immunoglobulin. the finding of neoplastic plasma cells in blood samples or smears is rare. dogs are affected more frequently than other species, but multiple myeloma has also been reported in horses, cattle, cats, and pigs. diagnosis of multiple myeloma is based on finding a minimum of two or three (opinions vary) of the following abnormalities: • markedly increased numbers of plasma cells in the bone marrow ( fig. 13-32 , a) • monoclonal gammopathy • radiographic evidence of osteolysis • light chain proteinuria the classic laboratory finding in patients with multiple myeloma is hyperglobulinemia, which results from the excessive production of immunoglobulin or an immunoglobulin subunit by the neoplastic cells. this homogeneous protein fraction is often called paraprotein or m protein. paraproteins produced from the same clone of plasma cells have the same molecular weight and electric charge. therefore they have the same migration pattern using serum protein electrophoresis, which results in a tall, narrow spike in the globulins region, termed monoclonal gammopathy (see fig. 13-32, b) . the term gammopathy is used because most immunoglobulins migrate in the γ-region of an electrophoresis gel. however, some immunoglobulins, especially immunoglobulin a (iga) and igm, migrate to the β-region. occasionally, biclonal or other atypical electrophoretic patterns may be seen with multiple myeloma as a result of protein degradation, protein complex formation, binding to other proteins, or when the tumor includes more than one clonal population. it is important to note that monoclonal gammopathy is not specific to multiple myeloma but has also been reported with lymphoma, chronic lymphocytic leukemia, canine ehrlichiosis, and canine leishmaniasis. definitively distinguishing monoclonal from polyclonal gammopathy requires immunoelectrophoresis or immunofixation using species-specific antibodies recognizing different immunoglobulin subclasses and subunits. occasionally, multiple myeloma cells produce only the immunoglobulin light chain. an immunoglobulin monomer consists of two heavy chains and two light chains connected by disulfide bonds. these light chains may deposit in tissues and cause organ dysfunction, especially renal failure. when the light chains form amyloid deposits, the disease is called amyloid light chain amyloidosis. but if marrow is dark red as a result of replacement of fat by hematopoietic tissue; the extent of replacement is an indication of the duration of the anemia. the severity of microscopic lesions is dependent on the chronicity of the disease, and they are most significant in the spleen, liver, and bone marrow. as would be anticipated, microscopic findings of the spleen are predominantly influenced by the number and activity of macrophages, which is a reflection of the duration of the disease and the frequency of hemolytic episodes. hemosiderin-laden macrophages persist for months to years; therefore large numbers are consistent with chronicity. kupffer cell hyperplasia with hemosiderin stores and periportal infiltrates of lymphocytes are the most significant changes in the liver. bone marrow histologic findings vary depending on the duration of the disease. in most animals the marrow is cellular because of the replacement of fat by intense, orderly erythropoiesis. granulocytes are relatively less numerous, and plasma cells are increased. as in the spleen, hemosiderin-laden macrophages are present in large numbers in chronic cases. emaciated animals with chronic disease have serous atrophy of fat (see efig. 13-1) . clinical findings with viremic episodes include fever, depression, icterus, petechial hemorrhages, lymph node enlargement, and dependent edema. equine infectious anemia infection is diagnosed on the basis of the coggins test, an agarose gel immunodiffusion test for the presence of the antibody against the virus. congenital dyserythropoiesis in polled herefords. a syndrome of congenital dyserythropoiesis and alopecia occurs in polled hereford calves. the cause and pathogenesis of this often fatal disease are unknown. early in disease there is hyperkeratosis and alopecia of the muzzle and ears, which progresses to generalized alopecia and hyperkeratotic dermatitis. histologically, there is orthokeratotic hyperkeratosis with dyskeratosis, as well as erythroid hyperplasia, dysplasia, and maturation arrest in the bone marrow. ineffective erythropoiesis results in nonregenerative to poorly regenerative anemia. erythrocyte band 3 is integral membrane protein that connects to the cytoskeleton and aids in erythrocyte stability. a hereditary deficiency of this protein has been identified in japanese black cattle, resulting in increased erythrocyte fragility, spherocytosis, intravascular hemolytic anemia, and retarded growth. affected calves show lesions consistent with hemolytic anemia, including pale mucous membranes, icterus, and splenomegaly. histologically, there are bilirubin accumulations in the liver, and hemosiderin in renal tubules. bovine leukemia virus. bovine leukemia virus is discussed in the later section on lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). bovine viral diarrhea virus. bvdv infection may cause thrombocytopenia in cattle, and a thrombocytopenic hemorrhagic syndrome has been specifically caused by type ii bvdv infection. investigations of the mechanism of bvdv-induced thrombocytopenia have resulted in varying, sometimes conflicting, conclusions. more than one study has shown viral antigen within bone marrow megakaryocytes and circulating platelets. evidence of impaired thrombopoiesis (megakaryocyte necrosis, megakaryocyte pyknosis, osteoclasts support the growth of myeloma cells, and that direct contact between the two cell types increases the myeloma cell proliferation and promotes osteoclast survival. increased osteoclast activity causes osteolysis, but the exact mechanism is not known. osteolysis often results in bone pain, lytic bone lesions on radiographs, hypercalcemia, and increased serum alkaline phosphatase activity. later in disease, osteolysis may cause pathologic fractures. morphologically, myeloma cells tend to grow in sheets that displace normal hematopoietic cells in the bone marrow. a proposed diagnostic criterion of multiple myeloma is that plasma cells constitute 30% or more of the nucleated cells in the marrow. welldifferentiated plasma cells are round with abundant basophilic cytoplasm (due to increased rough endoplasmic reticulum) and a perinuclear pale zone (enlarged golgi apparatus for the production of immunoglobulin); anisocytosis and anisokaryosis are often mild but may be marked. some plasma cell neoplasms have a bright eosinophilic fringe due to accumulated iga (see fig. 13-32, a) . nuclei are round with clumped chromatin and often peripherally placed with the cytoplasm; binucleation and multinucleation are common. poorly differentiated myeloma cells may lack and/or display less characteristic features. osteolysis of bone may be present microscopically. common sites of metastasis include the spleen, liver, lymph nodes, and kidneys. flavin adenine dinucleotide deficiency. flavin adenine dinucleotide (fad) is a cofactor for cytochrome-b 5 reductase, the enzyme that maintains hemoglobin in its functional reduced state, and for glutathione reductase, an enzyme that also protects erythrocytes from oxidative damage. reported in a spanish mustang mare and a kentucky mountain saddle horse gelding, erythrocyte fad deficiency is a result of an abnormal riboflavin kinase reaction, which is the first reaction in converting riboflavin to fad. clinicopathologic changes include persistent methemoglobinemia of 26% to 46%, eccentrocytosis, a slightly decreased or normal hematocrit, and erythroid hyperplasia in the bone marrow. equine infectious anemia virus. equine infectious anemia virus (eiav), the agent of equine infectious anemia, is a lentivirus that infects cells of the monocyte-macrophage system in horses (also ponies, donkeys, and mules). the virus is mechanically transmitted by biting flies, such as horseflies and deer flies. less common routes of transmission include blood transfusions, contaminated medical equipment, and transplacentally. disease may present in acute, subacute, and chronic forms and is potentially fatal. after an acute period of fever, depression, and thrombocytopenia that lasts 1 to 3 days, there is a prolonged period of recurrent fever, thrombocytopenia, and anemia. in most cases, clinical disease subsides within a year, and horses become lifelong carriers and reservoirs of eiav. eiav causes anemia by both immune-mediated hemolysis and decreased erythropoiesis. hemolysis is typically extravascular but may have an intravascular component during the acute phase. decreased erythropoiesis may result from direct suppression of earlystage erythroid cells by the virus, as well as anemia of inflammation. thrombocytopenia likely results from immune-mediated platelet destruction and suppressed platelet production. animals dying during hemolytic crises are pale with mucosal hemorrhages and dependent edema. the spleen and liver are enlarged, dark, and turgid, and they and other organs have superficial subcapsular hemorrhages. petechiae are evident beneath the renal capsule and throughout the cortex and medulla. the bone affected animals are not necessarily anemic. however, acute intravascular hemolytic episodes may occur with hyperventilationinduced alkalemia. lesions are typical of hemolytic anemia and include pale mucous membranes, icterus, hepatosplenomegaly, and dark red urine with microscopic emh and marrow erythroid hyperplasia. a single dna-based test is available to detect the common mutation. erythrocyte structural abnormalities. congenital erythrocyte structural abnormalities may occur with abnormal membrane composition or defective proteins within the membrane or cytoskeleton. some of these morphologic changes occur concurrently with clinical disease, but others do not. hereditary stomatocytosis is recognized in alaskan malamutes, drentse patrijshonds, and schnauzers. the specific defects are not known, but they are likely different in the various dog breeds. however, all affected dogs have stomatocytes on blood smear evaluation, as identified by their slit-shaped area of central pallor. erythrocytes also have increased osmotic fragility and decreased survival. schnauzers are clinically healthy and not anemic but do have reticulocytosis, suggesting that the hemolytic anemia is compensated by erythroid hyperplasia. mild to marked hemolytic anemia is documented in alaskan malamutes and drentse patrijshonds. alaskan malamutes have concurrent short-limb dwarfism, and drentse patrijshonds have hypertrophic gastritis and polycystic kidney disease. other (presumably heritable) erythrocyte abnormalities in dogs that do not have clinical signs include elliptocytosis caused by band 4.1 deficiency or β-spectrin mutation, and familial macrocytosis and dyshematopoiesis in poodles. scott's syndrome. an inherited thrombopathy resembling scott's syndrome in human beings, in which platelets lack normal procoagulant activity, has been recognized in a family of german shepherd dogs. the specific defect in these dogs has not been identified on the molecular level but involves impaired expression of phosphatidylserine on the platelet surface. affected dogs have a mild to moderate clinical bleeding tendency characterized by epistaxis, hyphema, intramuscular hematoma formation, and increased hemorrhage with surgery. macrothrombocytopenia. macrothrombocytopenia is an inherited condition in cavalier king charles spaniels in which there are lower than normal concentrations of platelets with enlarged and giant platelets. the condition is caused by defective β 1 -tubulin, which results in impaired microtubule assembly. affected dogs are asymptomatic but may have abnormal platelet aggregation in vitro. canine distemper. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells and is presented in greater detail in the lymphoid section. canine distemper virus may also infect other hematopoietic cells, including erythrocytes, nonlymphoid leukocytes, and platelets ( fig. 13-33) , and can cause decreased peripheral blood concentrations of neutrophils, lymphocytes, monocytes, and platelets during viremia. the thrombocytopenia is a result of virus-antibody immune complexes on platelet membranes and direct viral infection of megakaryocytes. increased erythrocyte osmotic fragility. a condition characterized by increased erythrocyte osmotic fragility has been described in abyssinian and somali cats. the specific defect has and degeneration) and increased thrombopoiesis (megakaryocytic hyperplasia, increased numbers of immature megakaryocytes) in the bone marrow has been reported in type ii bvdv-infected animals, including concurrent megakaryocyte necrosis and hyperplasia in some experimental subjects. calves infected with type ii bvdv also have impaired platelet function. cattle with the hemorrhagic syndrome are severely thrombocytopenic and neutropenic with multisystemic hemorrhages, particularly of the digestive tract, spleen, gallbladder, urinary bladder, and lymph nodes. histologic lesions include hemorrhage, epithelial necrosis of enterocytes, intestinal erosions, crypt proliferation with microabscesses, and lymphoid depletion of the gut-associated lymphoid tissue, peyer's patches, and spleen. lesions of the bone marrow are variable, as previously described. bovine neonatal pancytopenia. bovine neonatal pancytopenia (bnp) is caused by alloantibodies absorbed from colostrum, resulting in a hemorrhagic syndrome in calves. the syndrome was first recognized in europe in the early 2000s and has since been experimentally correlated with prior vaccination of affected calves' dams with a commercial bvdv vaccine (pregsure bvd; pfizer animal health). the vaccine has since been voluntarily recalled from the market. it is thought that vaccination induces alloantibody formation by the dam. the alloantibodies are ingested by the calf and bind to the calf's hematopoietic progenitor cells, resulting in functional compromise of those cells. acutely affected calves are less than a year of age and have peripheral thrombocytopenia and neutropenia. death results from thrombocytopenia-induced hemorrhages or neutropenia-induced secondary infections, including pneumonia, enteritis, and septicemia. within the bone marrow there is erythroid, myeloid, and megakaryocytic hypoplasia. cyclic hematopoiesis. cyclic hematopoiesis (also known as lethal gray collie disease) is an autosomal recessive disorder of pluripotent hscs in gray collie dogs. a defect in the adaptor protein complex (ap3) results in defective intracellular signaling and predictable fluctuations in concentrations of blood cells that occur in 14-day cycles. the pattern is cyclic marked neutropenia, and in a different phase, cyclic reticulocytosis, monocytosis, and thrombocytosis. production of key cytokines involved in regulation of hematopoiesis is also cyclic. neutropenia predisposes affected animals to infection, and many die of infectious causes. affected animals have dilute hair coats and lesions with acute or chronic infectious disease, especially of the lungs, gastrointestinal tract, and kidneys. dogs older than 30 weeks of age have systemic amyloidosis, which occurs because of cyclic increases in concentration of acute phase proteins during phases of monocytosis. phosphofructokinase deficiency. inherited autosomal recessive deficiency of the erythrocyte glycolytic enzyme, phosphofructokinase (pfk), is described in english springer spaniel, american cocker spaniel, and mixed-breed dogs. there are three genes encoding pfk enzymes, designated m-pfk in muscle and erythrocytes, l-pfk in liver, and p-pfk in platelets. a point mutation in the gene coding for m-pfk results in an unstable, truncated molecule. erythrocytes in pfk-deficient dogs have decreased atp and 2,3-diphosphoglycerate (2,3-dpg) production and increased fragility under alkaline conditions. the disease is characterized by chronic hemolysis with marked reticulocytosis. the marked regenerative response may compensate for the ongoing hemolysis; therefore erythrophagocytosis, thrombosis, and histologic changes of ischemia are common, especially within the spleen, liver, and lungs. affected cats typically become acutely ill with fever, pallor, and icterus and usually die within 2 to 3 days. for many years, cytauxzoonosis was considered to be almost always fatal. however, a recent not been identified, but pk deficiency (which has been reported in these breeds) was excluded as the cause. affected cats have chronic intermittent severe hemolytic anemia and often have other lesions secondary to hemolytic anemia (e.g., splenomegaly and hyperbilirubinemia). cytauxzoonosis. cytauxzoonosis is a severe, often fatal disease of domestic cats caused by the protozoal organism, cytauxzoon felis. disease is relatively common in the south central united states, particularly during summer months. bobcats (lynx rufus) and other wild felids are thought to be wildlife reservoirs of disease. c. felis is transmitted by a tick vector, dermacentor variabilis, which is probably essential for infectivity of the organism. cytauxzoonosis has a schizogenous phase within macrophages throughout the body (especially liver, spleen, lung, lymph nodes, and bone marrow) that causes systemic illness. these schizontcontaining macrophages enlarge and accumulate within the walls of veins, eventually causing vessel occlusion, circulatory impairment, and tissue hypoxia. later in disease, merozoites released from schizonts enter erythrocytes, resulting in an erythrocytic phase of infection. infected domestic cats often have nonregenerative anemia, but the pathogenesis for the anemia is unclear. however, it likely represents preregenerative hemolytic anemia because erythrocyte phagocytosis is a prominent finding in many organs. infected cats often also develop neutropenia and thrombocytopenia, which likely result from inflammation and disseminated intravascular coagulation, respectively. on blood smear evaluation, signet ring-shaped erythrocytic inclusions (piroplasms) may be observed during the erythrocytic phase of disease ( fig. 13-34) . these inclusions closely resemble small-form babesia (see fig. 13-24, a) and some theileria organisms. postmortem examination typically shows pallor, icterus, splenomegaly, enlarged and red lymph nodes, diffuse pulmonary congestion and edema, and multisystemic petechiae and ecchymoses. vascular obstruction may cause marked distention of abdominal veins. cavitary effusions are present in some cats. microscopically, large, schizont-laden macrophages accumulate within venous and sinusoidal lumens and often completely occlude the lumens (fig. 13-35) . which the b and t lymphocytes proliferate, differentiate, and mature. in mammals, lymphocytes arise from hscs in the bone marrow, and b lymphocytes continue to develop at this site. ruminants also have b lymphocyte proliferation and maturation within their peyer's patches. progenitor t lymphocytes migrate from bone marrow to mature and undergo selection in the thymus. the spleen, lymph nodes, and lymph nodules are secondary lymphoid organs and are responsible for the immune responses to antigens, such as the production of antibody and cell-mediated immune reactions. at these sites, lymphocytes are activated by antigens and undergo clonal selection, proliferation, and differentiation (see also chapter 5). in addition, the spleen and lymph nodes contain cells of the monocyte-macrophage system and thus also participate in the phagocytosis of cells and materials. the bone marrow is described in the first section of this chapter. the remaining primary lymphoid organ, the thymus, is described first in this section, followed by the secondary lymphoid organs: spleen, lymph nodes, and diffuse and nodular lymphatic tissues. errors from selection of inappropriate sampling sites and artifacts from compression and incorrect fixation for histopathologic and immunohistochemical examinations are common in routine veterinary pathologic analysis. the identification and remedies for these problems are discussed in e-appendix 13-2. the thymus is essential for the development and function of the immune system, specifically for the differentiation, selection, and maturation of t lymphocytes generated in the bone marrow (see also chapter 5). the basic arrangement of the thymus in domestic animals consists of paired cervical lobes (left and right), an intermediate lobe at the thoracic inlet, and a thoracic lobe, which may be bilobed. the cervical lobes are positioned ventrolateral to the trachea, adjacent to the carotid arteries, and extend from the intermediate lobe at the thoracic inlet as far cranially as the larynx. the intermediate lobe bridges between the cervical and the thoracic lobe. the right thoracic lobe is usually small or completely absent. the left lobe lies in the ventral aspect of the cranial mediastinum (except in the ruminant, where it is dorsal) and extends caudally as far as the pericardium. horse-the cervical lobes in foals are small, and the thoracic lobe constitutes the bulk of the thymus. ruminant-the cervical lobes are large. the left and right thoracic lobes are fused and unlike other domestic animals, lie in the dorsal aspect of the cranial mediastinum. pig-the cervical lobes are large. dog-the cervical lobes regress very early and thus appear absent. the thoracic lobe extends caudally to the pericardium. cat-the cervical lobes are small, and the thoracic lobe, which forms the majority of the thymus, extends caudally to the pericardium and molds to its surface. the thymus is referred to as a lymphoepithelial organ and hence is composed of epithelial and lymphoid tissue. formed from the endoderm of the third pharyngeal pouch in the fetus, the thymic epithelium is infiltrated by blood vessels from the surrounding mesoderm, resulting in the development of the thymic epithelial reticulum. the lymphocyte population consists of bone marrow-derived progenitor cells, which fill spaces within the epithelial network. a connective tissue capsule surrounds the thymus, and attached thin septa subdivide the tissue into partially separated lobules. each report, in which numerous cats from a subregion of the endemic area in the united states survived infection with an organism with greater than 99% homology to cytauxzoon felis, suggests the emergence of a less virulent strain. felv is an oncogenic, immunosuppressive lentivirus that causes hematologic abnormalities of widely varying types and severity. manifestations of disease caused by felv infection vary depending on dose, viral genetics, and host factors, but normal hematopoiesis is probably suppressed to some degree in all cases. felv infects hematopoietic precursor cells soon after the animal is exposed and continues to replicate in hematopoietic and lymphatic tissue of animals that remain persistently viremic. the virus disrupts normal hematopoiesis by inducing genetic mutations, by other direct effects of the virus on infected hematopoietic cells, or by an altered host immune system. hematologic changes include dysmyelopoiesis with resultant cytopenias or abnormal cell morphologic features, and neoplastic transformation of hematopoietic cells (leukemia). a notable form of dysplasia is the presence of macrocytic erythrocytes (macrocytes) and metarubricytosis in the absence of erythrocyte regeneration (inappropriate metarubricytosis). the relatively uncommon subgroup c viruses cause erythroid hypoplasia, probably because of infection of early-stage erythroid precursors. felv may be detected in megakaryocytes and platelets in infected cats and may result in platelet abnormalities, including thrombocytopenia, thrombocytosis, increased platelet size, and decreased function. proposed mechanisms of felv-induced thrombocytopenia include direct cytopathic effects, myelophthisis, and immunemediated destruction. platelet life span and function have been shown to be decreased in felv-positive cats. persistently viremic cats are immunosuppressed and are prone to developing other diseases, including infectious diseases, bone marrow disorders, and lymphoma. cbc abnormalities attributed to felv infection include various cytopenias, especially nonregenerative anemia, which may be persistent or cyclical. regenerative anemia may also occur with felv infection, often because of coinfection with m. haemofelis. hematopoietic cell dysplasia or neoplasia may also be evident. grossly, infected cats are often pale, but other lesions are dependent upon the presence of other cytopenias or concurrent disease. microscopically, the bone marrow is hypocellular, normocellular, or hypercellular. there may be erythroid hypoplasia, erythroid hyperplasia with maturation arrest, or acute leukemia. feline immunodeficiency virus. feline immunodeficiency virus (fiv), another feline lentivirus, causes anemia in a minority of infected cats. immunosuppressive effects of fiv from thymic depletion are discussed elsewhere. it is generally accepted that anemia does not result directly from fiv infection but instead develops because of concurrent disease such as coinfection with felv or hemotropic mycoplasma, other infection, or malignancy. the severity and type of anemia in fiv-infected cats depends on the other specific disease processes involved. the thymus, spleen, lymph nodes, and lymph nodules, including malt, are classified as part of both the lymphoid and immune systems. the lymphoid system (also known as lymphatic system in some texts) is broadly categorized into primary and secondary lymphoid organs. the main primary lymphoid organs include thymus, bone marrow, and bursa of fabricius in birds and are the sites at 761.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system because the thymus involutes after sexual maturity, evaluation of whether it is smaller than normal is difficult to discern unless the change is extreme or age-matched control animals are available. before sexual maturity the thymus is easily identified as a lobular white to gray organ with a thin capsule. after sexual maturity the gland is often grossly indistinguishable from adipose connective tissue within the cranial mediastinum, although microscopic remnants may remain. an extremely small thymus in a neonatal animal should be considered abnormal and may indicate a primary immunodeficiency or secondary lymphoid depletion caused by extreme stress, often due to infectious diseases. enlargement of the thymus is most often due to neoplasia. serial sectioning of the thymus allows for gross identification of neoplasms, cysts, or hematomas. spleens vary in size within the same species and among the different species of domestic animals. the spleen can be enlarged (splenomegaly), normal in size, or small (atrophy), and the surface can be smooth, wrinkled, or nodular. the appearance of the cut surface of the spleen in normal animals depends on the amount of stroma (e.g., trabeculae are prominent in ruminants); the size and visibility of the white pulp, which reflects the amount of lymphoid tissue; and whether the red pulp is congested with blood. during an autopsy (syn: necropsy), the spleen is dissected free and checked for torsion of the gastrosplenic ligament (in nonruminants). the spleen is then sliced transversely at approximately 5-mm intervals (serial sectioning), and the cut surfaces are checked for lesions. specimens are taken for tests that require fresh tissue (e.g., bacteriologic and virologic examinations), and the remaining cross-sections are placed in fixative (10% buffered neutral formalin). a diffusely enlarged spleen should be serially sectioned to determine if the splenomegaly is due to congestion. the cut surface of severely congested spleens is red to bluish-black and exudes blood (bloody spleens), whereas cut surfaces of noncongested enlarged spleens ooze little blood (meaty spleens) and the color depends on how much of the normal parenchyma is replaced by inflammatory cells, stored materials, or neoplastic cells (see splenomegaly and table 13 -5). the spleen may be measured and weighed, but because of the wide variation in the dimensions and weight of normal spleens and the amount of blood stored, this information is difficult to interpret. it is essential that spleens with one or more nodules also be serially sectioned and the nodules evaluated for size, shape, and consistency. nodules may be dark red and ooze blood on cut surface, white-tan with a more firm texture, or a mixture of both. multiple wedge sections that include the interface between a nodule and the adjacent nonmass spleen should be collected, because the center of the nodules often consists only of hemorrhage and necrosis, and neoplasms may be missed. the color of the capsular surface of the spleen also varies among species of domestic animals and depends on the opacity or translucence of the splenic capsule. the degree of opacity of the capsule is a function of its thickness and the amount of collagen. the splenic capsules of horses and ruminants are thick and usually appear gray because the color of the red pulp is not visible through the capsule. in the pig, dog, and cat, the splenic capsule is thin, and thus the surface of the spleen is red. the tenseness of the capsule depends on how much the splenic parenchyma is distended; storage spleens devoid of blood usually have a wrinkled surface. irregular contraction of storage spleens is common, especially in dogs, and consists of nonuniform areas of congestion with intermingled contracted and wrinkled regions. lymph nodes should be dissected free of fat and connective tissue, and any firm attachment to adjacent tissues should be noted because these attachments may indicate neoplastic infiltration through the capsule. gross examination includes evaluating size (measurement or weight), shape, and whether the capsule is intact. the cut surface is examined for the presence of bulging tissue, edema, congestion, exudate, discoloration (see pigmentation), obscuration of the normal architecture, and masses such as abscesses, granulomas, and discrete neoplasms. cytologic evaluation of superficial lymph nodes through fine-needle aspirates provides excellent cellular detail and often yields a diagnosis. however, diagnosis of certain diseases (including lymphomas for complete world health organization [who] classification) requires architectural assessments, and therefore cytologic or small histologic samples are not sufficient. tru-cut biopsies are not ideal, but a 2-mm tru-cut needle may provide adequate tissue. the surgeon or pathologist must handle lymph nodes carefully to minimize artifacts. compression (e.g., squeezing with forceps) may cause crush artifacts, usually resulting in nuclear "streaming." immediately after removal, imprints/impression smears should be prepared and then kept away from formalin fumes. formalin fixation (in this case by formaldehyde fumes) destroys the differential staining seen with romanowsky stains such as wright's and giemsa and results in diffuse blue staining. prompt transfer of biopsy or postmortem specimens into fixative is crucial because delayed fixation can lead to numerous artifacts, including an artificial decrease in mitotic index (up to 40% reduction with a more than 12-hour delay in fixation); this reduction can alter tumor classification and grade. the current recommendation for the duration of formalin fixation is 16 to 32 hours; complete fixation of 2-to 4-mm thick tissues is likely to be achieved after 24 to 48 hours. both underfixation and overfixation may lead to difficulties with antigen retrieval for immunohistochemistry, though underfixation is considered the more common and serious problem. thinly slicing some nodes may be difficult, and allowing the node to fix for 1 hour before slicing may help. some pathologists prefer not to incise very small lymph nodes to avoid compression artifacts, but instead nick the capsule to allow formalin penetration. however, fixation of unincised lymph nodes can also cause compression artifacts because the fibrous capsule contracts in the fixative. once fixed, nodes should be cut in uniformly thick cross section to include both the cortex and medulla. transverse sections are usually sufficiently small to allow the entire cross section of most lymph nodes to fit on one microscopic slide, which facilitates histologic interpretation. the longitudinal plane is preferred for porcine lymph nodes because the location and amount of cortex and medulla vary at different sites in transverse sections. molecules) but not self-antigens are permitted to mature by a process called positive selection. cells that do not recognize mhc molecules are removed by apoptosis. those t lymphocytes that recognize both mhc molecules and self-antigens are removed by macrophages at the corticomedullary junction, a process called negative selection. because of the rigid differentiation requirements attributable to mhc restriction and tolerance (positive and negative selection, respectively), only a small fraction (<5%) of the developing t lymphocytes that arrive at the thymus from the bone marrow survive. mature naïve t lymphocytes exit the thymus through postcapillary venules in the corticomedullary region, enter the circulation, and recirculate through secondary lymphoid tissues, primarily located in the paracortex of lymph nodes and the periarteriolar sheaths of the spleen. in these specialized sites, the mature naïve t lymphocytes are activated upon exposure to their specific antigens and undergo additional phases of development to differentiate into effector and memory cells. the thymus attains its maximal mass relative to body weight at birth and involutes after sexual maturity; the rate of involution may vary among domestic species. the lymphoid and epithelial components are gradually replaced by loose connective tissue and fat, although remnants remain histologically, even in aged animals. lobule is composed of a central medulla and surrounding cortex (fig. 13-36) . the thymic cortex consists mainly of an epithelial reticulum and lymphocytes ( fig. 13-37) . the stellate cells of the epithelial reticulum have elongate branching cytoplasmic processes that connect to adjacent epithelial cells through desmosomes, thus forming a supportive network (cytoreticulum). the lymphoid component is composed of differentiating lymphocytes derived from progenitor (also known as precursor) t lymphocytes in the bone marrow. the medulla is composed of similar epithelial reticular cells, many of which are much larger than those in the cortex and have a more obvious epithelial structure. some of the epithelial reticular cells form thymic corpuscles, also called hassall's corpuscles, which are distinctive keratinized epithelial structures (see fig. 13-37 ). interdigitating dendritic cells (dcs) are also present within the medulla, but there are far fewer lymphocytes than in the cortex. the progenitor t lymphocytes released from the bone marrow into the blood enter the thymus in the subcapsular zone of the cortex and begin the differentiation and selection processes, developing into mature naïve t lymphocytes as they traverse the thymic cortex to the medulla. in the cortex, t lymphocytes that recognize self-molecules (major histocompatibility complex [mhc] the responses of the thymus to injury and causes are listed in boxes 13-4 and 13-5. the most common change is lymphoid atrophy caused by physical and physiologic stresses, toxins, drugs, and viral infections. atrophy. because the thymus does not contain any lymphopoietic tissue, it depends on the bone marrow for the supply of progenitor t lymphocytes. thus thymic lymphoid atrophy can be the result of either an inadequate supply of lymphocytes from the bone marrow or lysis of lymphocytes (lymphocytolysis) in the thymus. thymic aims to define the terms used in this chapter. the term splenic sinusoid is used to describe a vascular structure present in the sinusal spleen (also known as sinusoidal spleen); dogs are the only domestic animal with true splenic sinusoids. the term red pulp vascular spaces is used (as opposed to "sinus") to describe the vascular spaces in the red pulp of both the nonsinusal and nonsinusoidal spleens of all domestic animals. 4 the other terms used here include marginal sinus, marginal zone, periarteriolar lymphoid sheath (pals), periarteriolar macrophage sheath (pams), and splenic lymphoid follicles. the spleen is located in the left cranial hypogastric region of the abdomen, where it is typically suspended in the gastrosplenic ligament between the diaphragm, stomach, and the body wall. the exception is in domestic ruminants, where it is closely adhered to the left dorsolateral aspect of the rumen. the gross shape and size of the spleen vary markedly among domestic animals, but generally it is a flattened, elongated organ. some species, notably birds, demonstrate seasonal variation in splenic shape and size. the spleen is covered by a thick capsule composed of smooth muscle and elastic fibers, from which numerous intertwining fibromuscular trabeculae extend into the parenchyma. these trabeculae and reticular cells form a spongelike supportive matrix for the parenchyma of the mammalian spleen in all domestic species. in cattle and horses the three muscular layers of the capsule lie perpendicular to each other, forming a capsule thicker than that of carnivores. carnivores, small ruminants, and pigs have interwoven smooth muscle within the splenic capsule, and pigs also have abundant elastic fibers within the capsule. the spleen differs from many other organs in the organization of its parenchyma. instead of a cortex and medulla, the spleen is divided into two distinct structural and functional components: the red pulp and white pulp (fig. 13-38) . with hematoxylin and eosin (h&e) staining, red pulp appears red-pink because of the abundance of red blood cells, whereas white pulp appears blue-purple because of the heavy concentration of lymphocytes. the white pulp consists of splenic follicles, populated by b lymphocytes; the pals, inhabited by t lymphocytes; and the marginal zone at the periphery of follicles. macrophages, antigen-presenting cells, and trafficking b and t lymphocytes populate the marginal zone. the radial arteries, branches of the central artery (also known as central arteriole), and capillaries from both red and white pulp drain into the marginal sinus of the marginal zone, although the latter has not been shown to be the case in all species to the same degree (e.g., the cat has a small marginal sinus but a well-developed pams) (figs. 13-39 and 13-40). the red pulp consists of cells of the monocyte-macrophage system, pams, sinusoids (dogs, rats, and human beings only), red pulp vascular spaces, and associated stromal elements such as reticular cells, fibroblasts, and trabecular myocytes. the labyrinth of the splenic red pulp vascular spaces serves as both a functional and physical filter for circulating blood cells. the blood circulation of the spleen is particularly suited to enable its functions, namely, (1) filtering and clearing the blood of atrophy must be differentiated from involution, which normally begins at sexual maturity. this distinction is difficult to make, unless the change is extreme or age-matched control animals are available for comparison. inflammation. inflammation of the thymus is rare. neutrophils and macrophages are often present within keratinized hassall's corpuscles during involution and should not be mistaken for a true thymitis. thymitis has been reported in salmon poisoning disease of dogs (see chapter 7), epizootic bovine abortion (see chapter 18), and in pigs infected with porcine circovirus type 2 (pcv2). necrosis and secondary infiltrates of neutrophils and macrophages may be seen in other infectious diseases (e.g., equine herpesvirus 1 [ehv-1]). the main portal of entry to the thymus is hematogenous. portals of entry used by microorganisms and other agents and substances to access the lymphatic system are summarized in box 13-6. these portals include the blood vessels (hematogenous spread by microorganisms free in the plasma or within circulating leukocytes or erythrocytes), afferent lymphatic vessels (lymphatic spread), direct penetration, or through m (for "microfold") cells and dcs in malt. defense mechanisms used by the thymus to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. viruses, bacteria, and particles arriving in the lymph and blood interact with cells of the monocyte-macrophage system through phagocytosis and antigen processing and presentation. hyperplasia of the macrophages often occurs concurrently. antigen processing and presentation are followed by an immune response resulting in proliferation of b lymphocytes, plasma cells, and the subsequent production of antibody; proliferation of t lymphocytes may also occur. the relationships between anatomic structures and the different functions of the spleen are complicated. there are also anatomic differences among domestic animal species and confusion about the correct and up-to-date terminology. the following brief discussion malt, mucosa-associated lymphoid tissue. 4 there are numerous synonyms and misuse of terms within the literature, which have contributed to the confusion over terminology for red pulp vascular spaces. these terms include reticular space, red pulp, splenic cords, sinuses, red pulp sinuses, sinus spaces, pulp spaces, mesh space of the spleen, reticular cell-lined meshwork, interstices of the reticulum network, bloodfilled reticular meshwork of the red pulp, chordal spaces, splenic cords, and cords of billroth. the latter two terms are defined as the red pulp between the sinusoids, which most domestic animals do not have (except the dog). therefore the term red pulp vascular spaces is more appropriate. as a result of this pattern of blood flow, macrophages in the marginal sinus have the first opportunity to phagocytize antigens, bacteria, particles, and other material before macrophages in the sinusoids (in the dog) or in the pams and red pulp vascular spaces (all other domestic animals). in the dog the marginal sinus drains into the sinusoids, but in other domestic animals it drains into the red pulp vascular spaces. the central arteries leave the white pulp, enter the red pulp, and branch into smaller penicillar arterioles. each arteriole is surrounded by a sheath of macrophages known as periarteriolar macrophage sheaths (pams, previously known as ellipsoids), which are notably prominent in pigs, dogs, and cats. in horses, cattle, pigs, and cats the terminal branches of the penicillar arterioles empty into the red pulp vascular spaces lined by reticular cells. because the red pulp vascular spaces are not lined by endothelium, this type of circulation is known as an open system. this system is in contrast to the sinusoidal spleen of the dog (also of the rat and human beings), where the branches of the central artery of the white pulp and vessels from the marginal sinus enter into the sinusoids, which are lined by a discontinuous endothelium, and these empty into splenic venules. this type of circulation is known as a closed system because the blood flow is through blood vessels (arterioles, capillaries, sinusoids, and venules), all of which are lined by endothelium. although circulation in the red pulp is anatomically open in nonsinusoidal spleens, under certain conditions (e.g., during splenic contraction) the circulation is functionally closed, and the blood in the red pulp is particulate matter and senescent cells; (2) transporting recirculating lymphocytes and naïve b and t lymphocytes to the follicle and pals, respectively, to fulfill their specific immune functions; and (3) storage of blood in some domestic animal species (dog, cat, and horse) (fig. 13-41) . phagocytosis is particularly effective in the spleen because blood flows through areas within the red pulp that are populated with increased concentrations of macrophages, namely, within the marginal sinuses, in cuffs around the penicillar arteries (pams), diffusely on the reticular walls of the red pulp vascular spaces, and along the sinusoids in dogs. trafficking of naïve and recirculating lymphocytes is facilitated by the proximity of the marginal sinus to the follicular germinal centers and pals. maps of the vascular blood flow in sinusoidal and nonsinusoidal spleens are illustrated in figures 13-41 to 13-43. the celiac artery is the major branch of the abdominal aorta from which the splenic artery arises. the splenic artery enters the splenic capsule at the hilus, where it branches and enters the fibromuscular trabeculae as trabecular arteries to supply the splenic parenchyma. trabecular arteries become the central arteries of the white pulp and are surrounded by cuffs of t lymphocytes forming the pals. the splenic follicles, populated by b lymphocytes, are eccentrically embedded within or just adjacent to the pals. the central arteries send branches-the radial arteries-to supply the marginal sinus surrounding the splenic follicles. thus the cells at the circumferences of the follicles are brought into intimate contact with blood-borne antigens and trafficking b and t lymphocytes in the marginal sinus. diverted into "channels" lined by reticular cells. because the dog has both sinusoids and red pulp vascular spaces, it has both open and closed splenic circulations, which may allow for both fast and slow flows of blood depending on the physiologic need of the animal. blood flowing through the sinusoids or red pulp vascular spaces is under the surveillance of macrophages. in dogs the pseudopodia of these perisinusoidal macrophages project into the sinusoidal lumen through the spaces in the discontinuous endothelium. in all domestic animals, blood in the red pulp vascular spaces is under surveillance of macrophages attached to the reticular walls. blood from the red pulp vascular spaces and sinusoids then drains into the splenic venules, splenic veins, and ultimately into the portal vein, which empties into the liver. the spleen filters blood and removes foreign particles, bacteria, and erythrocytes that are senescent, have structural membrane abnormalities, or are infected with hemotropic parasites. as a secondary lymphoid organ, its immunologic functions include the activation of macrophages to process and present antigen, the proliferation of b lymphocytes and production of antibody and biologic molecules, and the interaction of t lymphocytes and antigens. in some species the spleen stores significant quantities of blood (box 13-7). the functions of the spleen are best considered on the basis of the two main components of the spleen: the red and white pulp and the anatomic systems contained within them (monocyte-macrophage system, red pulp vascular spaces, and hematopoiesis in the red pulp, and the b and t lymphocyte systems within the white pulp). monocyte-macrophage system. within the red pulp, macrophages are located in the marginal sinus, pams, and attached to the reticular walls of the red pulp vascular spaces. in the dog, macrophages are also located perisinusoidally. the supportive reticular network of the red pulp vascular spaces is composed of a fine meshwork of reticular fibers made of type iii collagen, on which macrophages are dispersed. exactly in which of these concentrations of macrophages phagocytosis of blood-borne particles takes place depends upon (1) the sequence in which they are exposed to the incoming blood, (2) the concentration of macrophages in these areas (e.g., the cat marginal sinus is small and thus not a major site of clearance; there is a compensatory increase in pams for phagocytosis), and (3) the functions of the macrophages. some of the macrophages in the marginal sinus and marginal zone are responsible for phagocytosis of particulate matter and others for the trapping and ingestion of antigens and antigen-antibody complexes. macrophages responsible for phagocytosis of blood-borne foreign material (fig. 13-44) , bacteria, and senescent and/or damaged erythrocytes (e.g., as seen in immune-mediated anemias and infections with hemotropic parasites) are also found in the red pulp. in the dog, sinusoidal macrophages remove entire erythrocytes (erythrophagocytosis), as well as portions of an erythrocyte's membrane and cytoplasmic inclusions, such as nuclear remnants like heinz bodies, by a process called pitting. as such, the presence of large numbers of nuclear remnants in erythrocytes in canine blood smears may indicate malfunction of the sinusoidal system. the normal rate of removal of senescent erythrocytes from the circulating blood does not cause an increase in size of the spleen; however, splenomegaly can be observed when large numbers of defective erythrocytes must be removed, as in cases of severe acute hemolytic anemia. nonsinusoidal spleens lack the fenestrated endothelium and perisinusoidal macrophages of canine sinusoids that allow for slow processing of red blood cells to determine which are to be returned to the equine, canine, and feline spleens all have considerable storage and contractile capacity because of their muscular capsule, increased numbers of trabeculae, and the relatively small amount of splenic parenchyma devoted to white pulp. the storage capacity in dogs and horses is remarkable: it has been claimed that the canine spleen can store one-third of the dog's erythrocytes while the animal sleeps and the equine spleen holds one-half of the animal's circulating red cell mass (which is considered advantageous because it reduces the viscosity of the circulating blood). storage spleens expand and contract quickly under the influence of the autonomic nervous system, via sympathetic and vagal fibers in the trabeculae and reticular walls of the red pulp vascular spaces and other circulatory disruptions, such as hypovolemic and/or cardiogenic shock. thus storage spleens may be either grossly enlarged and congested or small with a wrinkled surface and a dry parenchyma depending on whether the spleen is congested from stored blood or shrunken from contraction (see uniform splenomegaly and small spleens). hematopoietic tissue. in the developing fetus the liver is the primary site of hematopoiesis, with the spleen making a minor contribution. shortly before or after birth, hematopoiesis ceases in the liver and spleen, and the bone marrow becomes the primary hematopoietic organ. under certain conditions, such as severe demand due to prolonged anemia, splenic hematopoiesis can be reactivated; this outcome is called extramedullary hematopoiesis (emh). studies have indicated that splenic emh in dogs and cats most commonly occurs with degenerative or inflammatory circulation, pitted, or phagocytized. instead, the macrophages of the red pulp perform these functions, and phagocytized cells remain in the red pulp vascular spaces. the location of the primary sites of pitting in nonsinusoidal spleens is unclear, but it is likely that most erythrophagocytosis takes place in the red pulp vascular spaces. the cat's spleen is deficient in pitting, and removal of heinz bodies is slow; however, some erythrophagocytosis does occur in the marginal sinus. the macrophages of the sinusoids, marginal sinus, and red pulp vascular spaces are of bone marrow origin. from the bone marrow these cells circulate in the blood as monocytes and migrate into the spleen. some macrophages are replenished by local proliferation. for example, after phagocytizing large amounts of material from the blood, the macrophages of the pams migrate through the wall of the cuff into the adjacent red pulp, denuding the pams of macrophages. after 24 hours, local residual macrophages have proliferated to repopulate the pams. the fixed macrophages elsewhere in the body, namely, those in connective tissue, lymph nodes (sinus histiocytes), liver (kupffer cells), lung (pulmonary intravascular macrophages and pulmonary alveolar macrophages), and brain (resident and perivascular microglial cells), are also derived from bone marrow (see chapters 5, 8, 9, and 14) . storage or defense spleens. spleens are also classified as either storage or defense spleens, based on whether or not they can store significant volumes of blood. the ability to store blood in the spleen depends on the fibromuscular composition of the splenic capsule and trabeculae. splenic capsules and trabeculae with a low percentage of smooth muscle and elastic fibers cannot expand and contract and are designated as defense spleens. these are found in rabbits and human beings. the spleens of other domestic animal species have distention of the red pulp from stored blood separates the foci of white pulp (pals and lymphoid follicles), making white pulp appear sparser. splenic white pulp is organized around central arteries in the form of pals, which are populated primarily by t lymphocytes (see . primary splenic follicles are located eccentrically in pals and are primarily composed of b lymphocytes. when exposed to antigen, the splenic lymphoid follicles develop germinal centers (see lymphoid/ lymphatic system, lymph nodes, function) . macrophages in the white pulp follicles remove apoptotic b lymphocytes not selected for expansion because of low binding affinity for antigen. failure of these macrophages to phagocytize has been experimentally correlated with decreased production of growth factors like tgf-β and increased production of inflammatory cytokines that predispose the animal to autoimmune conditions. the marginal zone surrounds the marginal sinus at the interface of the white and red pulp and consists of macrophages, dcs, and t and b lymphocytes. the blood supply of the marginal sinus is from conditions (e.g., hematomas, thrombosis) and may occur without concomitant hematologic disease (see uniform splenomegaly with a firm consistency). it is also found in splenic nodular hyperplasia (see splenic nodules with a firm consistency). in some species, such as the mouse, emh is a normal function of the adult spleen and not necessarily a response to disease or hypoxic challenge. the splenic red pulp also contains large numbers of monocytes, which function as a reserve for generating tissue macrophages in response to ongoing tissue inflammation in the body. white pulp. white pulp consists of pals, each with a splenic lymphoid follicle surrounded by a marginal zone. normally these foci of white pulp are so small that they may not be visible on gross examination of a cross section of the spleen. however, if nodules are enlarged either by lymphoid hyperplasia, amyloid deposits, or a neoplastic process (e.g., lymphoma), they can become grossly visible on the cut surface, initially as 0.5-to 1.0-mm white circular foci scattered through the red pulp. in animals with storage spleens, the the splenic artery enters at the hilus and divides into arteries, which enter the trabeculae. when a trabecular artery emerges from a trabecula it becomes the central artery and is encased in a periarteriolar lymphoid sheath (pals), which is composed of t lymphocytes. it then enters the splenic follicle and gives off branches-the radial arteries, which supply the marginal sinus and marginal zone. the central artery emerges from the splenic follicle to enter the red pulp and branches into the penicillary arterioles, which are enclosed in a cuff of macrophages-the periarteriolar macrophage sheath (pams). the emerging penicillar arteries branch into arterioles and capillaries that supply the red pulp vascular spaces (see fig. 13-43) . the red pulp vascular spaces also receive blood from capillaries draining from the marginal sinus and drain into the splenic venules and then into the trabecular veins and splenic vein. b, sinusoidal spleen, dog. the blood flow is essentially the same but with the additional feature that arterioles from the marginal sinus drain into the sinusoids and some blood from the red pulp vascular space passes through slits in the sinusoidal wall to enter the sinusoid (see fig. 13-42) . this is the site of pitting and erythrophagocytosis. note that the major flow in a is sequentially past concentrations of macrophages in the marginal sinus, pams, and red pulp vascular spaces. in b there is the additional route from the marginal zone into the sinusoids. dog. red pulp vascular spaces macrophages in the marginal zone are phenotypically distinct from those in the red pulp. the red pulp macrophages function primarily to filter the blood by phagocytizing particles and by removing senescent or infected erythrocytes and pathogenic bacteria and fungi. marginal zone macrophages are divided into two types based on their location and the type of cell surface receptors they possess. the first group is positioned toward the periphery of the marginal zone, whereas the second group, the marginal metallophilic macrophages (so called for their silver staining positivity), is at the inner margin of the marginal zone closer to the splenic follicle and pals. it has been difficult to generate mammalian models that eliminate one of the two classes of marginal zone macrophages, so the degree to which one group specializes in a particular function is not clear. some marginal zone macrophages actively phagocytize particulate matter or bacteria (e.g., septicemias caused by streptococcus pneumoniae, listeria monocytogenes, campylobacter jejuni, or bacillus anthracis) in the blood (see fig. 13-40) . they also play a similar role in limiting the spread of viral infections. other marginal zone macrophages phagocytize and process antigens. thus macrophages of the marginal zone serve to bridge the innate and adaptive immune responses by secreting inflammatory cytokines to activate other immune cells and providing receptor-based activation of marginal zone lymphocytes. studies have shown that a loss of marginal zone macrophages coincides with decreased antigen trapping by resident b lymphocytes of the marginal zone and consequently a decrease in the early igm response to antigens. the responses of the spleen to injury (box 13-8) include acute inflammation, hyperplasia of the monocyte-macrophage system, hyperplasia of lymphoid tissues, atrophy of lymphoid tissues, storage of blood or contraction to expel reserve blood, and neoplasia. these responses are also best considered on the basis of the two main components of the spleen, the red and white pulp, and the anatomic systems associated with each. monocyte-macrophage system. the distribution and function of macrophages in the spleen is described earlier in the section on structure and function. these interactions are complex, and their relationships to both innate and adaptive immunity are areas of intense study (see also chapter 5). to facilitate filtering, all of the blood in the body passes through the spleen at least once a day, and 5% of the cardiac output goes to the spleen. in dogs, blood flow and transit time depend on whether the spleen is contracted or distended; blood flow is slower in the distended spleen. the extent to which macrophages of the monocyte-macrophage system phagocytize particles depends to a large degree on the sequence in which they receive blood. in most species, macrophages of the marginal sinus are the first to receive blood, and consequently phagocytized particles and bacteria tend to be more concentrated here initially. however, there are differences among domestic animal species; the cat, for instance, has a comparatively small marginal sinus, and thus the pams play a larger role in phagocytosis. the spleen is able to mount a strong response to blood-borne pathogens, which has been demonstrated in several studies. the blood of immunized rabbits injected intravenously with pneumococci cleared 98% of those bacteria within 15 minutes and 100% within an hour. the blood of dogs injected with 1 billion pneumococci per pound of body weight into the splenic artery was cleared of all bacteria in 65 minutes. after splenectomy, blood-borne the radial branches of the central artery, and it serves as the portal of entry into the spleen for recirculating b and t lymphocytes. from here, t lymphocytes migrate to the pals and b lymphocytes to the germinal centers. macrophages in the marginal zone capture bloodborne antigens, process them, and present them to the lymphocytes. senescent erythrocytes damaged erythrocytes (e.g., immune-mediated anemias) parasitized erythrocytes (e.g., hemotropic parasites) storage of blood (in storage spleens) extramedullary hematopoiesis severe demand (e.g., anemias) degenerative/inflammatory conditions without concomitant hematologic disease incidental (e.g., within nodules of hyperplasia) monocytes within splenic cords reserve for generating tissue macrophages in response to inflammation homing of circulating lymphocytes in the blood phagocytosis and processing of antigen macrophage activation inflammation, which may be diffuse or multifocal/focal (e.g., blastomycosis and tuberculosis, respectively). red pulp vascular spaces. the main response to injury of the red pulp vascular spaces is congestion (see uniform splenomegaly with a bloody consistency), as well as the storage of blood or contraction to expel reserve blood. white pulp. the responses to injury within the white pulp are most pronounced in the splenic lymphoid follicles. lymphoid follicular hyperplasia is a response to antigenic stimuli and results in the formation of secondary follicles; marked hyperplasia may be grossly evident. hyperplasia of splenic lymphoid follicles follows a similar sequence of events and morphologic changes as seen in other secondary lymphoid organs and is discussed in more detail in lymphoid/lymphatic system, lymph nodes, dysfunction/responses to injury. similarly, atrophy of splenic lymphoid follicles has similar causes as lymphoid atrophy in other lymphoid organs (see box 13-5). briefly, atrophy occurs in response to lack of antigenic stimulation (e.g., from regression after antigenic stimulation has ceased), from the effects of toxins, antineoplastic chemotherapeutic agents, microorganisms, radiation, malnutrition, wasting/cachectic diseases, or aging, or when the bone marrow and thymus fail to supply adequate numbers of b and t lymphocytes, respectively. the follicles are depleted of lymphocytes, and with time, germinal centers and follicles disappear. the amount of the total lymphoid tissue is reduced, and the spleen may be smaller. the response to injury of the monocyte-macrophage system in the marginal sinus and marginal zone is also phagocytosis and proliferation. capsule and trabeculae. lesions in the capsule and trabeculae are uncommon and include splenic capsulitis secondary to peritonitis, and complete or partial rupture of the splenic capsule, usually due to trauma. the two main portals of entry to the spleen for infectious agents are hematogenous spread and direct penetration. the splenic capsule is thick, and thus direct penetration is less common. inflammation from an adjacent peritonitis is unlikely to penetrate the capsule into the splenic parenchyma. cattle with traumatic reticulitis may have foreign objects migrate into the ventral extremity of the spleen, causing a splenic abscess. splenic abscesses also develop secondary to perforation of the gastric wall in horses, due to foreign body penetration, gastric ulcers, or gastric inflammation. portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box 13-6. defense mechanisms used by the spleen to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsule of the spleen. lymph nodes are soft, pale tan, round, oval or reniform organs with a complex three-dimensional structure. on gross examination of a cross section of lymph nodes, two main areas are visible: an outer rim of cortex and an inner medulla (fig. 13-45) . to understand the pathologic response of the lymph node, it is important to consider its anatomic components and their relationship with antigen processing (fig. 13-46) : organisms multiply rapidly and may disseminate widely in the body to cause an overwhelming postsplenectomy infection. studies have also shown that the phagocytic function of the spleen is critical in the control of plasmodium (causative agent of malaria) in human beings and babesiosis in cattle. if the number of pathogenic bacteria in the circulation exceeds the capacity of the splenic macrophages, as in cases of severe septicemia, it may result in acute splenic congestion (see uniform splenomegaly with a bloody consistency). this may be followed by inflammation with areas of necrosis, fibrin deposition, and infiltration by neutrophils in bacteremias of pyogenic bacteria. the marginal zone can be the initial site of response to blood-borne antigens and bacteria delivered by the radial branches of the central arteries to the marginal sinus. similar to the response of the red pulp vascular spaces, the marginal zone can become congested and with time (only hours with highly pathogenic organisms) may contain aggregates of neutrophils and macrophages. histologically, the congestion and inflammation form a complete or partial concentric ring around the circumference of the splenic nodule (see anthrax). hyperplasia of the red pulp macrophages is also seen in chronic hemolytic diseases, because there is a prolonged need for phagocytosis of erythrocytes. similarly, chronic splenic congestion, usually the result of portal or splenic vein hypertension, can lead to proliferation of the macrophages present on the walls of the red pulp vascular spaces and results in thickening of the reticular walls between the red pulp vascular spaces. macrophages in the red pulp also proliferate in response to fungi and facultative intracellular pathogens (e.g., mycobacterium bovis) arriving hematogenously to the spleen. the number of red pulp macrophages may be augmented by monocytes recruited from the blood to form granulomatous acute inflammation with fibrin and necrosis abscesses, microabscesses granulomatous inflammation (diffuse, multifocal, focal) fibroblastic reticular cells and fibers. besides providing structural support, this reticulum helps form a substratum for the migration of lymphocytes and antigen-presenting cells to the follicles and facilitates the interaction with b and t lymphocytes. cortex. the outer/superficial cortex contains the lymphoid follicles (also referred to as lymphoid nodules) (see . the follicles are designated as primary if they consist mainly of small lymphocytes: mature naïve b lymphocytes expressing receptors for specific antigens exit the bone marrow and circulate through the bloodstream, lymphatic vessels, and secondary lymphoid tissues. on their arrival at lymph nodes, b lymphocytes exit through hevs in the paracortex and home to a primary follicle (which also contains follicular dcs in addition to the resting b lymphocytes). lymphoid follicles with germinal centers are designated as secondary follicles: b lymphocytes that recognize the antigen for which they are expressing receptors are activated and proliferate to form the secondary lymphoid follicles characterized by prominent germinal centers. germinal centers are areas with a specialized • stroma-capsule, trabeculae, and reticulum • cortex-"superficial" or "outer" cortex (lymphoid follicles, b lymphocytes) • paracortex-"deep" or "inner" cortex (t lymphocytes) • medulla-medullary sinuses and medullary cords • blood vessels-arteries, arterioles, high endothelial venules (hevs), efferent veins • lymphatic vessels-lymphatic afferent and efferent vessels; lymphatic sinuses (subcapsular, trabecular, and medullary) • monocyte-macrophage system-sinus histiocytes stroma. the lymph node is enclosed by a fibrous capsule penetrated by multiple afferent lymphatic vessels, which empty into the subcapsular sinus (see also . at the hilus, efferent lymphatic vessels and veins exit, and arteries enter the node. fibrous trabeculae extend from the capsule into the parenchyma to provide support to the node and to house vessels and nerves. the lymph node is also supported by a meshwork of medulla. the medulla is composed of medullary cords and medullary sinuses (see . the medullary cords contain macrophages, lymphocytes, and plasma cells. in a stimulated node the cords become filled with antibody-secreting plasma cells. the medullary sinuses are lined by fibroblastic reticular cells and contain macrophages ("sinus histiocytes"), which cling to reticular fibers crossing the lumen of the sinus. these macrophages phagocytize foreign material, cellular debris, and bacteria from the incoming lymph. vasculature: blood vessels, lymphatic vessels, and lymphatic sinuses. the blood vessels of the lymph node include arteries, arterioles, veins, and postcapillary venules (hevs) lined by specialized cuboidal endothelium (see figs. 13-45 and 13-46) . microenvironment that support the proliferation and further development of b lymphocytes to increase their antigen and functional capacity (see lymphoid/lymphatic system, lymph nodes, function). the mantle cell zone surrounds the germinal center and consists of small inactive mature naïve b lymphocytes and a smaller population of t lymphocytes (approximately 10%). paracortex. the diffuse lymphoid tissue of the paracortex (also referred to as the deep or inner cortex) consists mainly of t lymphocytes, as well as macrophages and dcs (see . this region contains the hevs through which b and t lymphocytes migrate from the blood into the lymphoid follicles and paracortex, respectively. t and b lymphocytes may also enter the lymph node via the lymphatic vessels. and larger molecules, small molecules and free antigens, and antigen within dcs. it is helpful to consider the paths taken by particles, molecules, antigens, and cells arriving at a lymph node. the following account describes the journey of an antigen as it enters a lymph node to trigger an immune response. antigen in the lymph arriving in the afferent lymphatic vessels empties into the subcapsular sinus. hydrostatic pressure here is low, and reticular fibers crossing the sinus impede flow, and thus particles tend to settle, which facilitates phagocytosis by the sinus macrophages. lymph then flows down the trabecular sinuses that line the outer surface of fibrous trabeculae, to the medullary sinus, and eventually exits via efferent vessels. as antigens within the lymph travel through the sinuses, they are captured and processed by macrophages and dcs. alternatively, dcs charged with antigen can migrate within blood vessels to the node and enter the paracortex via the hevs. circulating b lymphocytes also enter across the hevs, and if they encounter antigen-bearing dcs, there is a local reaction involving the appropriate t helper lymphocytes, b lymphocytes, and dcs. this results in the migration of the activated b lymphocytes to a primary follicle, where they initiate formation of a germinal center. germinal centers, upon migration of antigen-activated b lymphocytes, develop a characteristic architecture. distinct polarity composed of a superficial or light zone and a deep dark zone is present in cases of antigenic stimulation. the light zone, orientated at the source of antigen, consists mainly of small lymphocytes, called centrocytes, which have moderate amounts of pale eosinophilic cytoplasm. the cells of the dark zone, called centroblasts, are large, densely packed lymphocytes with scant cytoplasm, giving this area a darker appearance on h&e staining. the centroblasts undergo somatic mutations of the variable regions of the immunoglobulin gene, followed by isotype class switching (from igm to igg or iga). during this process most centroblasts undergo apoptosis, and cell fragments are phagocytized by macrophages, which are then termed tingible (stainable) body macrophages. the cells that have survived the affinity maturation process are now called centrocytes and along with t lymphocytes and follicular dcs, populate the germinal center light zone. these post-germinal center b lymphocytes leave the follicle as plasma cell precursors (immunoblasts or plasmablasts) and migrate from the cortex to the medullary cords, where they mature and excrete antibody into the efferent lymph. some of these cells may colonize the region surrounding the mantle cell zone to form a marginal zone. marginal zones are apparent only in situations of prolonged and intense immune stimulation and serve as a reservoir of memory cells. the elliptical mantle cell cuff is wider over the light pole of the follicle, though in instances of strong antigenic stimulation, the cuffs can completely encircle the germinal center. responses to injury are listed in box 13-9, and the responses are discussed on the basis of the following systems: sinus histiocytes of the monocyte-macrophage system, cortex, paracortex, and medulla (medullary sinuses and medullary cords). generally, enlarged lymph nodes can be distributed in several different patterns in the body. first, all lymph nodes throughout the body (systemic or generalized) may be enlarged (lymphadenopathy or lymphadenomegaly). this pattern is usually attributed to systemic infectious, inflammatory, or neoplastic processes. if a single lymph node or regional chain of nodes is enlarged, then the area drained by that node should be checked for lesions (e.g., evaluate the oral cavity if the mandibular lymph nodes are enlarged). thus it is important to know the area drained by specific lymph nodes. mesenteric lymph nodes are normally larger because of follicular approximately 90% to 95% of lymphocytes enter lymph nodes through the hevs, which also play an important role in lymph fluid balance. the lymphatic vasculature consists of afferent lymphatic vessels, which pierce the capsule and drain into the subcapsular sinus. lymph continues to drain through the trabecular sinuses to the medullary sinuses and finally exits at the hilus via efferent lymphatic vessels. all lymph nodes receive afferent lymphatic vessels from specific areas of the body. the term lymphocenter is often used in veterinary anatomy to describe a lymph node or a group of lymph nodes that is consistently present at the same location and drains from the same region in all species. for example, the popliteal lymph node, caudal to the stifle, drains the distal hind limb. the tracheobronchial nodes (bronchial lymphocenter), located at the tracheal bifurcation, collect lymph from the lungs and send it to the mediastinal nodes or directly to the thoracic duct. because lymph from a single afferent lymphatic vessel drains into a discrete region of a lymph node, only these regions of the node may be affected by the contents of a single draining lymph vessel (e.g., antigen, infectious organisms, or metastatic neoplasms [ fig. 13-47] ). the lymph node of the pig has a different structure. the afferent lymphatic vessels enter at the hilus instead of around the periphery of the node and empty lymph into the center of the node. the lymph drains to the "subcapsular" sinus (the equivalent of the medullary sinuses of other domestic animals) and then into several efferent lymphatic vessels, which pierce the outer capsule. this reversal of flow is the result of an inverted nodal architecture, with the cortex in the middle of the node surrounded by the medulla at the periphery. thus a pig lymph node that is draining an area of hemorrhage will have blood accumulate in the periphery (subcapsular) instead of in the center of the node (which may be grossly visible). the functions of the lymph node are (1) to filter lymph of particulate matter and microorganisms, (2) to facilitate the surveillance and processing of incoming antigens via interactions with b and t lymphocytes, and (3) to produce b lymphocytes and plasma cells. material arriving in the lymph can be subdivided into free particles that is undergoing follicular hyperplasia is enlarged and has a taut capsule, and the cut surface may bulge. histologically, the follicles contain active germinal centers with antigenic polarity (light and dark zones) (figs. 13-49 and 13-50; also see fig. 13-46) . depending on the duration and continued exposure to the antigen, there may also be concomitant paracortical hyperplasia and medullary cord hyperplasia and sinus histiocytosis, because these nodes continuously receive and respond to barrages of antigens and bacteria from the intestinal tract. sinus histiocytes (macrophages) are part of the monocyte-macrophage system and the first line of defense against infectious and noninfectious agents in the incoming lymph. in response to these draining agents, there is hyperplasia of the macrophages ("sinus histiocytosis"), most notable in the medullary sinuses ( fig. 13-48) . leukocytes, often monocytes, may harbor intracellular pathogens (e.g., mycobacterium spp., cell-associated viruses such as parvovirus), arrive in the blood or lymph, infect the lymph node, and then are disseminated throughout the lymphoid tissues of the body via the efferent lymph and circulating blood. cortex (lymphoid follicles). follicular hyperplasia of the cortex is discussed in the section lymphoid/lymphatic system, lymph node, function. an antigenically stimulated lymph node the two main portals of entry to the lymph node for infectious agents and antigens are afferent lymphatic vessels (lymphatic spread) and blood vessels (hematogenous spread). portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box 13-6. infectious microorganisms, either free within the lymph or within lymphocytes or monocytes, are transported to regional lymph nodes through lymphatic vessels. agents may escape removal by phagocytosis in one lymph node and be transported via efferent lymphatic vessels to the next lymph node in the chain and cause an inflammatory or immunologic response there. this process can continue serially down a lymph node chain, and if the agent is not removed, it may eventually be transported via the lymphatic vessels to either the cervical or thoracic ducts and then disseminated throughout the body. although most pathogens are transported to lymph nodes via afferent lymphatic vessels, bacteria can be transported to lymph nodes hematogenously (free or within leukocytes such as monocytes) in septicemias and bacteremias. direct penetration of a lymph node is uncommon, because it is protected by a thick fibrous capsule. occasionally, inflammatory cells or neoplasms can extend directly into nodal parenchyma from adjacent tissues. defense mechanisms used by the lymphatic system to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsules of lymph nodes. hemal nodes are small, dark red to brown nodules found most commonly in ruminants, mainly sheep, and have also been reported in horses, primates, and some canids. their architecture resembles that of a lymph node with lymph follicles and sinuses, except that in the hemal node, sinuses are filled with blood (efig. 13-8) . because erythrophagocytosis can be present, it is presumed that hemal nodes can filter blood and remove senescent erythrocytes, but as their blood supply is small, their functional importance is not clear. malt is the initial site for mucosal immunity and is crucial in the protection of mucosal barriers. malt is composed of both diffuse lymphoid tissues and aggregated lymphoid (also known as lymphatic) nodules, which can be subcategorized based on their anatomic location: (1) bronchus-associated lymphoid tissue (balt), which is often at the bifurcation of the bronchi and bronchioles; (2) tonsils (pharyngeal and palatine) form a ring of lymphoid tissue at the oropharynx; (3) nasal-, larynx-, and auditory tube-associated lymphoid tissues (nalt, lalt, and atalt, respectively) within the nasopharyngeal area; (4) gut-associated lymphoid tissue (galt), which includes peyer's patches and diffuse lymphoid tissue in the gut wall; (5) conjunctiva-associated lymphoid tissue (calt); (6) other lymphoid nodules (e.g., genitourinary tract) (fig. 13-51) . diffuse lymphoid tissue consists of lymphocytes and dcs within the lamina propria of the mucosa of the alimentary, respiratory, and genitourinary tracts. these cells intercept and process antigens, which then travel to regional lymph nodes to initiate the immune response, leading ultimately to the secretion of iga, igg, and igm. plasmacytosis. less florid follicular reactions will have smaller separated germinal centers, whereas nodes receiving persistent high levels of antigen stimulation may have coalescing germinal centers (termed "atypical benign follicular hyperplasia"). in such cases of chronic strong antigenemia, the highly reactive nodes may also exhibit colonization of lymphocytes into perinodal fat, and germinal centers may contain irregular lakes of eosinophilic material, known as follicular hyalinosis. as the immune response declines, there is follicular lymphoid depletion and the concentration of lymphocytes in the germinal centers is reduced, allowing the underlying follicular stroma (including dcs and macrophages) to become visible. with ongoing lymphocyte depletion, the mantle cell zones are thinned, less populated, and discontinuous. eventually, residual mantle cells collapse into the follicular stroma, forming clusters of small dark cells within the bed of dcs and macrophages, referred to as fading follicles. paracortex. paracortical atrophy may result from a variety of causes, including deficiency in lymphocyte production in the bone marrow, reduced differential selection of lymphocytes in the thymus, or destruction of lymphocytes in the lymph node by viruses, radiation, and toxins directly on the lymphocytes in the lymph node (see box 13-5). examination of h&e-stained sections allows evaluation of follicular activity in the cortex and the concentration of plasma cells in the medullary cords, which serve as a reasonable estimate of b lymphocyte activity for comparison. paracortical hyperplasia may have a nodular or diffuse appearance depending on which and how many afferent lymphatic vessels are draining antigen. this reaction may precede or be concurrent with the germinal center reaction of follicular hyperplasia. proliferation of t lymphocytes has been reported in the paracortex (and pals of the spleen) in malignant catarrhal fever (mcf) in cattle and in pigs with porcine reproductive and respiratory syndrome. pcv2 can cause a diffuse proliferation of macrophages within the paracortex. responses to injury by the medullary sinuses are dilation of the sinuses and proliferation of histiocytes ("sinus histiocytosis"). sinus macrophages proliferate in response to a wide variety of particulate matter in the lymph, including bacteria and erythrocytes (erythrophagocytosis) draining from a hemorrhagic area (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, pigmentation of lymph nodes). dilation of the sinuses due to edema occurs with many underlying conditions, including chronic cardiac failure or drainage from an acutely inflamed area. as the inflammation progresses, the sinuses become filled with neutrophils, macrophages, and occasionally fibrin, in addition to the hyperplastic resident sinus histiocytes (see fig. 13-48) . depending on the intensity of the inflammation, the adjacent parenchyma may become affected (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly], acute lymphadenitis). as pointed out in the section on lymph nodes, function, after activation and proliferation of b lymphocytes in the follicle, the immunoblasts formed there move to and mature in the medullary cords, which as a result are distended with plasma cells that secrete antibody into the efferent lymphatic vessels ("medullary plasmacytosis"). the concentration of medullary plasma cells correlates with the activity of the germinal centers. as the immune response subsides, the number of plasma cells decreases and the medullary cords return to their resting state populated by few lymphocytes and scattered plasma cells. chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system composition. for instance, m cells increase in animals transferred from pathogen-free housing to the normal environment. m cells may also be exploited as a portal for entry by some microbes (see lymphoid/lymphatic system, portals of entry/pathways of spread). table 13 -5 lists the interactions of the malt with different microorganisms. the responses of malt to injury are similar to those of other lymphoid tissues: hyperplasia, atrophy, and inflammation (box 13-10). hyperplasia. hyperplasia of lymphoid nodules is a response to antigenic stimulation and consists of activation of germinal centers with subsequent production of plasma cells (see fig. 13-51, b) . lymphoid nodule hyperplasia is often present in chronic disease conditions, such as balt hyperplasia in chronic dictyocaulus spp. (horses, cattle, sheep, and goats) or metastrongylus spp. (pigs) associated bronchitis or bronchiolitis. mycoplasma spp. pneumonias of sheep and pigs display marked balt hyperplasia that can encircle bronchioles and bronchi ("cuffing pneumonia"). hyperplastic lymphoid nodules can be so enlarged that they become grossly visible as discrete white plaques or nodules (see fig. 13 -51, a). they can be seen in the conjunctiva of the eyelids and the third eyelid in chronic conjunctivitis, the pharyngeal mucosa in chronic pharyngitis, the gastric mucosa in chronic gastritis, and the urinary bladder in chronic cystitis (follicular cystitis). the normal fetus has no detectable balt, though it may be present in fetuses aborted due to infectious disease. atrophy. atrophy of the diffuse lymphoid tissue and lymphoid nodules has the same causes as atrophy affecting other lymphoid tissues (see box 13-5) and includes lack of antigenic stimulation, cachexia, malnutrition, aging, viral infections, or failure to be repopulated by b lymphocytes from the bone marrow or t lymphocytes from the thymus. lymphocytolysis of germinal center lymphocytes of peyer's patches is a characteristic lesion in bvdv infection in ruminants and canine and feline parvovirus infections ("punchedout peyer's patches) (see chapters 4 and 7). the main portals of entry to malt for infectious agents are hematogenous spread and through migrating macrophages, dcs , and m cells. pathogenic bacteria such as escherichia coli, yersinia pestis, mycobacterium avium ssp. paratuberculosis (map), l. monocytogenes, salmonella spp., and shigella flexneri can invade the host from the lumen of the intestine through dendritic or m cells. some viruses (e.g., reovirus) may be transported by m cells. the scrapie prion protein (prp sc ) may also accumulate in peyer's patches. many viruses, such as bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus, cause lymphocyte depletion within the malt. portals of entry used by microorganisms and other agents and substances to access the lymphoid system are summarized in box 13-6. defense mechanisms used by malt to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. congenital disorders of the thymus are discussed in detail in chapter 5. summaries of the gross and microscopic morphologic changes are solitary lymphoid nodules are localized concentrations of lymphocytes (mainly b lymphocytes) in the mucosa and consist of defined but unencapsulated clusters of small lymphocytes (primary lymphoid nodule). they are usually not grossly visible in the resting or antigenically unstimulated state, but upon antigenic stimulation, they proliferate and form germinal centers and surrounding mantle cell zones (secondary lymphoid nodules). aggregated lymphoid nodules consist of groups of lymph nodules, the most notable of which are the tonsils and peyer's patches. the aggregated lymphoid follicles of the peyer's patches are most obvious in the ileum. the latter are covered by a specialized epithelium, the follicle-associated epithelium (fae). the fae is the interface between the peyer's patches and the luminal microenvironment and consists of enterocytes and interdigitated m cells. m cells transport (via endocytosis, phagocytosis, pinocytosis, and micropinocytosis) antigens, particles, bacteria, and viruses from the intestinal lumen to the underlying area rich in dcs , which deliver the material to the lymphoid tissue of the peyer's patches. m cells also express iga receptors, which allows for the capture and transport of bacteria entrapped by iga. the proportion of enterocytes and m cells within the fae is modulated by the luminal bacterial a b mercury have a suppressive effect on the immune system. halogenated aromatic hydrocarbons cause dysfunction of dcs through several mechanisms that lead to atrophy of the primary and secondary lymphoid organs. heavy metals, such as lead, mercury and nickel, are immunosuppressive and generally affect the levels of b and t lymphocytes, nk cells, and inflammatory cytokines. other metals, such as selenium, zinc, and vanadium, may be immunostimulatory at low doses. the immunotoxic mechanisms may differ and include chelation of molecules and effects on protein synthesis, cell membrane integrity, and nucleic acid replication. the toxic effects of mycotoxins such as fumonisins b 1 and b 2 (secondary fungal metabolites produced by members of the genus fusarium) and aflatoxin (produced by aspergillus flavus) include lymphocytolysis in the thymic cortex. box 13-10 responses of mucosa-associated lymphoid tissue to injury described in the sections on disorders of horses and disorders of dogs. thymic cysts can be found within the developing and mature thymus and in thymic remnants in the cranial mediastinum. thymic cysts are often lined by ciliated epithelium and represent developmental remnants of branchial arch epithelium and are usually of no significance. thymitis is an uncommon lesion and may be seen in pcv2 infection (see disorders of pigs and also chapter 4), enzootic bovine abortion (see chapter 18), and salmon poisoning disease of dogs (see chapter 7). infectious agents more commonly cause thymic atrophy. variable degrees of acquired immunodeficiency can be also be caused by toxins, chemotherapeutic agents and radiation, malnutrition, aging, and neoplasia. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv-1 in aborted foals (fig. 13-52) , classic swine fever virus, bvdv, canine distemper virus, canine and feline parvovirus, and fiv; severe thymic lymphoid depletion is an early lesion in fiv-infected kittens. environmental toxins, such as halogenated aromatic hydrocarbons (e.g., polychlorinated biphenyls and dibenzodioxins), lead, and thymic hyperplasia. asymptomatic hyperplasia may occur in juvenile animals in association with immunizations and results in symmetrical increase in the size of the thymus. autoimmune lymphoid hyperplasia of the thymus has germinal center formation and occurs with myasthenia gravis. asplenia or the failure of a spleen to develop in utero occurs rarely in animals, and the effect on the animal's immune status is uncertain. (splenic aplasia is present in certain strains of mice, but because these are usually maintained under either germ-free or specific pathogen-free [spf] conditions, the effect of asplenia cannot be evaluated.) congenital immunodeficiency diseases are described in detail in chapter 5, and in the sections on disorders of horses and disorders of dogs. gross examination of the spleen involves deciding whether the spleen is enlarged (splenomegaly), normal, or small (see e-appendix 13-2). diffuse enlargement of the spleen may be due to congestion (termed bloody spleen) or other infiltrative disease (termed meaty spleen). the cut surface of congested spleens will exude blood, whereas meaty spleens are more firm and do not readily ooze blood. the diseases and disorders having splenomegaly are discussed using the following categories, which list the common causes of uniform splenomegaly (table 13-6): • uniform splenomegaly with a bloody consistency (bloody spleen) ( fig. 13-54 , a) • uniform splenomegaly with a firm consistency (meaty spleen) (see fig. 13-54 , b) • splenic nodules with a bloody consistency • splenic nodules with a firm consistency uniform splenomegaly with a bloody consistency-bloody spleen. the common causes of a bloody spleen are (1) congestion (due to gastric volvulus with splenic entrapment, splenic volvulus chemotherapeutic drugs inhibit the cell cycle through various mechanisms, and thus all dividing cells, including lymphocytes, bone marrow cells, and enterocytes, are sensitive to their effects. as such, bone marrow suppression, immunosuppression, and gastrointestinal disturbances are common side effects of anticancer drugs. purine analogues (e.g., azathioprine) compete with purines in the synthesis of nucleic acids, whereas alkylating agents like cyclophosphamide cross-link dna and inhibit the replication and activation of lymphocytes. cyclosporin a specifically inhibits the t lymphocyte signaling pathway by interfering with the transcription of the il-2 gene. methotrexate, a folic acid antagonist, blocks the synthesis of thymidine and purine nucleotides. the immunosuppressive effects of some of these agents is desirable for the treatment of immune-mediated disease (e.g., immune-mediated hemolytic anemia) or to prevent allograft rejection after transplantation. corticosteroids may be given at an immunosuppressive dose, though the degree of suppression is highly variable among species. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissue is within the therapeutically irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. malnutrition and cachexia, which may occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. thymic function may be impaired in young malnourished animals, resulting in a decrease in circulating t lymphocytes and subsequent depletion of t lymphocyte regions of secondary lymphoid organs. lymphoid atrophy may result from physiologic and emotional stress, which can cause the release of catecholamines and glucocorticoids. as part of the general effects of aging in cells (see chapter 1), all lymphoid organs decrease in size (atrophy) with advancing age. in the case of the thymus this reduction in size occurs normally after sexual maturity and is more appropriately termed thymic involution. the term involution should be reserved for normal physiologic processes in which an organ either returns to normal size after a period of enlargement (e.g., postpartum uterus) or regresses to a more primitive state (e.g., thymic involution). because the thymus has both lymphoid and epithelial components, neoplasms may arise from either component. thymic lymphoma arises from the t lymphocytes in the thymus (and very rarely b lymphocytes). it is most often seen in young cats and cattle and less frequently in dogs (fig. 13-53 ) (see hematopoietic neoplasia). thymomas arise from the epithelial component and are usually benign neoplasms that occupy the cranial mediastinum of older animals. histologically, these neoplasms consist of clustered or individualized neoplastic epithelial cells, often outnumbered by nonneoplastic small lymphocytes ("lymphocyte-rich thymoma"). thymomas are common in goats and often contain large cystic structures. immunemediated diseases, including myasthenia gravis and immunemediated polymyositis, occur with thymomas in dogs, and also rarely in cats. myasthenia gravis is caused by autoantibodies directed toward the acetylcholine receptors, which lead to destruction of postsynaptic membranes and reduction of acetylcholine receptors at neuromuscular junction. megaesophagus and aspiration pneumonia are common sequelae to this condition. (syn: necropsy) in horses and dogs that have been euthanized or anesthetized with barbiturates. grossly, the spleen is extremely enlarged (fig. 13-55) , and the cut surface bulges and oozes copious blood. because of the splenic distention, the splenic capsule can be fragile and easily ruptured. histologically, the red pulp is distended by erythrocytes, and the lymphoid tissues of the white pulp are small and widely separated (fig. 13-56 ). electric stunning of pigs at slaughter may result in a large congested spleen; the mechanism is unknown, but it should not be confused with a pathologically congested spleen. splenic congestion in acute cardiac failure is rarely seen in animals. acute congestion/hyperemia. acute septicemias may cause acute hyperemia and concurrent acute congestion of marginal zones and splenic red pulp. microbes are transported hematogenously to these sites, where they are rapidly phagocytized by macrophages. enormous numbers of intravenous bacteria can be cleared by the spleen from the blood in 20 to 30 minutes, but when this defensive mechanism is overwhelmed, the outcome is usually fatal. the response of the spleen depends on the duration of the disease. in acutely fatal cases, such as anthrax and fulminating salmonellosis, distention by blood may be the only gross finding. if the animal survives longer, as in swine erysipelas and the less virulent forms of [all of which compress the splenic vein], and barbiturate euthanasia, anesthesia, or sedation), (2) acute hyperemia (due to septicemia), and (3) acute hemolytic anemia (due to an autoimmune disorder or an infection with a hemotropic parasite). splenic torsion. torsion of the spleen occurs most commonly in pigs and dogs; in dogs this usually involves both spleen and stomach and is seen more often in deep-chested breeds (see chapter 7). in contrast to ruminants, in which the spleen is firmly attached to the rumen, the spleens of dogs and pigs are attached loosely to the stomach by the gastrosplenic ligament. it is the twisting of the spleen around this ligament that results initially in occlusion of the veins, causing splenic congestion, and later in occlusion of the artery, causing splenic infarction. in dogs the spleen is uniformly and markedly enlarged and may be blue-black from cyanosis. it is often folded back on itself (visceral surface to visceral surface) in the shape of the letter "c." treatment for this condition is most often splenectomy. barbiturate euthanasia, anesthesia, or sedation. intravenous injection of barbiturates induces acute passive congestion in the spleen due to relaxation of smooth muscle in the capsule and trabeculae. this phenomenon is seen most dramatically at autopsy only histologic lesion may be marked congestion of the marginal sinuses and the splenic red pulp vascular spaces. at low magnification, congestion of the marginal sinus may appear as a circumferential red ring around the splenic follicle, and there is marked lymphocytolysis of follicles and pals. intravascular free bacilli are noted and may be seen in impression smears of peripheral blood, presumably because death is so rapid from the anthrax toxin that there is insufficient time for phagocytosis to take place. if the animal lives longer, scattered neutrophils are present in the marginal sinuses and red pulp vascular spaces (fig. 13-57 ). anthrax cases are not normally autopsied because exposure to air causes the bacteria to sporulate-anthrax spores are extremely resistant and readily contaminate the environment. acute hemolytic anemias. hemolytic diseases, including acute babesiosis, hemolytic crises in equine infectious anemia, and immune-mediated hemolytic anemia, can cause marked splenic congestion. the splenic congestion is due to the process of removal (phagocytosis) and storage of large numbers of sequestered parasitized and/or altered erythrocytes from the circulation. histologically, there is dilation of the red pulp vascular spaces with erythrocytes and erythrophagocytes. with chronicity there is hyperplasia of the red pulp macrophages, hemosiderosis, and reduced congestion because the number of sequestered diseased erythrocytes is diminished. spleen. the three general categories of conditions leading to uniform splenomegaly with a firm meaty consistency are (1) marked phagocytosis of cells, debris, or foreign agents/material; (2) proliferation or infiltration of cells as occurs in diffuse lymphoid and histiocytic hyperplasia, diffuse granulomatous disease (e -table 13 -2), emh, and neoplasia; (3) storage of materials in storage diseases or amyloidosis. it is important to recognize that more than one of these processes can occur in the same patient (e.g., dogs with immunemediated hemolytic anemia may have both marked erythrophagocytosis and emh). the appearance of the cut surface of a meaty spleen depends on the underlying cause. in diffuse marked lymphoid hyperplasia, large, disseminated, discrete, white, bulging nodules are visible. spleens with diffuse infiltrative neoplasms, such as lymphoma, are pink-light purple on cut surface. diffuse lymphoid hyperplasia. lymphoid hyperplasia has been described in detail in the section on dysfunction/responses to injury. in cases of prolonged antigenic stimulation the lymphoid salmonellosis, there may be sufficient time for neutrophils and macrophages to accumulate in the marginal sinuses, marginal zones, and splenic red pulp vascular spaces. anthrax. b. anthracis, the causative agent of anthrax, is a grampositive, large, endospore-forming bacillus, which grows in aerobic to facultative anaerobic environments. anthrax is primarily a disease of ruminants, especially cattle and sheep (see chapters 4, 7, 9, and 10) . once the spores are ingested, they replicate locally in the intestinal tract, spread to regional lymph nodes, and then disseminate systemically through the bloodstream, resulting in septicemia. b. anthracis produces exotoxins, which degrade endothelial cell membranes and enzyme systems. grossly, the spleen is uniformly enlarged and dark red to bluishblack and contains abundant unclotted blood. in peracute cases the 782.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system diffuse granulomatous disease. chronic infectious diseases may cause a uniformly firm and enlarged spleen, mostly due to macrophage hyperplasia and phagocytosis, diffuse lymphoid hyperplasia, or diffuse granulomatous disease. diffuse granulomatous diseases (see etable 13 -2) occur in (1) intracellular facultative bacteria that infect macrophages (e.g., mycobacterium spp., brucella spp., and francisella tularensis); (2) systemic mycoses (e.g., blastomyces dermatitidis, histoplasma capsulatum) (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly]) ( fig. 13-59, a and b) , and (3) protozoal infections that infect macrophages (e.g., leishmania spp.). some of these organisms may also produce nodular spleens with the formation of discrete to coalescing granulomas (e.g., m. bovis) (see splenic nodules with a firm consistency). follicles throughout the splenic parenchyma can become enlarged and visible on gross examination (fig. 13-58 ), leading to diffuse splenomegaly. in contrast to b lymphocyte hyperplasia of the lymphoid follicles, certain diseases (e.g., malignant catarrhal fever in cattle) may lead to t lymphocyte hyperplasia of the pals. diffuse histiocytic hyperplasia and phagocytosis. splenomegaly from hyperplasia and increased phagocytosis of splenic macrophages is a response to the need to engulf organisms in prolonged bacteremia or parasitemia from hemotropic organisms. whereas acute hemolytic anemias cause splenomegaly with congestion (bloody spleen), with chronicity there is decreased sequestration of diseased erythrocytes and hence less congestion. therefore in cases of chronic hemolytic disease, splenomegaly is attributed to diffuse proliferation of macrophages, phagocytosis, and concurrent hyperplasia of the white pulp due to ongoing antigenic stimulation. for example, equine infectious anemia has cyclical periods of viremia, with immune-mediated damage to erythrocytes and platelets, and phagocytosis to remove altered erythrocytes and platelets. these cycles result in proliferation of red pulp macrophages, hyperplasia of hematopoietic cells (emh) to replace those lost, and hyperplasia of lymphocytes in the white pulp. in animals less than 1 year of age. in general, these substrates are lipids and/or carbohydrates that accumulate in the cells, the result of the lack of normal processing within lysosomes. major categories of stored materials include mucopolysaccharides, sphingolipids, glycolipids, glycoproteins, glycogen, and oligosaccharides. macrophages are commonly affected by storage diseases, and thus extramedullary hematopoiesis. emh is the development of blood cells in tissues outside the medullary cavity of the bone (efig. 13-9 ). the formation of single or multiple lineages of hematopoietic cells is often observed in many tissues and commonly in the spleen. the ability of blood cell precursors to home, proliferate, and mature in extramedullary sites relies on the presence of hscs and pathophysiologic changes in the microenvironment (i.e., extracellular matrix, stroma, and chemokines). in the spleen, hscs have been found within vessels and adjacent to endothelial cells to form a vascular niche; thus splenic emh occurs in the red pulp, both within the red pulp vascular spaces and sinusoids (of the dog). the predilection for emh to occur varies among species (for instance, splenic emh persists throughout adulthood in mice), and the underlying mechanisms are not completely understood, but four major theories to explain the causes of emh are (1) severe bone marrow failure; (2) myelostimulation; (3) tissue inflammation, injury, and repair; and (4) abnormal chemokine production. because splenic emh is often observed in animals without obvious hematologic abnormalities, tissue inflammation, injury, and repair is the most likely mechanism of emh in this organ. in dogs and cats emh occurs most frequently with degenerative and inflammatory disorders, such as lymphoid nodular hyperplasia, hematomas, thrombi, histiocytic hyperplasia, inflammation (e.g., fungal splenitis), and neoplasia. emh in multiple tissues may be observed in chronic cardiovascular or respiratory conditions, chronic anemia, or chronic suppurative diseases in which there is an excessive tissue demand for neutrophils that exceeds the supply available from the marrow (e.g., canine pyometra). primary neoplasms. primary neoplastic diseases of the spleen arise from cell populations that normally exist in the spleen and include hematopoietic components, such as lymphocytes, mast cells, and macrophages, and stromal cells, such as fibroblasts, smooth muscle, and endothelium. the primary neoplasms that result in diffuse splenomegaly are the round cell tumors, including lymphoma ( fig. 13-60) , leukemia, visceral mast cell tumor, and histiocytic sarcoma. it is important to note that all of these types of neoplasms can produce nodular lesions instead of-or along with-a diffusely enlarged spleen. the different types of lymphoma in domestic animals are discussed in the section on hematopoietic neoplasia. secondary neoplasms of the spleen are due to metastatic spread and most often form nodules in the spleen, not a uniform splenomegaly. amyloid. the accumulation of amyloid in the spleen may occur with primary (al) or secondary (aa) amyloidosis (see chapters 1 and 5). rarely, severe amyloid accumulation may cause uniform splenomegaly ( fig. 13-61) , in which the spleen is firm, rubbery to waxy, and light brown to orange. microscopically, amyloid is usually in the splenic follicles, which if large enough, are grossly visible as approximately 2-mm-diameter gray nodules. amyloid deposition can also be seen within the walls of splenic veins and arterioles. plasma cells tumors within the spleen may also be associated with amyloid (al) deposits. lysosomal storage diseases. storage diseases are a heterogeneous group of inherited defects in metabolism characterized by accumulation of storage material within the cell (lysosomes). genetic defects, which result in the absence of an enzyme, the synthesis of a catalytically inactive enzyme, the lack of activator proteins, or a defect in posttranslational processing, can lead to a storage disease. acquired storage diseases are caused by exogenous toxins, most often plants that inhibit a particular lysosomal enzyme (e.g., swainsonine toxicity due to indolizidine alkaloid found in astragalus and oxytropis plant spp.). storage diseases typically occur diagnosis, it may be difficult (and futile) to determine the primary site. histologically, hemangiosarcomas are composed of plump neoplastic endothelial cells, which wrap around stroma to form haphazardly arranged and poorly defined blood-filled vascular spaces ( fig. 13-66 ). splenic nodules with a firm consistency. the most common disorders of the spleen with firm nodules are (1) lymphoid nodular hyperplasia, (2) complex nodular hyperplasia, (3) primary neoplasms, (4) secondary metastatic neoplasms, (5) granulomas, and (6) abscesses. lymphoid and complex nodular hyperplasia. see lymphoid/ lymphatic system, disorders of dogs. primary neoplasms. the primary neoplastic diseases of the spleen that result in firm nodules include lymphoma (multiple subtypes), histiocytic sarcoma, leiomyoma, leiomyosarcoma, fibrosarcoma, myelolipomas, liposarcomas, myxosarcomas, undifferentiated pleomorphic sarcomas, solid hemangiosarcomas, and rare reports of primary chondrosarcomas. these locally extensive neoplasms may be solitary or multiple, raised above the capsular surface, but usually confined by the capsular surface. the consistency and cut surface appearance varies depending on the type of neoplasm; spindle cell tumors like leiomyosarcomas and fibrosarcomas will be white and firm, liposarcomas and myelolipomas are soft and bulging, and myxomatous neoplasms are gelatinous. it is important to remember that many round cell neoplasms, such as lymphoma, mast cell tumors, plasma cell tumors, myeloid neoplasms, and histiocytic sarcomas, can form nodules or diffuse splenic enlargement (or both). metastatic neoplasms. neoplasms that metastasize to the spleen usually result in enlarged nodular spleens (fig. 13-67 ) and include any number of sarcomas, carcinomas, or malignant round cell tumors. metastatic sarcomas can include fibrosarcomas, leiomyosarcomas, chondrosarcomas, and osteosarcomas. mammary, prostatic, pulmonary, anal sac gland and neuroendocrine carcinomas may metastasize widely to abdominal viscera, including the spleen. granulomas and abscesses. microorganisms that cause diffuse granulomatous splenitis and uniform splenomegaly may also cause focal to multifocal nodular lesions (e.g., mycobacterium spp., fungal organisms) (see diffuse granulomatous diseases of the spleen and also enlarged lymph nodes). although there are a large number of abscesses bulge from the capsule and cut surfaces, and the exudate can vary in amount, texture, and color depending on the inciting organism and the age of the lesion. the most common diseases or conditions that have small spleens are (1) developmental anomalies, (2) aging changes, (3) wasting and/or cachectic diseases, and (4) splenic contraction. splenic hypoplasia. primary immunodeficiency diseases can result in splenic hypoplasia, as well as small thymuses and lymph nodes (which may be so small as to be grossly undetectable in some diseases). these diseases affect young animals and involve defects in t and/or b lymphocytes (fig. 13-70) . spleens are exceptionally small, firm, and pale red and lack lymphoid follicles and pals. these diseases and their pathologic findings are discussed in chapter 5 and in the sections on disorders of horses and disorders of dogs. congenital accessory spleens. accessory spleens can be either congenital or acquired (see splenic rupture). congenital accessory spleens are termed splenic choristomas, which are nodules of normal splenic parenchyma in abnormal locations. these are usually small diseases and conditions commonly caused by bacteremia (e.g., navel ill, joint ill, chronic respiratory infections, bacterial endocarditis, chronic skin diseases, castration, tail docking, and ear trimming and/ or notching), these rarely result in visible splenic abscesses. pyogranulomas and abscesses in the spleen (multifocal chronic suppurative splenitis) that do develop after septicemia and/or bacteremia are usually caused by pyogenic bacteria such as streptococcus spp., rhodococcus equi (fig. 13-68) , trueperella pyogenes ( fig. 13-69) , and corynebacterium pseudotuberculosis. cats with the wet or dry form of feline infectious peritonitis virus may have nodular pyogranulomatous and lymphoplasmacytic inflammatory foci throughout the spleen. splenic abscesses due to direct penetration by a migrating foreign body are reported in cattle (from the reticulum) and less commonly in the horse (from the stomach). perforating gastric ulcers in horses due to gasterophilus and habronema spp. have also reportedly led to adjacent splenic abscesses. granulomas and a b and may be located in the gastrosplenic ligament, liver, or pancreas (see fig. 13-74, b) . splenic fissures. fissures in the splenic capsule are elongated grooves whose axes run parallel to the borders of the spleen. this developmental defect is seen most commonly in horses but also occurs in other domestic animals and has no pathologic significance. the surface of the fissure is smooth and covered by the normal splenic capsule. aging changes. as part of the general aging change of cells as the body ages, there is reduction in the number of b lymphocytes produced by the bone marrow and decline of naïve t lymphocytes due to age-related thymic involution. consequently, there is lymphoid atrophy in secondary lymphoid organs. the spleen is small, and its capsule may be wrinkled. microscopically, the white pulp is atrophied, and splenic follicles, if present, lack germinal centers. sinuses may also collapse from a reduced amount of blood, possibly because of anemia, which makes the red pulp appear fibrous. wasting/cachectic diseases. any chronic disease, such as starvation, systemic neoplasia, and malabsorption syndrome, may produce cachexia. starvation has a marked effect on the thymus, which results in atrophy of the t lymphocyte areas in the spleen and lymph nodes, which is in part mediated by leptin. b lymphocyte development is also diminished, because b lymphocytes require accessory signals from helper t lymphocytes to undergo somatic hypermutation and immunoglobulin isotype switching. splenic contraction. contraction of the spleen is a result of contraction of the smooth muscle in the capsule and trabeculae of storage spleens. it can be induced by the activation of the sympathetic "fight-or-flight" response and is seen in patients with heart failure or shock (cardiogenic, hypovolemic, and septic shock) and also occurs in acute splenic rupture that has resulted in massive hemorrhage (hemoabdomen/hemoperitoneum). the contracted spleen is small, its surface is wrinkled, and the cut surface is dry. hemosiderosis. hemosiderin is a form of storage iron derived chiefly from the breakdown of erythrocytes, which normally takes place in the splenic red pulp. thus some splenic hemosiderosis is to be expected, and the amount varies with the species (it is most extensive in the horse). excessive amounts of splenic hemosiderin are seen when erythropoiesis is reduced (less demand for iron) or from the rapid destruction of erythrocytes in hemolytic anemias (increased stores of iron), such as those caused by immune-mediated hemolytic anemias or hemotropic parasites. excess splenic hemosiderin may also occur in conditions such as chronic heart failure or injections of iron dextran or as focal accumulations at the sites of old hematomas, infarcts, or trauma-induced hemorrhages. hemosiderin is also present in siderofibrotic plaques. siderofibrotic plaques. siderofibrotic plaques are also known as siderocalcific plaques and gamna-gandy bodies. grossly, they are gray-white to yellowish, firm, dry encrustations on the splenic capsule. usually they are most extensive along the margins of the spleen but can be elsewhere on the capsule (fig. 13-71 ) and sometimes in the parenchyma. with h&e staining these plaques are a multicolored mixture of yellow (hematoidin), golden brown (hemosiderin), purple-blue (hematoxylinophilic calcium mineral), and pink (eosinophilic fibrous tissue) (fig. 13-72 the diseases or conditions with small lymph nodes are (1) congenital disorders, (2) lack of antigenic stimulation, (3) viral infections, (4) cachexia and malnutrition, (5) aging, and (6) radiation. congenital disorders. primary immunodeficiency diseases are described in detail in chapter 5 and in the sections on disorders of horses and disorders of dogs. neonatal animals with primary immunodeficiency diseases often have extremely small to undetectable lymph nodes. in dogs and horses with severe combined immunodeficiency disease (scid), lymphoid tissues, including lymph nodes from affected animals, are often grossly difficult to identify and characterized by an absence of lymphoid follicles. congenital may represent sequelae to previous hemorrhages from trauma to the spleen. splenic rupture. splenic rupture is most commonly caused by trauma, such as from an automobile accident or being kicked by other animals. thinning of the capsule from splenomegaly can render the spleen more susceptible to rupture, and this may occur at sites of infarcts, hematomas, hemangiosarcomas, and lymphoma. in acute cases of splenic capsular rupture, the spleen is contracted and dry and the surface wrinkled from the marked blood loss (fig. 13-73) . in more severe cases the spleen may be broken into two or more pieces, and small pieces of splenic parenchyma may be scattered throughout the omentum and peritoneum (sometimes called splenosis) (fig. 13-74, a) . clotted blood, fibrin, and omentum may adhere to the surface at the rupture site. if the rupture is not fatal, the spleen heals by fibrosis, and there may be a capsular scar. occasionally there are two or more separate pieces of spleen adjacent to each other and sometimes joined by scar tissue in the gastrosplenic ligament. the functional capabilities of the small accessory spleens are questionable, although erythrophagocytosis, hemosiderosis, hyperplastic nodules, emh, and neoplasia can be present in these nodules. accessory spleens due to traumatic rupture should be distinguished from peritoneal seeding of hemangiosarcoma and the developmental anomaly splenic choristomas (see fig. 13-74, b) , which are nodules of normal splenic parenchyma in abnormal locations (such as liver and pancreas). chronic splenic infarcts. in the early stage, splenic infarcts are hemorrhagic and may elevate the capsule (see splenic nodules with a bloody consistency). however, as the lesions age and fibrous connective tissue is laid down, they shrink and become contracted and often depressed below the surface of the adjacent capsule. conditions causing lymphadenomegaly include (1) lymphoid hyperplasia (follicular or paracortical), (2) hyperplasia of the sinus histiocytes (monocyte-macrophage system), (3) acute or chronic lymphadenitis, (4) lymphoma, and (5) metastatic neoplasia. macrophage system. detailed descriptions of lymphoid follicular hyperplasia, paracortical hyperplasia, and hyperplasia of sinus histiocytes are in the sections on lymph nodes, function, and lymph node, dysfunction/responses to injury. follicular lymphoid hyperplasia can involve large numbers of lymph nodes, as in a systemic disease, or can be localized to a regional lymph node draining an inflamed or antigenically stimulated (e.g., vaccine injection) area. acute lymphadenitis. lymph nodes draining sites of infection and inflammation may develop acute lymphadenitis (e.g., retropharyngeal lymph nodes draining the nasal cavity with acute rhinitis, tracheobronchial lymph nodes in animals with pneumonia ( fig. 13-75 ), and mammary [supramammary] lymph nodes in animals with mastitis). grossly, affected lymph nodes in acute lymphadenitis are red and edematous, have taut capsules, and may have necrotic areas ( fig. 13-76 ). in some instances the afferent lymphatic vessels may also be inflamed (lymphangitis). the material draining to the regional lymph node may be microorganisms (bacteria, parasites, protozoa, and fungi), inflammatory mediators, or a sterile irritant. in septicemic diseases, such as bovine anthrax, the lymph nodes are markedly congested and the sinuses filled with blood. examination of these lymph nodes should include culturing for bacteria and the examination of smears and histologic sections for bacteria and fungi. pyogenic bacteria, such as streptococcus equi ssp. equi in horses , streptococcus porcinus in pig, and trueperella pyogenes in cattle and sheep, cause acute suppurative lymphadenitis (see disorders of horses and disorders of pigs). hereditary lymphedema has been reported in certain breeds of cattle and dogs. grossly, the most severely affected animals have generalized subcutaneous edema (see fig. 2 -10) and effusions. in severe cases the peripheral and mesenteric lymph nodes are hypoplastic and characterized by an absence of follicles. nodes draining an edematous area may be grossly enlarged from marked sinus edema. lack of antigenic stimulation. the size of the lymph node depends on the level of phagocytosis and antigenic stimulation; lymph nodes that are not receiving antigenic stimuli (e.g., spf animals) will be small with low numbers of primary lymphoid follicles and few, if any, secondary follicles or plasma cells in the medullary cords. conversely, nodes receiving constant antigenic material (such as those draining the oral cavity or intestines) are large with active secondary lymphoid follicles. the number of follicles increases or decreases with changes in the intensity of the antigenic stimuli, and the germinal centers go through a cycle of activation, depletion, and rest, as described previously (see lymph nodes, function). as the antigenic response wanes, germinal centers become depleted of lymphocytes, and lymphoid follicles become smaller. viral infections. many viral infections of animals target lymphocytes and cause the destruction of lymphoid tissue. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv-1 in aborted foals, classic swine fever virus, bvdv, canine distemper virus, and canine and feline parvovirus. although some viruses destroy lymphoid tissue, others can lead to lymph node hyperplasia (e.g., follicular b lymphocyte hyperplasia in fiv and paracortical t lymphocyte hyperplasia in malignant catarrhal fever virus) or cause neoplasia (e.g., felv, blv, and marek's disease). cachexia and malnutrition. malnutrition and cachexia, which occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. starvation has marked effect on the thymus with resultant atrophy of the t lymphocyte areas in the spleen and lymph nodes and may also affect b lymphocyte development. lymphoid atrophy may result from physiologic and emotional stress and the concurrent release of catecholamines and glucocorticoids. glucocorticoids reduce b and t lymphocytes via redistribution of these cells and glucocorticoidinduced apoptosis. t lymphocytes are more sensitive to glucocorticoid-induced apoptosis than are b lymphocytes. aging. as part of the general aging change of cells as the body ages, there is reduction in the number of lymphocytes produced by the bone marrow and regressed thymus, and consequently a reduction in the b and t lymphocytes in secondary lymphoid organs, resulting in lymphoid atrophy. consequently, lymph nodes are small, with loss of b and t lymphocytes and plasma cells in the cortical follicles, paracortex, and medullary cords, respectively. radiation. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissues are within the irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. fibrosis of tissues within the irradiated field also occurs as mainly a late effect of chronic radiation. bouts of chronic lymphadenitis (e.g., regional lymph node draining chronic mastitis in cows) lead to fibrosis and lymphoid hyperplasia, in addition to chronic abscesses. the classic example of chronic suppurative lymphadenitis with encapsulated abscesses is caseous lymphadenitis, a disease of sheep and goats caused by c. (also see disorders of ruminants). it is also the cause of ulcerative lymphangitis in cattle and horses and pectoral abscesses in horses. classic examples of focal to multifocal granulomatous lymphadenitis are mycobacterium tuberculosis complex, which includes m. bovis among others. members of m. avium complex cause similar lesions and have been described in a number of species, including dogs, cats, primates, pigs, cattle, sheep, horses, and human beings. infection may begin by inhalation of aerosol droplets containing the bacilli, which may spread via the lymphatic vessels to regional lymph nodes, resulting in granulomatous lymphangitis and lymphadenitis (fig. 13-81) . initially lesions in the lymphatic system are confined to the lymphatic vessels (granulomatous lymphangitis) and regional lymph nodes (e.g., the tracheobronchial lymph nodes in the case of pulmonary histologically, the subcapsular, trabecular, and medullary sinuses and the parenchyma of the cortex and medulla have focal to coalescing foci of neutrophilic inflammation, necrosis, and fibrin deposition ( fig. 13-78 ). if inflammation in the lymph node continues for several days or longer, the lymph node is further enlarged by follicular hyperplasia and plasmacytosis of the medullary cords from the expected immune response. chronic lymphadenitis. the types of chronic lymphadenitis include chronic suppurative lymphadenitis, diffuse granulomatous inflammation, and discrete granulomas. in chronic suppurative inflammation, abscesses range in size from small microabscesses to large abscesses that occupy and obliterate the whole node. recurrent figure 13 -76 acute lymphadenitis, lymph node, dog. acute lymphadenitis usually occurs when a regional lymph node drains a site of inflammation caused by microorganisms and subsequently becomes infected. the lymph node is firm and enlarged with a tense capsule. the cut surface bulges and is wet with blood, edema, and an inflammatory cell infiltrate. histoplasmosis (see disorders in dogs). in feline cryptococcosis (most often cryptococcus neoformans), the inflammatory response may be mild due to the thick polysaccharide capsule, which has strong immunomodulatory properties and promotes immune evasion and survival within the host. therefore the nodal enlargement is due mainly to a large mass of organisms (see efig. 13-12) . pigs with pcv2 infection may have a multifocal to diffuse infiltrate of macrophages and multinucleated giant cells of varying severity (see disorders of pigs). secondary (metastatic) neoplasms. carcinomas typically metastasize via lymphatic vessels to the regional lymph node. other common metastatic neoplasms include mast cell tumor and malignant melanoma. although sarcomas most often metastasize hematogenously, some more aggressive sarcomas (e.g., osteosarcoma) may spread to regional lymph nodes. histologically, single cells or clusters of neoplastic cells travel via the afferent lymphatic vessels and are deposited in a sinus, usually the subcapsular sinus (see fig. 13 -47). here the cells proliferate and can ultimately occupy the whole lymph node, as well as drain to the next lymph node in the chain. tuberculosis), but once disseminated in the lymph or blood, lymph nodes throughout the body will have lesions. well-organized granulomas consist of a central mass of macrophages with phagocytized mycobacteria, surrounded by epithelioid and foamy macrophages and occasional multinucleated giant cells (langhans type). these inflammatory nodules are surrounded by a layer of lymphocytes enclosed in a fibrous capsule. over time the center of the granuloma may undergo caseous necrosis due to the high lipid and protein content of the dead macrophages (see chapter 3). in bovine johne's disease the mesenteric lymph nodes draining the infected intestine can have noncaseous granulomas (fig. 13-82 ). diffuse granulomatous lymphadenitis. coalescing to diffuse granulomatous lymphadenitis is seen in disseminated fungal infections such as blastomycosis, cryptococcosis (efig. 13-12) , and extent of the emphysema. in severe cases the lymph node is light, puffy, and filled with discrete gas bubbles, and the cut surface may be spongy. histologically, the sinuses are distended with gas and lined by macrophages and giant cells. this change has been considered a foreign body reaction to the gas bubbles. macrophages and giant cells are also seen in afferent lymphatic vessels (granulomatous lymphangitis). vascular transformation of lymph node sinus (nodal angiomatosis). vascular transformation of the sinuses is a nonneoplastic reaction to blocked efferent lymphatic vessels or veins. this pressure-induced lesion results in the formation of anastomosing vascular channels and may be confused with a nodal vascular neoplasm. these proliferative but noninvasive masses usually begin in the subcapsular sinuses and may be followed by lymphoid atrophy, erythrophagocytosis/hemosiderosis, and fibrosis. the blockage may be caused by malignant neoplasms of the tissues that the lymph node drains (e.g., thyroid carcinoma with nodal angiomatosis of the mandibular lymph node). see section on bone marrow, disorders of domestic animals, hematopoietic neoplasia for a discussion of the who classification of hematopoietic neoplasia that predominantly arise and proliferate within bone marrow. this section will cover neoplasms of lymphoid tissue(s) arising outside of bone marrow. lymphoma. the term lymphoma (also known as lymphosarcoma) encompasses a diverse group of malignancies arising in lymphoid tissue(s) outside of bone marrow. grossly, there may be diffuse to nodular enlargement of one or more lymph nodes (fig. 13-83) , and the cut surface is soft, white, and bulging with loss of normal corticomedullary architecture. there is great variation in the clinical manifestations and cytopathologic features of lymphoma, which underlie the importance of classification to better predict the clinical behavior and outcome. an understanding of lymphocyte maturation is crucial, because the who classification of lymphoma postulates a normal cell counterpart for each type of lymphoma (when possible). in other words, lymphoma can arise at any stage in the development/maturation of a lymphocyte-from precursor lymphocytes (b or t lymphoblasts) to mature lymphoid b and t, lymphocytes and nk cells (table 13 -7 and box 13-11). pathologists use gross features, histomorphologic features, immunophenotype (b or t lymphocyte), and clinical characteristics to red discoloration is caused by (1) draining erythrocytes from hemorrhagic or acutely inflamed areas, (2) acute lymphadenitis with hyperemia and/or hemorrhage, (3) acute septicemias with endotoxininduced vasculitis or disseminated intravascular coagulation, and (4) dependent areas in postmortem hypostatic congestion. blood in pig lymph nodes is especially obvious due to the inverse anatomy (the equivalent of the medullary sinuses are subcapsular and thus readily visible in the unsectioned node). initially erythrocytes fill trabecular and medullary sinuses and then rapidly undergo erythrophagocytosis by proliferating sinus macrophages. hemosiderin deposition occurs within 7 to 10 days in these macrophages, imparting a brown discoloration of the node. black discoloration is often present in the tracheobronchial lymph nodes due to draining of carbon pigment (pulmonary anthracosis, see chapter 9). black ink from skin tattoos will drain to the regional lymph node. these pigments are usually noted within the medullary sinus macrophages. brown discoloration may be due to melanin, parasitic hematin, or hemosiderin. melanin pigment is seen in animals with chronic dermatitis when melanocytes are damaged and their pigment is released into the dermis and phagocytized by melanomacrophages (pigmentary incontinence) and drained to the regional lymph node. the mandibular lymph nodes often contain numerous melanomacrophages in animals with heavily pigmented oral mucosa, presumably due to chronic low levels of inflammation. this must be distinguished from metastatic malignant melanomas. lymph nodes draining areas of congenital melanosis may have melanin deposits. parasitic hematin pigment is produced by fascioloides magna (cattle) and fasciola hepatica (sheep) in the liver and then transported via the lymphatic vessels to the hepatic lymph nodes. hemosiderin, an erythrocyte breakdown product, may form in a hemorrhagic node or arrive in hemosiderophages draining from congested, hemorrhagic, or inflamed areas. drainage of iron dextran from an intramuscular injection may also cause hemosiderin pigment accumulation within the draining lymph node. green discoloration is rare and may be caused by green tattoo ink (often used in black animals); ingestion of blue-green algae, which drain to mesenteric lymph nodes; massive eosinophilic inflammation; and in mutant corriedale sheep, which have a genetic defect that results in a deficiency in the excretion of bilirubin and phylloerythrin by the liver. the phylloerythrin or a metabolite stains all the tissues of the body a dark green, except for the brain and spinal cord, which are protected by the blood-brain barrier. miscellaneous discolorations of lymph nodes may be seen with intravenously injected dyes (e.g., methylene blue or trypan blue) or subcutaneous drug injections. lymph nodes may be yellow in severely icteric patients. the pigmented strain of map (johne's disease) may impart an orange discoloration in the mesenteric lymph nodes of sheep. inclusion bodies. many viruses produce inclusion bodies, and some of these occur in lymph nodes. these viruses include ehv-1 in horses, bovine adenovirus, cytomegalic virus in inclusion body rhinitis and pcv2, herpesvirus of pseudorabies in pigs, and rarely parvovirus in dogs and cats. emphysema. emphysema in lymph nodes is a consequence of emphysema in their drainage fields and is seen most frequently in tracheobronchial lymph nodes in bovine interstitial emphysema and in porcine mesenteric lymph nodes in intestinal emphysema (see chapter 7). the appearance of the lymph node varies with the immunohistochemistry (mum1/irf4 is particularly sensitive and specific for plasma cell neoplasms). histiocytic disorders. histiocytic disorders are frequently diagnosed in dogs and occur less often in cats. briefly, histiocytes are categorized as macrophages and dcs, the latter of which are subdivided into langerhans cells (lcs), found in skin, gastrointestinal, respiratory, and reproductive epithelia (mucosae), and interstitial dcs (idc), located in perivascular spaces of most organs. the term interdigitating dcs describes dcs (either resident or migrating) found in t lymphocyte regions of lymph nodes (paracortex) and spleen (pals); interdigitating dcs consist of both lcs and idcs. these lineages can be differentiated using immunohistochemical stains. histiocytic disorders that are diagnosed in veterinary medicine at this time include the following: canine cutaneous histiocytoma, canine lc histiocytosis, canine cutaneous and systemic histiocytosis, feline pulmonary lc histiocytosis, feline progressive histiocytosis, dendritic cell leukemia in the dog, and histiocytic sarcoma and hemophagocytic histiocytic sarcoma in both dogs and cats. lymph node involvement is seen in many of these conditions. rare reports of regional lymph node metastasis in cases of solitary canine cutaneous histiocytoma have been published. lymphatic invasion with subsequent regional nodal involvement may be seen in dogs with lc histiocytosis, which is a poor prognostic indicator and likely reflects systemic infiltration. the normal architecture of tracheobronchial lymph nodes is often effaced in cats with pulmonary lc histiocytosis. canine reactive histiocytoses are not clonal neoplastic proliferations but likely reflect an immune dysregulation consisting of activated dermal idcs (and t lymphocytes). they are categorized as cutaneous histiocytosis (ch), involving skin and draining lymph nodes, and a more generalized systemic histiocytosis (sh), affecting skin and other sites (e.g., lung, liver, bone marrow, spleen, lymph nodes, kidneys, and orbital and nasal tissues). histiocytic sarcoma complex. histiocytic sarcomas (hss) are neoplasms of idcs and therefore can arise in almost any tissue, frequently the spleen, lung, skin, meninges, lymph nodes, bone marrow, and synovium. secondary involvement of the liver is common as the disease progresses. this neoplasm is most commonly diagnosed in dogs, and a lower incidence is seen in cats. localized histiocytic sarcoma may be a focal solitary lesion or multiple nodules within a single organ. disseminated histiocytic sarcoma describes classify lymphomas. the morphologic features used in histopathologic classification are the following: • histologic pattern-nodular or diffuse. • cell size-the nuclei of the neoplastic lymphocytes are compared to the diameter of a red blood cell (rbc ≅ 5 µm). small is less than 1.5 times the diameter of an rbc; intermediate is 1.5 to 2.0 times the diameter of an rbc; large is more than 2.0 times the diameter of an rbc. • grade-mitotic figures are counted in a single high-power (400×) field. indolent is 0 to 1; low is 2 to 5; mid is 6 to 10; high is more than 10. although there are numerous subtypes of lymphoma recognized under the who system, a detailed discussion of each subtype is outside the scope of this textbook. however, a select number of subtypes are more commonly seen in domestic animals (see table 13 -7) and currently best described in the dog (see disorders of dogs, neoplasms, lymphomas). the most common types in dogs are large cell lymphomas and include diffuse large b cell lymphoma and peripheral t cell lymphoma. t cell-rich large b cell lymphoma is thought to be a variant of diffuse large b cell lymphoma with a distinctive reactive t lymphocyte infiltrate. intermediate cell lymphomas include b or t lymphocyte lymphoblastic lymphomas and burkitt-like lymphoma (both high grade), marginal zone lymphoma, and the intermediate cell variant of t zone lymphomas (both indolent and nodular). small cell lymphomas most commonly diagnosed in domestic animal species include enteropathy-associated t cell lymphoma, commonly seen in the cat, t zone lymphoma (small cell variant), and small cell lymphoma. cutaneous lymphomas are most often of t lymphocyte origin and may be epitheliotropic or nonepitheliotropic, and a distinct entity of inflamed t cell lymphoma has been recently described in dogs (see chapter 17). plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone. multiple myeloma. see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, plasma cell neoplasia. extramedullary plasmacytomas. extramedullary plasmacytomas are most commonly diagnosed in the skin of dogs (also cats and horses), where they constitute 1.5% of all canine cutaneous tumors (see chapter 17). the pinnae, lips, digits, and chin are the most commonly affected locations, and most lesions are solitary, though multiple plasmacytomas are infrequently diagnosed. other tissues affected include the oral cavity, intestine (colorectal in particular), liver, spleen, kidney, lung, and brain; of these, the oral cavity and intestine (colorectal) are involved most often. in one study, extramedullary plasmacytomas represented 5% of all canine oral tumors and 28% of all extramedullary plasmacytomas diagnosed. most cutaneous extramedullary plasmacytomas are benign, and complete excision is usually curative; oral cavity and colorectal extramedullary plasmacytomas are likely to behave in a similar manner. more aggressive forms may occur at any site. as with multiple myeloma, the neoplastic cells composing the tumor may vary from well differentiated to pleomorphic, often within the same tumor. the cells often have a characteristic perinuclear golgi clearing or "halo," and the more pleomorphic cells exhibit karyomegaly and binucleation (fig. 13-84) . extramedullary plasmacytomas may produce monoclonal immunoglobulins with resulting monoclonal gammopathy. amyloid deposition (which may mineralize) is also observed in a proportion of cases. differentiation from other round cell tumors may be aided by severe combined immunodeficiency disease of arabian foals is an autosomal recessive primary immunodeficiency disorder characterized by the lack of functional t and b lymphocytes caused by a genetic mutation in the gene encoding for dna-dependent protein kinase catalytic subunit (dna-pkcs). this enzyme is required for receptor gene rearrangements involved in the maturation of lymphocytes, and the resulting loss of functional t and b lymphocytes leads to a profound susceptibility to infectious diseases. though normal at birth, these foals develop diarrhea and pneumonia by approximately 10 days of age, often due to adenovirus, cryptosporidium parvum, and pneumocystis carinii infections. affected foals often die before 5 months of age. lymph nodes and thymus are small and often grossly undetectable, and the spleen is small and firm due to the absence of white pulp (see fig. 13-70) . the development of genetic tests to identify carriers of the disorder has led to a decrease in the prevalence of severe combined immunodeficiency disease. recently, severe combined immunodeficiency disease was diagnosed in a single caspian filly, though the exact genetic defect was not determined. congenital immunodeficiency diseases are also discussed in detail in chapter 5. streptococcus equi ssp. equi, the etiologic agent of equine strangles, is inhaled or ingested after direct contact with the discharge from infected horses or from a contaminated environment. the bacteria attach to the tonsils, penetrate into deeper tissues, enter the lymphatic vessels, drain to regional lymph nodes (mandibular, retropharyngeal, and occasionally parotid and cervical lymph nodes), and cause large abscesses (see fig. 13 -77). retropharyngeal enlargement from abscesses may lead to compression of the pharynx and subsequent respiratory stridor and dysphagia. abscesses may rupture and discharge pus through a sinus to the skin surface or spread medially into guttural pouches, where residual pus dries and hardens to form chondroids (which serve as a nidus for live bacteria to persist in carrier animals). in up to 20% of these cases, ruptured abscess material may spread via blood or lymph to other organs (metastatic abscess formation, bastard strangles), including lung, liver, kidney, synovia, mesenteric and mediastinal lymph nodes, spleen, and occasionally brain. purpura hemorrhagica, a type iii hypersensitivity reaction, may result in necrotizing vasculitis in some horses with repeated natural exposure to s. equi ssp. equi or after vaccination in horses that have had strangles. the typical manifestation of r. equi infection is chronic suppurative bronchopneumonia with abscesses (see chapter 9). approximately 50% of foals also develop intestinal lesions characterized by pyogranulomatous ulcerative enterotyphlocolitis, often over peyer's patches, and pyogranulomatous lymphadenitis of mesenteric and colonic lymph nodes (see chapter 7; see fig. 13-68) . large abdominal abscesses may be the only lesion in the abdomen and presumably originate from an infected mesenteric lymph node. the diffuse lymphatic tissue in the lamina propria may contain granulomatous inflammation with the phagocytized bacteria. mediastinal pyogranulomatous lymphadenitis may compress the trachea, causing respiratory distress. r. equi lesions also can develop in the liver, kidney, spleen, or nervous tissue. lymphoma is the most common malignant neoplasm in horses and mostly affects adult animals (mean age 10 to 11 years) with no lesions that involve distant sites and has replaced the term malignant histiocytosis. breed predispositions to histiocytic sarcoma complex are seen in bernese mountain dogs, rottweilers, golden retrievers, and flat-coated retrievers, though the disease can occur in any breed. histiocytic sarcoma complex is considered to have a rapid and highly aggressive course, and the clinical signs depend on the particular organ(s) involved. grossly, affected organs may be uniformly enlarged and/or contain multiple coalescing white-tan nodules. tissue architecture is effaced by sheets of pleomorphic round to spindle-shaped cells. there is marked cellular atypia with numerous karyomegalic and multinucleated neoplastic cells (fig. 13-85) . hemophagocytic histiocytic sarcoma. hemophagocytic histiocytic sarcoma is seen in dogs and cats and is a neoplasm of macrophages of the spleen and bone marrow. clinically, dogs present with hemolytic regenerative anemia and thrombocytopenia, thus mimicking evans's syndrome, though they are coombs negative. this form of histiocytic sarcoma carries the worst prognosis of the histiocytic sarcomas, which is likely in part related to the severe anemia and coagulopathy. it is characterized by a non-mass forming infiltrate of histiocytes within the bone marrow and splenic red pulp, causing diffuse splenomegaly. the neoplastic cells exhibit marked erythrophagocytosis, but the severe cellular pleomorphism seen in the histiocytic sarcoma complex may be lacking. the neoplastic cells are often intermixed with emh and plasma cells. metastasis is frequently to the liver, where the cells concentrate within the sinuses. tumor emboli within the lung are often present. malt is involved in a variety of ways with bacteria and viruses, and these are summarized for large animals in table 13 -5. these interactions include being a portal of entry for pathogens (e.g., salmonella spp., yersinia pestis, map, and l. monocytogenes); a site of replication for viruses (e.g., bvdv); a site for hematogenous infection (e.g., panleukopenia virus and parvovirus); and a site of gross or microscopic lesions in some viral diseases. bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus cause lymphocyte depletion within the malt. anthrax is caused by b. anthracis, a gram-positive bacillus found in spore form in soil. cattle, sheep, and goats become infected when grazing on infected soil, and infection causes fulminant septicemia. the spleen in infected animals is markedly enlarged and congested (see uniform splenomegaly with a bloody consistency; see also chapter 4). bovine viral diarrhea is caused by bvdv, a pestivirus. cattle are the natural host, but other animals such as alpacas, deer, sheep, and goats are also affected. bvdv preferentially infects cells of the immune system, including macrophages, dcs, and lymphocytes. the associated lesions in lymphoid tissues are severe lymphoid depletion in mesenteric lymph nodes and peyer's patches, whose intestinal surface may be covered by a fibrinonecrotic membrane. histologically, there is marked lymphocytolysis and necrosis of germinal centers in peyer's patches and cortices of lymph nodes. there is thymic atrophy because the thymus is markedly depleted of lymphocytes and may consist of only collapsed stroma and few scattered lymphocytes. bvd is discussed in detail in chapters 4 and 7. apparent breed or sex predisposition. the most frequent anatomic locations of equine lymphoma are multicentric, cutaneous, and gastrointestinal tract. multicentric lymphoma, defined as involving at least two organs (excluding the regional lymph nodes), is the most common manifestation, followed by skin and gastrointestinal tract types. solitary locations have been reported in the mediastinum, lymph nodes, ocular/orbital region, brain, spinal cord, oral cavity, and spleen. of the multicentric lymphomas, the most frequently observed type is t cell-rich large b cell lymphoma (tcrlbcl), reportedly in one study affecting 34% of the cases. peripheral t cell lymphoma (ptcl) was the second most common, followed by diffuse large b cell lymphoma (dlbcl). the most common lymphoma type in the gastrointestinal tract is also t cell-rich large b cell lymphoma, followed by enteropathyassociated t cell lymphoma. cutaneous lymphomas in horses account for up to 3% of all equine skin tumors. t cell-rich large b cell lymphoma is again the most common lymphoma subtype in the skin, representing up to 84% of all cutaneous lymphomas, and most frequently presents clinically as multiple skin masses. cutaneous t cell lymphoma (ctcl) is the second most common form and arises as smaller solitary nodules. thoroughbreds may have a higher incidence of cutaneous t cell lymphoma compared to other breeds. overall, horses with cutaneous t cell-rich large b cell lymphoma appear to have a longer survival time than horses with other types of lymphoma of the skin. progesterone receptor-positive lymphomas have also been identified in horses, and there is one report of subcutaneous tumor regression following removal of an ovarian granulosa-theca cell tumor. there may be an increased frequency of lymphoma in horses diagnosed with equine herpesvirus 5 (ehv-5, gammaherpesvirus), when compared to healthy horses, although the exact cause-effect role of this observation in lymphomagenesis 5 is not yet known. histologically, the hallmark features of t cell-rich large b cell lymphoma include a majority of small (nuclei approximately the size of an rbc), reactive, mature t lymphocytes admixed with a neoplastic population of large b lymphocytes whose nuclei are two to three times the diameter of an equine rbc. these large atypical cells are often binucleated and have prominent eosinophilic nucleoli ( fig. 13-86 ). the large cells may be observed in mitosis or in necrosis as single cells with retracted cytoplasm and pyknotic nuclei. t cell-rich large b cell lymphoma is often accompanied by the presence of a dense fibrovascular network. johne's disease primarily affects domestic and wild ruminants (and rarely pigs and horses) and is due to infection by map. the characteristic lesions include granulomatous enteritis usually confined to the ileum, cecum, and proximal colon; lymphangitis; and lymphadenitis of regional lymph nodes (see fig. 13-82) . the bacteria are ingested, engulfed by the m cells overlying peyer's patches, and then transported to macrophages in the lamina propria and submucosa. among cattle, sheep, goats, and wild ruminants, there is wide variation in the severity, distribution of lesions, primary inflammatory cell type (lymphocytes, epithelioid macrophages, multinucleated giant cells), and numbers of bacteria within lesions (multibacillary or paucibacillary). histologically, the architecture of the ileocecal lymph nodes may be partially replaced by aggregates of epithelioid postweaning multisystem wasting syndrome pcv2, a small single-stranded dna virus, is highly prevalent in the domestic pig population. several clinical syndromes are attributed to pcv2 infection and collectively termed pcv-associated diseases (pcvads). these include postweaning multisystemic wasting syndrome (pmws), porcine respiratory disease complex (prdc), porcine dermatitis and nephropathy syndrome, and enteric disease (see chapters 4 and 9). the major postmortem findings of postweaning multisystemic wasting syndrome are poor body condition, enlarged lymph nodes, and interstitial pneumonia. the lesions of the lymphoid system are commonly observed in the tonsil, spleen, peyer's patches, and lymph nodes. some pigs have all lymphoid tissues affected, whereas others may have only one or two affected lymph nodes. the characteristic microscopic lesions are lymphoid depletion of both follicles and paracortex with replacement by histiocytes, mild to severe granulomatous inflammation with multinucleated giant cells, and intrahistiocytic sharply demarcated, spherical, basophilic cytoplasmic inclusion bodies. necrosis of prominent lymphoid follicles (necrotizing lymphadenitis) is occasionally observed, and pcv2 can be detected within the necrotic regions. the loss of lymphocytes may be due to reduced production in the bone marrow, decreased proliferation in the secondary lymphoid organs, or necrosis of lymphocytes. splenic abscesses can be the result of bacteremia (see fig. 13 -69) or direct penetration by a foreign body from the reticulum (see spleen and also portals of entry/pathways of spread). c. pseudotuberculosis is a gram-positive intracellular bacterium that causes caseous lymphadenitis, a chronic suppurative disease of sheep and goats. the bacterium may enter through skin wounds (e.g., shearing cuts in sheep, tagging, tail docking, or castration), drain to the regional lymph node, and then be disseminated in lymph and circulating blood to external and internal lymph nodes, as well as other internal organs, including lung. external abscesses are most often detected in the "jaw and neck" region, specifically in the mandibular and parotid lymph nodes. on gross examination the abscesses are encapsulated and filled with greenish semifluid pus due to an infiltrate of eosinophils (see fig. 13-79) . over time the abscesses lose the greenish hue, and contents become inspissated to form the characteristic concentric laminations (see fig. 13 -80); old abscesses may reach a diameter of 4 to 5 cm. bovine lymphoma is broadly classified into enzootic and sporadic forms. the enzootic form, called enzootic bovine leukosis (ebl), is caused by blv, a retrovirus common in cattle. there is a higher prevalence in dairy cattle compared to beef breeds. blv is transmitted horizontally (e.g., blood, milk/colostrum, saliva) or iatrogenically (e.g., rectal sleeves, instruments/equipment). following infection, blv invades and integrates into the genome of infected b lymphocytes, resulting in a polyclonal b lymphocyte lymphocytosis in approximately 30% of cattle. in approximately 1% to 5% of blv-infected cattle, a single clone will emerge, leading to the development of b lymphocyte leukemia/lymphoma. the average incubation period between infection and development of lymphoma is 7 to 8 years, and this low conversion rate suggests that the latency period may be longer than the life span of most animals (dairy cattle seldom live to the 7-to 8-year peak incidence of lymphoma occurrence). other contributing variables, such as genetic background, coinfections, and environmental factors, may also play a role in lymphomagenesis. the exact mechanism of blv-induced tumorigenesis is poorly understood. recently blv micrornas (mirnas) were identified in preleukemic and malignant b lymphocytes, which showed repression of structural and regulatory gene expression. these findings suggested that mirnas may play a key role in tumor onset and progression. grossly, multiple tissues may be affected in cattle that develop lymphoma, including peripheral lymph nodes (cephalic, cervical, sublumbar) ( fig. 13-87) , abdominal lymph nodes, retrobulbar region, abomasum, liver, spleen, heart, urogenital tract, bone marrow, vertebral canal ( fig. 13-88) , and spinal cord. one study indicates most of these high-grade lymphomas are diffuse large cell lymphomas (66%), and approximately 20% are intermediate cell lymphomas (burkitt-like and lymphoblastic lymphomas). the sporadic form of bovine lymphoma is most often of t lymphocyte immunophenotype and has three subcategories: cutaneous, calf, and thymic. there is no known viral cause for the sporadic form, and each subcategory has a much smaller prevalence compared to the enzootic form. of the three sporadic forms, the cutaneous form seems to be the most common and manifests itself as multiple skin nodules in 1-to 3-year-old cattle. the calf form presents as generalized lymphadenopathy with weight loss, lethargy, and weakness in calves less than 6 months old. the thymic form is reportedly more common in beef cattle, 6 to 24 months of age. lymphoma is the most frequently reported cancer of pigs based on abattoir surveys. affected pigs are typically less than 1 year of age, and there is no reported breed predisposition, although a hereditary basis is suspected in cases arising in inbred herds. the two main forms of porcine lymphoma are thymic/mediastinal and multicentric; the latter is more common. spleen, liver, kidney, bone marrow, and lymph nodes are affected in the multicentric form, with visceral lymph nodes reportedly more commonly involved than peripheral nodes. a recent study of lymphoma in 17 pigs found the majority to be multicentric, and subtypes included the following: b lymphoblastic leukemia/lymphoma, follicular lymphoma, diffuse and intestinal large b cell lymphoma, and peripheral t cell lymphoma. one case each of thymic b cell and t cell lymphomas were also described. several types of severe combined immunodeficiency diseases have been described in dogs. a mutation in dna-pkcs (similar to arabian horses) with an autosomal recessive mode of inheritance is seen in jack russell terriers. an x-linked form of severe combined immunodeficiency disease is well described in basset hounds and is caused by mutations in the common γ-chain (γc) subunit of the receptors for il-2, il-4, il-7, il-9, il-15, and il-21. a similar disease is seen in cardigan welsh corgi puppies, though it is an autosomal mode of inheritance in this breed. the mutation inhibits the signal transduction pathways initiated by any of these cytokines, which are critical for the proliferation, differentiation, survival, and function of b and t lymphocytes. affected dogs have normal numbers of circulating b lymphocytes that are unable to class switch to igg or iga and reduced numbers of t lymphocytes, which are nonfunctional due to the inability to express il receptors. affected puppies are remarkably susceptible to bacterial and viral infections and rarely survive past 3 to 4 months of age. the thymus of these dogs is small and consists of only small dysplastic lobules with a few of hassall's corpuscles. tonsils, lymph nodes, and peyer's patches are often grossly unidentifiable due to the severe lymphocyte hypoplasia. congenital immunodeficiency diseases are also discussed in detail in chapter 5. thymic hemorrhage and hematomas have been reported in dogs and are most often seen in young animals. a variety of causes are described, including ingestion of anticoagulant rodenticides (warfarin, dicumarol, diphacinone, and brodifacoum), dissecting aortic aneurysms, trauma (e.g., automobile accident), and idiopathic/ spontaneous. histologically, hemorrhage variably expands the thymic lobules and septa, and in severe cases the lobular architecture is obscured by hemorrhage. in cases of anticoagulant rodenticide toxicosis, the medulla appears to be the main site of hemorrhage. see uniform splenomegaly with a bloody consistency (also see figs. 7-72 and 7-73). see the section on splenic nodules with a bloody consistency for discussion on splenic hematomas (including those induced by nodular hyperplasia or occurring with hemangiosarcoma), incomplete splenic contraction, acute splenic infarcts, and hemangiosarcomas. porcine reproductive and respiratory syndrome (prrs) is caused by an arterivirus and causes two overlapping clinical syndromes: reproductive failure and respiratory disease. the virus is transmitted by contact with body fluids (saliva, mucus, serum, urine, and mammary secretions and from contact with semen during coitus), but often it first colonizes tonsils or upper respiratory tract. the virus has a predilection for lymphoid tissues (spleen, thymus, tonsils, lymph nodes, peyer's patches). viral replication takes place in macrophages of the lymphoid tissues and lungs, though porcine reproductive and respiratory syndrome virus antigen is found in resident macrophages in many tissues and may persist in tonsil and lung macrophages. the result of this infection is a reduction in the phagocytic and functional capacity of macrophages of the monocytemacrophage system. as a consequence, there is reduction in resistance to common bacterial and viral pathogens. most porcine reproductive and respiratory syndrome-infected pigs are coinfected with one or more pathogens, including streptococcus suis and salmonella choleraesuis. infection with bordetella bronchiseptica and mycoplasma hyopneumoniae appear to increase the duration and severity of the interstitial pneumonia. the major lesions are interstitial pneumonia and generalized lymphadenopathy, and tracheobronchial and mediastinal lymph nodes are most commonly affected. coinfections often complicate the gross and histopathologic changes. lymph nodes are enlarged, pale tan, occasionally cystic, and firm; some strains of virus also cause nodal hemorrhage. microscopically, the lesions in the lymph nodes, tonsils, and spleens consist of varying degrees of follicular and paracortical hyperplasia and lymphocyte depletion in follicular germinal centers. streptococcus porcinus causes jowl abscesses in pigs. the bacteria colonize the oral cavity and spread to infect tonsils and regional lymph nodes. the mandibular lymph nodes are the most often affected and have multiple, 1-to 10-cm abscesses; the retropharyngeal and parotid lymph nodes may also be involved (fig. 13-89 ). this once-prevalent disease is now rare, presumably due to improvements in husbandry, feeder design, and hygiene. it is occasionally isolated in pigs with bacteremia. disseminated histoplasmosis include wasting, emaciation, fever, respiratory distress, diarrhea with hematochezia or melena, and lameness. the clinicopathologic changes of disseminated histoplasmosis may include neutrophilia, monocytosis, nonregenerative anemia in chronic infections, changes in total serum protein level, and liver enzyme level elevations with hepatic involvement. the anemia is likely a result of chronic inflammation, histoplasma infection of the bone marrow, and/or intestinal blood loss in dogs with gi disease. cytologic examination is useful for the diagnosis of histoplasmosis (tracheal wash preparations, aspirates of bone marrow and lymph nodes), where organisms are often visible in macrophages (efig. 13-15) . grossly, there is hepatosplenomegaly, the intestines are thickened and corrugated, and the lymph nodes are uniformly enlarged (fig. 13-91) with loss of normal architecture (somewhat similar to siderofibrotic plaques, splenic rupture, and accessory spleens see the section on miscellaneous disorders of the spleen for discussions on siderofibrotic plaques, splenic rupture, and accessory spleens. splenic nodular hyperplasia is common in dogs and categorized based on their cellular components as lymphoid nodular hyperplasia or complex nodular hyperplasia. hematomas may arise within nodules of hyperplasia (see splenic nodules with a bloody consistency). lymphoid (or simple) nodular hyperplasia consists of a focal well-demarcated mass composed of discrete to coalescing aggregates of lymphocytes. the lymphocytes may form follicular structures with germinal centers and/or consist of a mixture of lymphocytes with mantle and marginal zone cell morphologic features. the intervening tissue is often congested and may contain plasma cells, but stroma is not observed (fig. 13-90; e-fig. 13-13) . complex nodular hyperplasia is a focal mass that contains two proliferative components: lymphoid and stroma (efig. 13-14) . the lymphoid component resembles lymphoid nodular hyperplasia described above. there is proliferation of the intervening stromal tissues with fibroplasia, smooth muscle hyperplasia, and histiocytic hyperplasia; emh and plasma cells may also be present. it has recently come to light that the entity splenic fibrohistiocytic nodule (sfhn), first described in 1998, is not a single condition, but in fact a complex group of diseases. our better understanding of the spectrum of diseases once described under the term splenic fibrohistiocytic nodule is due to increasing knowledge of histiocytic disorders and immunochemistry. the original definition of splenic fibrohistiocytic nodule is a nodule characterized by a stromal population of histiocytoid and spindle cells intermixed with lymphocytes. grading was based on the lymphocyte percentage of the population (e.g., > 70% lymphocytes = grade 1; < 40% lymphocytes = grade 3); dogs with grade 1 splenic fibrohistiocytic nodule had a much better 1-year survival rate, and dogs with grade 3 nodules may develop sarcomas (often malignant fibrous histiocytoma, a now outdated term). with our increasing knowledge of histiocytic disorders and additional immunohistochemical stains, diseases that likely were encompassed by the term splenic fibrohistiocytic nodule include the following: complex and lymphoid nodular hyperplasia (see earlier), stromal sarcoma, histiocytic sarcoma, marginal zone hyperplasia, marginal zone lymphoma, and diffuse large b cell lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). histoplasma capsulatum can cause a disseminated fungal disease that is widely endemic, particularly in areas with major river valleys and temperate or tropical climates (e.g., midwestern and southern united states). free-living organisms in the mycelial phase produce macroconidia and microconidia that are inhaled and converted to the yeast phase in the lung. yeasts are phagocytized and harbored by macrophages of the monocyte-macrophage system. in some dogs the disease is limited to the respiratory tract and causes dyspnea and coughing. however, in most dogs, the disease is disseminated throughout the body, predominantly affecting the liver, spleen, gastrointestinal tract, bone marrow, skin, and eyes; primary gastrointestinal disease is also reported. the clinical signs in cases of and hepatosplenomegaly (efig. 13-16) . histologically, the lymph node sinuses and splenic red pulp are filled with macrophages that contain intracytoplasmic, round, 2-µm-diameter organisms with a small kinetoplast. though there is an initial stage of lymphoid hyperplasia in the spleen and lymph node, subsequent lymphoid atrophy occurs with chronicity. the atrophy is due to impairment of follicular dcs, b lymphocyte migration, and germinal center formation. there may be lymphoid atrophy of the spleen and lymph nodes in severe chronic infections. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells (see chapter 14). dogs are exposed through contact with oronasal secretions, and the virus infects macrophages within the lymphoid tissue of the tonsil and respiratory tract (including tracheobronchial lymph nodes) and later disseminates to the spleen, lymph nodes, bone marrow, malt, and hepatic kupffer cells. the virus causes necrosis of lymphocytes (especially cd4 t lymphocytes) and depression of lymphopoiesis in the bone marrow, leading to severe immunosuppression. dogs are therefore susceptible to secondary infections, including bordetella bronchiseptica, toxoplasma gondii, nocardia, salmonella spp., and generalized demodicosis. canine parvovirus type 2 (cpv-2) is a highly contagious disease of dogs spread through the fecal-oral route or oronasal exposure to contaminated fomites. the virus has tropism for rapidly dividing cells, and replication begins in the lymphoid tissues of the oropharynx, thymus, and mesenteric lymph nodes and then is disseminated to the small intestinal crypt epithelium. by infecting lymphoid tissues, canine parvovirus type 2 causes immunosuppression directly through lymphocytolysis and indirectly though bone marrow depletion of lymphocyte precursors. there is marked lymphoid atrophy of thymus and follicles of the spleen, lymph nodes, and maltparticularly of peyer's patches to produce the classic gross lesion of depressed oval regions of the mucosa (so-called punched-out peyer's patches). thymomas. see lymphoid/lymphatic system, disorders of domestic animals: thymus, neoplasia. lymphomas. lymphoma is the most common hematologic malignancy in the dog. using the who classification scheme, several lymphoma subtypes are identified in dogs and clinically range from slow-growing indolent tumors to highly aggressive tumors. of all domestic animal species, lymphoma is the most extensively studied in dogs. the most common clinical presentation in dogs is generalized lymphadenopathy, with or without clinical signs such as lethargy and inappetence. the majority of lymphomas in dogs are large cell mid-to highgrade lymphomas, and up to half of all lymphoma cases are subtyped as diffuse large b cell lymphoma. diffuse large b cell lymphomas are further subdivided into centroblastic or immunoblastic based on nucleolar morphologic features (see table 13 -7 and box 13-11), although it is unclear if this difference has any prognostic significance. histologically, lymph node architecture is most often completely effaced by sheets of large neoplastic cells, which may invade through the capsule and colonize the perinodal tissue. these dogs are often treated with chemotherapy and achieve remission. the overall median survival time for dogs with diffuse large b lymphocyte lymphoma is approximately 7 months, although this number lymphoma, though the nodes tend to be more firm in histoplasmosis). histologically within the node, there is a multifocal to coalescing infiltrate of epithelioid macrophages with intracytoplasmic, small (2 to 4 µm in diameter) yeast organisms with spherical basophilic central bodies surrounded by a clear halo (fig. 13-92) . leishmaniasis is a disease of the monocyte-macrophage system caused by protozoa of the genus leishmania. it occurs in dogs and other animals and is endemic in parts of the united states, europe, mediterranean, middle east, africa, and central and south america. the protozoa proliferate by binary fission in the gut of the sand fly and become flagellated organisms, which are introduced into mammals by insect bites, where they are phagocytized by macrophages and assume a nonflagellated form. cutaneous and/or visceral forms of the disease are observed. in the visceral form, dogs are emaciated and have general enlargement of abdominal lymph nodes numerous other subtypes of lymphoma have been reported in dogs, including several forms of cutaneous lymphomas, most often of t lymphocyte origin and epitheliotropic (see chapter 17). hepatosplenic t cell lymphoma, thought to be of γ/δ t lymphocyte origin, affects the liver and spleen without significant nodal involvement. hepatocytotropic t cell lymphoma is a distinct form of lymphoma with tropism for the hepatic cords; clusters or individual neoplastic lymphocytes invade the hepatic cords, without hepatocyte degeneration. intravascular lymphoma is a proliferation of large neoplastic lymphocytes within blood vessels of many tissues, leading to progressive occlusion and subsequent thromboses and infarcts. this neoplasm does not form an extravascular mass, and neoplastic cells are not found in peripheral blood smears or bone marrow. indolent lymphomas constitute up to 29% of all canine lymphomas. indolent lymphomas in dogs, in descending order of frequency, include t zone lymphoma (tzl), marginal zone lymphoma (mzl), mantle cell lymphoma (mcl), and follicular lymphoma (fl). mantle cell lymphoma and follicular lymphoma are less commonly diagnosed than t zone lymphoma and marginal zone lymphoma; therefore the reader is referred to the suggested readings to learn more on mantle cell lymphoma and follicular lymphoma. t zone lymphoma. t zone lymphoma is the most common indolent lymphoma in dogs ( fig. 13-93) . it presents as a solitary or varies based on the study and the grade of the tumor (as determined by mitotic figures). peripheral t cell lymphomas-not otherwise specified are the second most common subtype in dogs. this category includes all t cell lymphomas that do not fit into the other categories (e.g., t zone lymphoma, enteropathy-associated t cell lymphoma, and hepatosplenic t cell lymphoma). peripheral t cell lymphoma also effaces nodal architecture, and when compared to diffuse large b cell lymphoma, there is more variation in nuclear size and morphologic features. dogs with this subtype tend to have shorter survival times. intermediate cell size, high-grade lymphomas are less common in dogs, and the two most frequently encountered subtypes are lymphoblastic lymphoma (lbl) and burkitt-like lymphoma (bll). lymphoblastic lymphoma may be of b or t lymphocyte origin, though t cell lymphoblastic lymphoma is more common of the two. it is important to recognize a common misuse of the term "lymphoblast" in lymphoblastic lymphoma-by definition in lymphoblastic lymphoma, lymphoblasts are intermediate-sized cells with a distinct dispersed chromatin pattern, and not the large lymphocytes seen in cases of diffuse large b cell lymphoma or peripheral t cell lymphoma. t lymphocyte lymphoblastic lymphoma is an aggressive disease that is often resistant to treatment. burkitt-like lymphoma is a high-grade lymphoma of b lymphocytes. consequently there is marked lymphoid atrophy of thymus, spleen, lymph node, and malt (particularly peyer's patches). see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, myeloid neoplasia, mast cell neoplasia and see efig. 13-6 . lymphoma is the most commonly diagnosed neoplasm in cats, and the incidence is reportedly the highest for any species. mediastinal or multicentric lymphomas are seen in young, felv-infected cats (see fig. 13 -53). with the advent of felv vaccine and routine testing, the prevalence of felv-associated lymphoma is decreased. currently the alimentary tract is the most commonly affected site, and typically occurs in cats greater than 10 years of age . other miscellaneous sites commonly affected are brain, spinal cord, eye, kidney, and nasopharynx. the retrovirus felv has long been recognized as a cause of lymphoma in cats-the risk for lymphoma is increased sixtyfold in infected cats. before the advent of a vaccine in 1985, approximately 70% of cats (mainly young animals) with lymphoma were felv positive. felv infects t lymphocytes and can cause myelodysplastic syndrome, acute myeloid leukemias (see myeloid neoplasia), and t lymphocyte leukemia/lymphoma. in the latter the mediastinum (thymus, mediastinal, and sternal lymph nodes) is the site most commonly involved, although a multicentric distribution also occurs. routine felv vaccination has led to a significant decrease in the prevalence of felv infection, which has resulted in a decrease in the proportion of mediastinal lymphomas. the risk for developing lymphoma in fiv-infected cats is fivefold to sixfold higher than in uninfected cats. cats that underwent kidney transplantation and thus received immunosuppressive drug therapy had a similar risk for developing lymphoma. both fivinfected and posttransplantation cats predominantly developed extranodal, high-grade, diffuse large b lymphocyte lymphomas. this form is also the most common subtype in human immunodeficiency virus and posttransplantation patients caused by the epstein-barr virus (ebv). therefore it is reasonable to question whether these two groups of immunosuppressed cats may be more prone to infection by a gammaherpesvirus similar to ebv, leading to lymphoma. recently a novel feline gammaherpesvirus (fcaghv1) was multiple peripheral lymphadenomegaly (often mandibular lymph nodes) in otherwise healthy-appearing dogs. the characteristic histopathologic architecture is a nodular expansion of the paracortex by neoplastic cells, which push atrophied "fading" cortical follicles against the thinned capsule and trabeculae. this unique architectural feature is best highlighted with immunohistochemical stains (often cd3 for t lymphocytes and cd79a, pax5, or cd20 for b lymphocytes). the neoplastic cells are small to intermediate in size with pale eosinophilic cytoplasm and oval nuclei with sharp shallow indentations. mitotic figures are rare. dogs with this lymphoma subtype tend to be diagnosed with an advanced stage of the disease, likely because they present clinically healthy, without loss of appetite or activity level. even so, dogs with t zone lymphoma have a relatively long survival time compared to other lymphomas: reports on median survival time range from 13 to 33 months, and data suggest that dogs who do not receive chemotherapy actually have longer median survival times. marginal zone lymphoma. marginal zone lymphoma is an indolent b lymphocyte neoplasm derived from the cells of the marginal zone of lymphoid follicles. most marginal zone lymphomas (and mantle cell lymphomas) are assumed to originate in the spleen with slow spread to lymph nodes and often present as a mottled white-red smooth spherical splenic mass. histopathologic assessment of tissue architecture is needed for a diagnosis of marginal zone lymphoma and is characterized by a distinct nodular pattern in which the lighter-staining neoplastic marginal zone cells form a dense cuff around small foci of darkly stained mantle cells (fading follicles). the neoplastic marginal zone lymphocytes are intermediate in size and have a single prominent central nucleolus. mitotic figures are often rare or absent early on and increase with disease progression. differentiating between marginal zone lymphoma and marginal zone hyperplasia (which refers to a proliferation of marginal zone cells and contains a mixture of small and intermediate lymphocytes) is challenging because marginal zone lymphoma arises on the background of marginal zone hyperplasia. additionally, lymphoid and complex nodular hyperplasia are common in the dog spleen (see disorders of dogs), and it is possible that many cases of nodular hyperplasia contain areas of marginal zone lymphoma. therefore immunophenotyping and molecular clonality are ultimately required for a definitive diagnosis of marginal zone lymphoma. the overall median survival time in dogs with splenic marginal zone lymphoma after splenectomy is approximately 13 months (even longer if it is diagnosed as an incidental finding). plasmacytomas. see disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia, extramedullary plasmacytomas (see fig. 13 -84). feline panleukopenia, caused by the single-stranded dna virus feline parvovirus (fpv), is a highly contagious and often lethal disease of cats and other felidae, as well as other species (including raccoons, ring-tailed cats, foxes, and minks). fpv is transmitted by the fecal-oral route through contact with infected body fluids, feces, or fomites. following intranasal or oral infection, the virus initially replicates in the macrophages in the lamina propria of the oropharynx and regional lymph nodes, followed by viremia, which distributes the virus throughout the body. because fpv requires rapidly multiplying cells in the s phase of division for its replication, replication occurs in mitotically active tissues (lymphoid tissue, bone marrow, and intestinal mucosa). by infecting lymphoid tissues, fpv causes immunosuppression directly through lymphocytolysis and of the small intestine. the neoplastic cells are small (nuclei are equal to the diameter of a feline rbc), mitotic figures are infrequent (low grade), and mucosal and crypt epitheliotropism is common (see fig. 13-95) . a diagnosis of this subtype of lymphoma may be difficult (particularly in endoscopic biopsy samples), because this disease often is multifocal and concurrent with or arises within lymphoplasmacytic inflammatory bowel disease (ibd). the neoplastic lymphocytes are morphologically similar to the inflammatory lymphocytes. early small cell mucosal t cell lymphomas often require additional diagnostic testing, namely, immunohistochemistry and molecular clonality testing (pcr for antigen receptor rearrangement [parr]) to confirm a clonal neoplasm. transmural t cell lymphomas also occur focally or multifocally in the small intestine of cats (best classified as enteropathy-associated t cell lymphoma type i) and by definition must extend into the submucosa and muscularis. some tumors invade the serosa and adjacent mesentery. t cell large granular lymphocyte (lgl) lymphoma is often diagnosed, and the intestinal segments orad and aborad to the transmural mass may also have mucosal lymphoma. gastrointestinal b cell lymphomas are less prevalent in cats but occur in the stomach, jejunum, and ileocecocolic region as transmural lesions. most are diagnosed as diffuse large b cell lymphomas. lymphomas in other sites also occur less frequently in cats. the upper respiratory tract (nasal and/or nasopharyngeal region) is a relatively rare site for lymphoma. however, lymphoma is the most common primary nasal tumor, and diffuse large b cell lymphomas (of immunoblastic type) are the predominant subtype. both cutaneous (cutaneous t cell lymphoma) and subcutaneous lymphomas (usually large cell lymphomas) are rare. presumed solitary ocular lymphomas have also been reported. t cell-rich large b cell lymphoma, also referred to as feline hodgkin-like lymphoma in some studies, is composed of a mixture of reactive small lymphocytes and large neoplastic b lymphocytes, many of which may be binucleated and/or have prominent nucleoli (thus resembling the reed-sternberg cells of human hodgkin's lymphoma). this disease is typically characterized by a distinctive clinical presentation of an indolent unilateral neoplasm of the cervical lymph nodes, which spreads slowly to adjacent nodes within the chain. however, a proportion of cases may go on to develop into a more aggressive multicentric large to anaplastic b lymphocyte lymphoma that can affect peripheral and central nodes and multiple organs. suggested readings are available at www.expertconsult.com. a b discovered in domestic cats with a 16% prevalence in north america, and further studies to investigate its role in lymphomagenesis are needed. the overall incidence of feline lymphomas has increased, mainly due to an increase in gastrointestinal lymphomas. mucosal t cell lymphoma, also known as enteropathy-associated t cell lymphoma (eatcl type ii), is the most common and arises from diffuse malt partial thromboplastin time (aptt or ptt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of a contact activator, calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in intrinsic and/or common coagulation pathway (all factors except for vii and xiii) causes prolongation of ptt • insensitive test-prolongation requires 70% deficiency. • other causes of prolongation include polycythemia (less plasma per unit volume, so excess amount of citrate is available to chelate calcium) and heparin therapy activated clotting time (act) • required sample: nonanticoagulated whole blood in special act tube (diatomaceous earth as contact activator) used in practice setting-performed by warming sample to body temperature, monitoring for clot formation; normal clotting times are within 60 to 90 seconds in dogs, 165 seconds in cats • less sensitive version of ptt-prolongation requires 95% deficiency severe thrombocytopenia may cause prolongation. • one-stage prothrombin time (ospt or pt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of tissue factor (tf; thromboplastin), calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in extrinsic (factor vii) and/or common coagulation pathway cause prolongation of pt • insensitive test-prolongation requires 70% deficiency proteins induced by vitamin k antagonism or absence (pivka) test • required sample: citrated plasma • essentially a version of the pt using an especially sensitive thromboplastin reagent • pivka are inactive (uncarboxylated) vitamin k-dependent factors; an increase in pivka is not specific for vitamin-k antagonism but may be an earlier and more sensitive detector than pt or ptt • thrombin time (tt) • required sample: citrated plasma • measures time for fibrin clot formation after thrombin (factor iia) is added • defects directly involving formation and/or polymerization of fibrin prolong this test (i.e., if the lesion is upstream of the conversion of fibrinogen to fibrin, the tt will be normal). hypofibrinogenemia or dysfibrinogenemia causes prolongation of the tt required sample: citrated plasma. • fibrinogen concentration measured based on time to clot formation after addition of thrombin; this is essentially the same as the tt mentioned earlier and is a more accurate method than the heat precipitation method • decreased fibrinogen may be because of increased consumption (disseminated intravascular coagulation) or decreased production (liver disease) increased fibrinogen is associated with inflammation, renal disease, and dehydration required sample: special fdp tube. • used in the practice setting. • performed by adding blood to a special tube containing thrombin and a trypsin inhibitor (sample clots almost instantly in normal dogs and cats) and incubating two dilutions of serum (1 : 5 and 1 : 20) with polystyrene latex particles coated with sheep anti-fdp antibodies (should be negative in normal dogs and cats required sample: citrated plasma. • latex agglutination test. • to date, only validated in dogs and horses assay detects a specific type of fdp resulting from breakdown of cross-linked fibrin; concentration of plasma d-dimer indicates the degree of fibrinolysis; often used as part of a disseminated intravascular coagulation panel • required sample: citrated plasma. • decreased because of decreased production (liver disease), loss (protein-losing nephropathy or enteropathy) • specific factor assays • required sample: citrated plasma. • performed at specialized laboratories intermediary spleen microvasculature in canis familiaris-morphological evidence of a closed and open type molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states characteristics, diagnosis, and treatment of inherited platelet disorders in mammals making sense of lymphoma diagnostics in small animal patients canine granulocytic anaplasmosis: a review lymphoid tissues and organs lymph nodes and thymus diagnostic cytology and hematology of the dog and cat two hundred three cases of equine lymphoma classified according to the world health organization (who) classification criteria hepcidin: a key regulator of iron metabolism and mediator of anemia of inflammation changes to bovine hematology reference intervals from 1957 to atlas of veterinary hematology: blood and bone marrow of domestic animals pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses feline leukemia virus infection and diseases tumors of the hemolymphatic system proposed criteria for classification of acute myeloid leukemia in dogs and cats immunobiology: the immune system in health and disease extramedullary hematopoiesis: a new look at the underlying stem cell niche, theories of development, and occurrence in animals lineage-specific hematopoietic growth factors hepatosplenic and hepatocytotropic t-cell lymphoma: two distinct types of t-cell lymphoma in dogs histology and cell biology: an introduction to pathology lymphoid neoplasms in swine jc: robbins & cotran pathologic basis of disease the spleen ehrlichiosis and related infections hemotropic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential clinical, laboratory, and histopathologic features of equine lymphoma classification and clinical features in 88 cases of equine cutaneous lymphoma histologic and immunohistochemical review of splenic fibrohistiocytic nodules in dogs feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality current state of knowledge on porcine circovirus type 2-associated lesions molecular pathology of severe combined immunodeficiency in mice, horses, and dogs hematologic abnormalities associated with retroviral infections in the cat pathology of the pig: a diagnostic guide pathologic and prognostic characteristics of splenomegaly in dogs due to fibrohistiocytic nodules: 98 cases splenic myeloid metaplasia, histiocytosis, and hypersplenism in the dog (65 cases) fundamentals of veterinary clinical pathology feline parvovirus infection and associated diseases novel gammaherpesviruses in north american domestic cats, bobcats, and pumas: identification, prevalence, and risk factors palmer's pathology of domestic animals veterinary comparative hematopathology histologic classification of hematopoietic tumors of domestic animals. in world health organization international histological classification of tumors in domestic animals, second series canine lymphomas: association of classification type, disease stage, tumor subtype, mitotic rate, and treatment with survival classification of canine malignant lymphomas according to the world health organization criteria canine indolent nodular lymphoma the 2008 revision of the world health organization (who) classification of myeloid neoplasms and acute leukemia: rationale and important changes the coombs' test in veterinary medicine: past, present, future a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital (1996-2004) schalm's veterinary hematology chronic lymphocytic leukemia in dogs and cats: the veterinary perspective grossly, incompletely contracted areas are characterized by multiple, variably sized and irregularly shaped, dark red to black, raised, soft, blood-filled "nodules." these areas are usually at the margins of the spleen, and the intervening tissues are depressed and pink-red, corresponding to the contracted portions of red pulp devoid of blood. incompletely contracted areas may be confused with acute splenic infarcts or hematomas on gross examination.acute splenic infarcts. splenic infarcts are wedge-shaped or triangular hemorrhagic lesions that occur primarily at the margins of the spleen. in dogs, splenic infarcts most often occur with hypercoagulable states (e.g., liver disease, renal disease, cushing's disease), neoplasia, and cardiovascular disease. splenic vein thrombi may occur in association with traumatic reticulitis, splenic abscesses, portal vein thrombosis, and arterial thrombosis in bovine theileriosis in cattle. valvular endocarditis may also lead to multiorgan infarcts, including the spleen. splenic infarcts are common in pigs with classical swine fever.acute splenic infarcts may not always be grossly visible in the early stages but develop into discrete, dark red and blood-filled, bulging, wedge-shaped foci with the base toward the splenic capsule ( fig. 13-64, a) . with chronicity the lesion becomes gray-white and contracted due to fibrosis (see fig. 13 -64, b).hemangiosarcoma. hemangiosarcoma is a malignant neoplasm of endothelial cells and is a common primary tumor of the spleen, especially in dogs. benign splenic hemangiomas are extraordinarily rare. grossly, hemangiosarcomas may appear as single, multifocal, or coalescing dark red-purple masses and cannot be easily differentiated from a hematoma ( fig. 13-65 ). on cut surface they are bloody with varying amounts of soft red neoplastic tissue; in more solid areas the neoplasm can be slightly more firm and whitetan. metastatic spread occurs early in the disease process. seeding of the peritoneum results in numerous discrete red-black masses throughout the omentum and serosa of abdominal organs, and hematogenous spread to liver and lung are common. hemangiosarcomas in dogs also occur in the right atrium of the heart, retroperitoneal fat, and skin (dermal and/or subcutaneous) and multiorgan hemangiosarcomas are described in horses, cats, and cattle. because hemangiosarcomas have often metastasized at the time of initial accumulations of macrophages within several organs, including splenic macrophages, kupffer cells of the liver, and macrophages in the brain are often observed.splenic nodules with a bloody consistency. the most common disorders of the spleen with bloody nodules are (1) hematomas, including those induced by nodular hyperplasia or occurring with hemangiosarcoma, (2) incompletely contracted areas of the spleen, (3) acute splenic infarcts, and (4) hemangiosarcomas. the term nodule has been applied rather loosely here. in some of these conditions, such as incompletely or irregularly contracted areas of the spleen, the elevated area of the spleen is not as well defined as the term nodule would imply.hematomas. bleeding into the red pulp to form a hematoma is confined by the splenic capsule, and produces a red to dark red, soft, bulging, usually solitary mass of varying size (2 to 15 cm in diameter) (fig. 13-62 ). resolution of a splenic hematoma progresses over days to weeks, through the stages of coagulation and breakdown of the blood into a dark red-brown soft mass (fig. 13-63, a) , infiltration by macrophages that phagocytize erythrocytes and break down hemoglobin to form hematoidin and hemosiderin (see fig. 13-63 , b), and repair leading to fibrosis. on occasion the capsule (splenic capsule and visceral peritoneum) over the hematoma can rupture, resulting in hemoperitoneum, hypovolemic shock, and death.the origin or cause of many hematomas is unknown. some are due to trauma, and others may also be induced by splenic nodular hyperplasia. it is postulated that as the splenic follicles become hyperplastic they distort the adjacent marginal zone and marginal sinus, which compromises their drainage into sinusoids and red pulp vascular spaces. the result is an accumulation of pooled blood surrounding the hyperplastic nodule, which leads to hematoma formation. splenic hematomas can also occur secondary to the rupture of hemangiosarcomas within the spleen.incompletely contracted areas of the spleen. incompletely or irregularly contracted areas of the spleen are caused by failure of the smooth muscle to contract in response to circulatory shock (hypovolemic, cardiogenic, or septic) or sympathetic "fight-or-flight" response, resulting in a lack of splenic evacuation of stored blood. key: cord-022754-ehq9qnoo authors: nan title: liver date: 2012-07-25 journal: canine and feline gastroenterology doi: 10.1016/b978-1-4160-3661-6.00061-4 sha: doc_id: 22754 cord_uid: ehq9qnoo nan the basic elements of the biliary tract are the hepatic canaliculi, bile ductules, intralobular ducts, interlobular ducts, hepatic ducts, cystic duct, gallbladder, common bile duct, and the pancreaticobiliary sphincter (of oddi). 2 there are many variations on this central theme, the most important of which are (a) the pancreaticobiliary sphincter is a common physiologic and anatomical channel at the duodenal papilla in the cat 3 and (b) there are many anatomic variations in the feline gallbladder, from single gallbladder to bilateral gallbladders, body duplication, fundic duplication, complete duplication, septate, and y-shaped gallbladder. 4 hepatocytes account for 60% to 80% of the liver cell mass (see table 61 -1) and contribute to a wide range of metabolic activity, including carbohydrate, protein, lipid, nucleic acid, porphyrin, metal, vitamin, glutathione, hormone, and xenobiotic metabolism; coagulation factor synthesis; biliary secretion; and immune surveillance. 1, 5 hepatocytes have an eosinophilic cytoplasm reflecting numerous mitochondria, and basophilic stippling caused by large amounts of rough endoplasmic reticulum and free ribosomes. hepatocyte nuclei are round with dispersed chromatin and prominent nucleoli. anisokaryosis is common and often reflects various degrees of polyploidy, a normal feature of more than 50% of hepatocytes. the average life span of the hepatocyte is 5 to 6 months reflecting their ability to regenerate. hepatocytes are organized into plates separated by vascular channels (sinusoids), an arrangement supported by a reticulin (collagen type iii) network. the sinusoids have a discontinuous, fenestrated endothelial cell lining. the endothelial cells have no basement membrane and are separated from the hepatocytes by the space of disse, which drains lymph into the portal lymphatics. hepatocytes are supported by a number of other cell types, which account for 40% of the liver cell mass. representing 3% to 10% of liver cell mass, cholangiocytes are also known as biliary epithelial cells. 6 they secrete water, bicarbonate, and cations into the bile in the physiologic state, but they may also participate in the immune response as antigen-presenting cells in disease states. the biliary tract is a convergent system of canals that begins in the canaliculi, followed by the bile ducts, and ending with the common bile duct. bile secretion depends on the function of membrane transport systems in hepatocytes and cholangiocytes and on the structural and functional integrity of the biliary tract. the hepatocytes, constituting the most abundant liver cell population, generate the so-called primary bile in their canaliculi. biliary canaliculi are blind tubular structures, with a very high surface-to-volume ratio that by means of osmotic gradients favors the formation of bile flow. cholangiocytes, which constitute 3% to 10% of the liver cells, modify the canalicular bile by secretory and reabsorptive processes as bile passes through the bile ducts, and they are responsible for approximately 30% of bile volume. in contrast to hepatocytes, where secretion is constant and poorly controlled, cholangiocyte secretion is broadly regulated. 5, 6 the immunoglobulin family, without which the liver cannot clear complement system-coated pathogens. in disease states, kupffer cells contribute to the pathology of ethanol and other toxic principles through production of inflammatory mediators, activation of toll-like receptors, and elaboration of tumor necrosis factor (tnf-α). 7 kupffer cell activation is responsible for early ethanol-induced liver injury, common in chronic alcoholics. ethanol activates the toll-like receptor 4 and cd14, receptors on the kupffer cell that internalize the endotoxin lipopolysaccharide. internalization activates the transcription of tnf-α and production of superoxide (a prooxidant). tnf-α then enters the stellate cell in the liver, leading to collagen synthesis and fibrosis. fibrosis eventually causes cirrhosis or loss of function of the liver (see the role of the stellate cell, which is discussed in "stellate cells" section that follows). hepatic stellate cells (hscs) (also referred to as vitamin a-storing cells, lipocytes, interstitial cells, fat-storing cells, and ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50% to 80% of vitamin a in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. [7] [8] [9] [10] in physiologic conditions, these cells play pivotal roles in the regulation of vitamin a homeostasis. in pathologic conditions, such as hepatic fibrosis or liver cirrhosis, hscs lose vitamin a and synthesize a large amount of ecm components, including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. the morphology of these cells also changes from that of the star-shaped stellate cell to that of the fibroblast or myofibroblast. hscs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. 11 activation of hscs, a key event in liver fibrosis, is caused by diminished adipogenic transcription. 12 wnt signaling inhibits antiadipogenic activation of hscs and liver fibrogenesis; wnt antagonism inhibits hsc activation and liver fibrosis. 9 also known as natural killer (nk) cells or large granular lymphocytes, pit cells represent 1% of liver cell mass, and serve as part of the immune surveillance mechanism in the hepatic sinusoids. pit also known as browicz-kupffer cells or stellate macrophages, these cells represent 2% to 5% of the liver cell mass, and are specialized macrophages localized to the sinusoids as part of the mononuclear phagocyte system. kupffer cells begin their development in the bone marrow with the genesis of promonocytes and monoblasts into monocytes, and then on to peripheral blood monocytes, completing differentiation into kupffer cells within the liver. in health, kupffer cells are involved in the metabolism of erythrocyte hemoglobin. during perfusion of the liver, senescent red blood cells are phagocytized by the kupffer cells, and the hemoglobin molecule is further metabolized into its component parts. globin chains and amino acids are reutilized; the iron-containing portion of heme is removed, transported, and stored; and heme is further oxidized into bilirubin, conjugated with glucuronic acid within hepatocytes, and secreted into the bile. kupffer cells also express a complement receptor of progenitor cells have multilineage potential and similar characteristics to stem cells. the late progenitor cells have differentiated further and produce progeny in only a single lineage. although they divide rapidly, they are capable of only a short-term tissue reconstitution and they do not self-renew. 15 early studies in hepatocyte turnover and liver regeneration showed that the parenchymal cell, the hepatocyte, was the primary and only cell involved in tissue renewal. however, new studies of liver regeneration, hepatocarcinogenesis, liver transplantation, and various cell lines show that a variety of cell types participate in maintaining hepatocyte number and mass. recent studies indicate the presence of both intrahepatic and extrahepatic stem/progenitor cell populations that serve to maintain the normal organ and to regenerate damaged parenchyma in response to a variety of insults. the intrahepatic compartment most likely derives primarily from the biliary tract, particularly the most proximal branches, that is, the canals of hering and smallest ductules. the extrahepatic compartment is at least in part derived from diverse populations of cells from the bone marrow. embryonic stem cells are considered as a part of the extrahepatic compartment. 16 the precise role(s) of each of these individual cells remains to be determined, but it is clear that in the aggregate they confer the vast regenerative capacity of the liver . the liver is involved in many aspects of intermediary metabolism. 1 the liver is at the center of carbohydrate metabolism through its role in maintaining normoglycemia. glucose is the primary energy source for most mammalian cells, and its metabolism is tightly regulated to guarantee that a sufficient supply is available to glucosedependent organs, particularly the brain. glucose can be made available from two sources: absorption of dietary glucose from the intestine, and release of glucose from organs such as the liver and kidney. early in fasting, the majority of endogenous glucose is generated by glycogenolysis where glycogen in the liver is converted to glucose-6-phosphate under the regulation of debranching enzyme, hepatic glycogen phosphorylase, and phosphorylase kinase. with more prolonged fasting, endogenous glucose is generated by gluconeogenesis from certain substrates such as amino acids, lactate, and glycerol. both processes generate glucose-6-phosphate, which must then be dephosphorylated in order to transport glucose out of the cell. • early fasting: glycogen → glycogenolysis → glucose → normoglycemia • prolonged fasting: amino acids → gluconeogenesis → glucose → normoglycemia the enzyme responsible for the dephosphorylation of glucose-6phosphate is glucose-6-phosphatase-α. alterations in quantity, location, or activity of glucose-6-phosphatase, such as those seen in type 1 glycogen storage diseases, effectively result in a lack of all endogenous glucose production and severe hypoglycemia develops during periods of fasting. 17 the liver is an important site of protein metabolism. amino acids and proteins absorbed from the intestine or produced in the body are delivered to the liver. the liver deaminates amino acids and cells belong to the group of sinusoidal cells, together with kupffer, endothelial, and fat-storing (stellate) cells. pit cells use the fasl fas ligand (fasl) and perforin/granzyme pathway to kill target cells. fasl on effector cells binds the fas that is present on the target cell membrane, which results in oligomerization of fas and activation of caspase 8. perforin and granzymes, of which granzyme b is the most potent, reside in granules of the cytotoxic lymphocytes and are released by exocytosis. intracellular delivery of granzyme b results in the initiation of the caspase cascade by proteolytic activation of caspase 3, either directly or through a mitochondrial-dependent pathway. caspases play a central role in the execution of apoptosis. lymphocyte recruitment from the circulation into tissue is dependent on the ability of the lymphocyte to recognize and bind molecules on the endothelial cell surface that promote transendothelial migration. a multistep model of leukocyte adhesion to vascular endothelium has been described and is broadly applicable, although the details of the signals involved differ between tissues. 13, 14 in a generally accepted model, tethering or rolling receptors expressed on endothelial cells capture free-flowing leukocytes. these receptors may be either selectins or members of the immunoglobulin superfamily. once captured, the leukocyte can receive activating messages presented by endothelial cells in the form of chemokines that activate specific g-protein-coupled receptors on the leukocyte surface. occupancy of these receptors triggers a cascade of intracellular signals that results in the presentation of high-affinity integrin receptors on the leukocyte surface that bind to immunoglobulin family of counterreceptors on the endothelium to promote leukocyte arrest on the vessel wall. in the presence of the appropriate migratory signals the leukocyte will migrate across the endothelium into tissue, where it follows a hierarchy of chemotactic signals toward the focus of inflammation. representing 2% to 5% of the liver cell mass, smooth muscle cells are located primarily in the hepatic artery and portal vein and their tributaries, and serve primarily to regulate the hepatic microcirculation. hepatic fibrosis is a common outcome of hepatic injury in the dog. activated fibroblasts that develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: (a) portal or septal myofibroblasts, (b) interface myofibroblasts, and (c) the perisinusoidally located hscs. it is difficult to arrive at a universally applicable definition of a stem cell because some of the defined properties of a stem cell can be exhibited by the stem cells in some tissues or organisms but not in others. in spite of that, a generally acceptable consensus defines a stem cell as an undifferentiated cell that has capacity to self-renew, for production of progeny in at least two lineages, for long-term tissue repopulation after transplantation, and for serial transplantability. in addition, stem cells exist in a mitotically quiescent form and clonally regenerate all of the different cell types that constitute the tissue in which they exist. they can undergo asymmetrical cell division, with production of one differentiated (progenitor) daughter and another daughter that is still a stem cell. the offspring of stem cells are referred to as progenitor cells, also named as transit amplifying cells and therefore cannot be serially transplanted, and are classified as early and late. the early progenitor or stem/ either removed from the circulation by the liver or undergo further transformation by lipoprotein lipase and/or hepatic lipase to form intermediate-density lipoproteins and ldls. ldls, which are relatively depleted of triglyceride and enriched in cholesteryl esters and phospholipid, circulate in the blood and bind to specific ldl receptors that are widely distributed throughout tissues in order to deliver cholesterol. hdls produced by the liver play an important role as donors and acceptors of apo c, apo e, and various lipids from other lipoproteins in the circulation. reverse transport. hdls play an important role in the reverse transport of cholesterol from the periphey to the liver. lecithin cholesterol acyl transferase esterifies hdl cholesterol and cholesteryl esters move to the core of the hdl molecule to allow more free cholesterol to be absorbed into the particle. continued absorption of free cholesterol and subsequent esterification by lecithin cholesterol acyl transferase leads to the formation of the larger, cholesteryl ester-rich hdl2s. hdl2 molecules continuously acquire cholesteryl esters, resulting in the formation of the hdl1 molecules. on hdl1, cholesteryl esters are transferred from tissues to the liver for disposal or reuse, and not to ldl or vldl molecules (as in humans), which transfer cholesterol to peripheral tissues. this function of hdl1s may account for the lower incidence of atherosclerotic disorders in dogs compared with humans. 23, 24 nucleic acids pyrimidine biosynthesis is one of the classic roles of the liver in nucleic acid metabolism. more recently, micrornas have been impugned in the normal development and regeneration of the liver, as well as in hepatic pathology. micrornas are small noncoding rnas that regulate both messenger rna and protein expression of target genes, which results in alterations in messenger rna stability or translation inhibition. micrornas influence at least one-third of all human transcripts and are known regulators of various important cellular growth and differentiation factors. micrornas recently emerged as key regulatory molecules in chronic liver disease. 25 porphyrins porphyrins are intermediates of the heme biosynthetic pathway. porphyrins are found in hemoglobin, myoglobin, cytochromes, catalase, and peroxidase enzyme. the liver and biliary tract serve as an excretory route for the porphyrins. the liver stores iron, which can be toxic in excessive amounts (hemochromatosis). the amount of iron in the body is largely determined by regulation of its absorption in the upper small intestine. iron is stored intracellularly as ferritin in a number of tissues, with the liver having a large storage capacity. when the capacity of the liver is exceeded, iron accumulates as hemosiderin. the liver incorporates copper into specific copper proteins such as cytochrome c oxidase, mitochondrial monoamine oxidase, and ceruloplasmin. mobilization of copper from hepatocytes takes place by at least two mechanisms: ceruloplasmin and bile secretion. cholestatic liver disease is associated with secondary copper retention, which may then induce hepatocyte injury. 26, 27 vitamins the liver is importantly involved in vitamin metabolism. the liver produces bile for absorption of fat-soluble vitamins (a, d, e, k), and the liver is an important site for vitamin storage. converts them to carbohydrates and lipids. [18] [19] [20] [21] deamination produces α-keto acids, which can be metabolized for energy or used for synthesis of monosaccharides and fatty acids. 20 the liver synthesizes amino acids from intermediates of carbohydrate and lipid metabolism by amination and transamination. 21 examples of amino acid transaminations include: • alanine + α-ketoglutarate ↔ pyruvate + glutamate • aspartate + α-ketoglutarate ↔ oxaloacetate + glutamate the liver synthesizes many proteins, including albumin and fibrinogen, most α globulins, and some of the β globulins. prothrombin and clotting factors v, vii, viii, ix, and x are produced in the liver, as well as ceruloplasmin, ferritin, and many serum enzymes. lipid metabolism and transport is organized into three basic transport systems: (a) exogenous transport, which is associated with the metabolism of exogenous (dietary) lipids, (b) endogenous transport, which is associated with the metabolism of endogenously produced lipids, and (c) reverse transport, which is associated with the transport of lipids from the periphery (e.g., skeletal muscle, adipose, connective tissue) to the liver. exogenous transport. triglyceride is the major dietary lipid, along with cholesterol, phospholipids, and fat-soluble vitamins. 22 the digestion of dietary lipids begins in the proximal gi tract with the action of lingual and gastric lipases, and is completed in the small intestine with the actions of pancreatic lipase, cholesterol ester hydrolase, and phospholipase a 2 . lipid digestion and absorption is more complicated than carbohydrate and protein digestion and absorption because of lipid solubility characteristics, and involves emulsification of lipids by bile salts, hydrolysis by pancreatic lipase and colipase, solubilization of fatty acids and monoglycerides into mixed micelles, absorption, reesterification, chylomicron formation, and transport into the intestinal lymphatics or portal capillaries. chylomicrons containing short-and long-chain triglycerides, and the newly incorporated b-100 apoprotein, are preferentially absorbed into the intestinal lymphatics where they are transported into the cisterna chyli, thoracic duct, and systemic circulation where they acquire apolipoproteins c and e from circulating high-density lipoproteins (hdls). apolipoprotein (apo) c-ii activates lipoprotein lipase in the capillary beds of adipose and skeletal muscle, where they are stored as is or hydrolyzed into free fatty acids, β-monoglyceride, and glycerol. the cholesterol-rich remaining particles (now referred to as chylomicron remnants), return apo c-ii molecule to hdl and are recognized by specific hepatic apo e and apo b-100 receptors that rapidly remove them from the circulation by endocytosis. the cholesterol found in chylomicron remnants can be used in very-low-density lipoprotein (vldl), lipoprotein synthesis, bile acid formation, or cholesteryl storage. endogenous transport. while chylomicrons are the apoprotein responsible for transport of dietary lipids, vldls, intermediatedensity lipoproteins, low-density lipoproteins (ldls), and hdls are instead involved in the metabolism of endogenously produced lipids. triglycerides and cholesterol produced by the liver combine with phospholipids, apo b-100, and apo b-48 to form vldls. when secreted from the liver, vldls acquire the apo c and apo e from hdl. vldl apo c-ii activates lipoprotein lipase located in the capillary beds, where once again triglyceride hydrolysis takes place with the production of free fatty acids and glycerol. the vldl molecules remaining after hydrolysis of vldl triglycerides are intestinal bacteria to produce the secondary bile acids, deoxycholic acid, and lithocholic acid. 2, 5, 28 prior to secretion, bile acids are conjugated with taurine and/or glycine to form tauro-and glycoconjugated bile salts (see figure 61 -3, c). conjugation lowers the pk a to well below the physiologic range of biliary and intestinal ph, and conjugated bile acids become ionized anions (referred to as bile salts) rather than undissociated bile acids. in the ionized form, they are less likely to be absorbed by the small intestine and so maintain a higher intraluminal concentration appropriate for emulsification, digestion, and absorption of lipids. dogs and cats conjugate primarily with taurine. dogs can convert to glycine conjugation if taurine is deficient, but cats cannot. cats are obligate taurine conjugators, and have an essential dietary taurine requirement. 2, 5, 28 bile salts are amphipathic molecules with polar and nonpolar domains imparting two important functions. bile salts have an initial detergent effect on fat particles in food permitting the breakup of fat globules into smaller sizes. this is the initial emulsification phase of bile salts that facilitates intraluminal lipid hydrolytic digestion. bile salts further assist in the absorption of fatty acids, monoglycerides, cholesterol, and other lipids through the formation mixed micelles. these micelles serve to transfer digested lipids across the unstirred layer of the mucosa. following emulsification and micellarization of fat, most of the secreted bile salts are transported along the gi tract to the ileum where they are absorbed into ileal enterocytes and portal blood flow via na + -bile salt cotransporters. 2, 28 the liver plays an important role in maintaining hemostasis. the liver produces procoagulant, anticoagulant, and fibrinolytic proteins, and also removes normal and abnormal clotting factors from the circulation. 29 hepatocytes synthesize most of the clotting factors including clotting factor i (fibrinogen), ii (prothrombin), v, vii, ix, x, xi, and xiii. the site of biosynthesis of factor viii remains controversial, but it is probable that the liver plays an important role in this factor, too. the liver is also responsible for the activation of the vitamin k-dependent factors ii, vii, ix, and x and protein c. in addition to the production and activation of coagulation factors, the liver is also essential for the clearance of activated coagulation water bilirubin -vitamin a is stored in both stellate cells and hepatocytes. approximately 95% of total body vitamin a is stored in the liver, representing a 1-to 2-year supply. the liver continues to release vitamin a to maintain normal blood concentrations despite reductions in its content. liver and plasma vitamin a concentrations are reduced by malnutrition, liver disease, and intestinal malabsorption, but signs of deficiency do not appear until abnormalities become severe. fat-soluble vitamins a, d, e, and k require normal bile secretion for absorption. vitamin k is particularly essential for synthesis of the prothrombin-complex clotting factors. water-soluble vitamins, with the exception of vitamin b 12 (cobalamin), are readily absorbed from the small intestine. these vitamins are used primarily as coenzymes in metabolic processes. vitamin phosphorylation, occurring primarily in hepatocytes, is required to produce some coenzymes. thiamine is phosphorylated to thiamine pyrophosphate, for example, primarily in the liver and kidney. nicotinic acid is a precursor in pyridine nucleotide synthesis, and an initial step in its conversion is nicotinamide synthesis in the liver. pyridoxine is phosphorylated to its active form in the liver, as is the transformation of pantothenic acid to coenzyme a. folic acid is converted to its active form in the liver. large amounts of all water-soluble vitamins except vitamin c are stored in the liver. glutathione is synthesized in most if not all mammalian cells. the liver is particularly active and has relatively high levels of glutathione. glutathione performs a variety of physiologic and metabolic functions, including thiol transfer reactions that protect cell membranes and proteins; thiol-disulfide reactions involved in protein synthesis, protein degradation, and catalysis; reduction of capacity; detoxification of hydrogen peroxide, organic peroxides, free radicals, and foreign compounds; and metabolism of various endogenous compounds. seen in dogs with chronic hepatitis plus cirrhosis, which may be a result of reduced synthesis rather than increased consumption of coagulation factors. numerous foreign compounds, including drugs, are so hydrophobic that they would remain in the body indefinitely were it not for hepatic biotransformation. cytochrome p450 (p450 or cyp) comprises a superfamily of enzymes that catalyze oxidation of a variety of xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens, as well as endobiotic chemicals including steroids, fatty acids, prostaglandins, and vitamins. the cytochrome p450 enzymes in families one to three mediate 70% to 80% of all phase i-dependent metabolism of clinically used drugs and participate in the metabolism of a huge number of xenobiotic chemicals. there are 57 known active p450 genes and 58 pseudogenes in the human genome. with 54 active genes, dogs are phylogenetically closest to the human. although there are many similarities between dogs and humans, there also are many important differences. [30] [31] [32] [33] dogs present an products and the production of clotting factor inhibitors, such as antithrombin and α 1 -antitrypsin, as well as fibrinolytic proteins like plasminogen. in liver disease, factor and inhibitor synthesis and clearance of activated factors in both the coagulation factors and fibrinolytic system may be impaired. the extent of coagulation abnormalities depends upon the degree of disturbed liver function. 29 patients with hepatic failure may present with the entire spectrum of factor deficiencies and may even develop disseminated intravascular coagulation. in a study of 42 dogs with histologically confirmed liver disease, one or more coagulation abnormalities were found in 57% of dogs with liver disease. 29 activated partial thromboplastin time was significantly prolonged in dogs with chronic hepatitis with or without cirrhosis. mean platelet numbers, antithrombin, and factor ix activity were significantly lower in dogs with chronic hepatitis with cirrhosis, compared to dogs with other hepatopathies. d-dimers were not significantly increased in any group. only three dogs, all with different histologic diagnoses, satisfied the criteria for disseminated intravascular coagulation. hemostatic abnormalities were primarily where nh3 = ammonia; adenosine triphosphate = adenosine triphosphate; adp = adenosine diphosphate; p i = inorganic phosphate; and amp = adenosine monophosphate. mineralocorticoids (aldosterone), glucocorticoids (cortisol, corticosterone), and sex steroids (androgens, estrogens, progesterone) are metabolized by the liver. changes in the concentrations of total and free cortisol and of the binding capacity of corticosteroid-binding globulin have been reported in canine liver disease. as a consequence of hypercortisolemia, dogs with liver disease and hepatoencephalopathy have clinical and biochemical characteristics of pdh, including polyuria, high basal cortisol levels, and α-melanotropin. 37, 38 chronic hypercortisolism is associated with impaired osmoregulation of the release of vasopressin and inadequate urinary concentration. 39 the multiple physiologic functions of the liver require an immune response that is locally regulated. pathogenic microorganisms must be efficiently eliminated, while the large number of antigens derived interesting challenge in the assessment of p450-mediated drug-drug interactions because most of the enzymes have not been completely characterized, diet and aging induce significant changes in gene expression, and dogs are often treated off-label with a number of human drugs with little idea of risk for drug-drug interaction. drug metabolism takes two general forms: phase i metabolism (modification reactions) and phase ii metabolism (conjugation reactions). phase i metabolism typically subjects a drug to oxidation or hydrolysis. it involves the cytochrome p450 (cyp) enzymes, which facilitate reactions that include n-, o-, and s-dealkylation; aromatic, aliphatic, or n-hydroxylation; n-oxidation; sulfoxidation; deamination; and dehalogenation. phase ii metabolism conjugates the drug to hydrophilic substances, such as glucuronic acid, sulfate, glycine, or glutathione. phase i metabolism usually precedes phase ii metabolism, but this is not always the case. 34 the liver is an important site of drug toxicity and oxidative stress because of its proximity and relationship to the gi tract. seventyfive percent to 80% of hepatic blood flow comes directly from the gi tract and spleen via the main portal vein. portal blood flow transports nutrients, bacteria and bacterial antigens, drugs, and xenobiotic agents absorbed from the gut to the liver in a more concentrated form. drug-metabolizing enzymes detoxify many xenobiotics but might activate the toxicity of others. hepatic parenchymal and nonparenchymal cells may all contribute to the pathogenesis of hepatic toxicity. the toxicity of drugs can be considered in five contexts: on-target toxicity, hypersensitivity and immunologic reactions, off-target pharmacology, bioactivation to reactive intermediates, and idiosyncratic drug reactions. 35, 36 ammonia ammonia is an important by-product of amino acid metabolism. organisms that cannot easily and quickly remove ammonia usually have to convert it to some other substance, like urea or uric acid, which are much less toxic. insufficiency of the urea cycle occurs in from the gi tract must be tolerated. the liver favors the induction of tolerance rather than the induction of immunity. although hepatocytes constitute the major cell population of the liver, direct interaction of hepatocytes with leukocytes in the blood is unlikely. sinusoidal endothelial cells, which line the hepatic sinusoids and separate hepatocytes from leukocytes in the sinusoidal lumen, and kupffer cells, the resident macrophage population of the liver, can directly interact with passenger leukocytes. in the liver, clearance of antigen from the blood occurs mainly by sinusoidal endothelial cells through very efficient receptor-mediated endocytosis. liver sinusoidal endothelial cells constitutively express all molecules necessary for antigen presentation (cd54, cd80, cd86, major histocompatibility complex [mhc] classes i and ii, and cd40) and can function as antigen-presenting cells for cd4 + and cd8 + t cells. 40, 41 thus, these cells probably contribute to hepatic immune surveillance by activation of effector t cells. antigen-specific t-cell activation is influenced by the local microenvironment. this microenvironment is characterized by the physiologic presence of bacterial constituents such as endotoxin and by the local release of immunosuppressive mediators such as interleukin-10, prostaglandin e 2 , and transforming growth factor-β. 42 regeneration liver regeneration after partial hepatectomy is a very complex and well-orchestrated phenomenon. it appears to be carried out by the participation of all mature liver cell types. 43, 44 the process is associated with signaling cascades involving growth factors, cytokines, matrix remodeling, and several feedbacks of stimulation and inhibition of growth related signals. 45, 46 the liver manages to restore any lost mass and adjust its size to that of the organism, while at the same time providing full support for body homeostasis during the entire regenerative process. in situations when hepatocytes or biliary cells are blocked from regeneration, these cell types can function as facultative stem cells for each other. gene expression in the regenerating liver is a multistep process with at least two critical steps: the transition of quiescent hepatocytes into the cell cycle ("priming"), and the progression beyond the restriction point in the g 1 phase of the cell cycle. hepatocytes must first be primed before they can fully respond to growth factors. as many as 70 different genes participate in the early response to hepatectomy, but tumor necrosis factor (tnf), interleukin (il)-6, and interleukin-22 appear to be the major cytokines involved in the priming of hepatocytes. 47 the proliferative effect of tnf on hepatocytes is further influenced by reactive oxygen species, nitric oxide, and glutathione content, and multiple transcription factors (e.g., nuclear factor kappa b, stat3, ap-1, and c/ebpβ) play major roles in the initiation of early liver regeneration. 47 progression through the cell cycle beyond the initiation phase requires growth factors, primarily hepatocyte growth factor and transforming growth factor-α (tgf-α). the subsequent expression of cell-cycle genes establishes the stage at which replication becomes growth factorindependent and autonomous. at this point, the hepatocyte is irreversibly committed to replicate and the cell cycle replication machinery takes over. the proliferation of hepatocytes advances from periportal to pericentral ares of the lobules, as a wave of mitoses. hepatocytes surrounding the central veins are the last ones to undergo cell replication. proliferation of biliary epithelial cells occurs a little later than hepatocytes. proliferation of endothelial cells starts at 2 to 3 days and ends around 4 to 5 days after partial hepatectomy. the kinetics of proliferation of stellate cells is incompletely understood. the regenerative capacity of the residual hepatocytes may restore liver mass and function after as much as 65% to 70% hepatectomy and it takes place over 7 to 14 days in most animal species. 48 a small wave of apoptosis in hepatocytes occurs at the end of regeneration. the liver is the second largest organ in the body and performs an estimated 1500 essential biochemical functions. 1 these diverse functions include drug metabolism; removal of exogenous and endogenous toxins (e.g., ammonia, food antigens); synthesis of vital substances such as albumin and blood clotting factors; protein, fat, and carbohydrate metabolism; vitamin storage and activation; glycogen, triglyceride, and mineral (e.g., copper, iron) storage; activation, conversion, secretion, deactivation, and excretion of various hormones; bile salt synthesis; conjugation and excretion of bilirubin in bile; among others. symptoms (defined here as abnormalities noted by the owner), clinical signs (defined here as abnormalities found during the physical examination), and diagnostic results reflect impairments in these functions. hepatitis represented approximately 1% of the clinical population of the companion animal teaching hospital of utrecht university. box 61-1 summarizes the most common liver diseases with their possible etiologies in dogs and cats. a properly taken history is pivotal to defining the most clinically relevant problems that need to be resolved. a structured interview process and understanding the basics of communication are important success factors to retrieve this crucial information. fortunately, the knowledge about communication in the medical profession and the focus on the veterinary curriculum, has increased considerably during the last few years. 2, 3 some basic principles should be kept in mind to understand the symptoms in dogs and cats with diseases affecting the hepatic parenchyma, portal vasculature, and the biliary system. first, for most of its functions, the liver has a tremendous (approximately 80%) reserve capacity and a remarkable potential to regenerate. 4 symptoms occur only when progressive disease exhausts hepatic reserves. diseases often remain subclinical for lengthy periods of time; symptoms may be relatively mild and nonspecific because the liver reserve prevents overt abnormalities. symptoms such as lethargy, vomiting, or mild polyuria and polydipsia (pu/pd) may alert the clinician that a liver disorder could be developing. serious symptoms may indicate loss of hepatic reserves. the onset of symptoms may be acute, but they may be the end result of a disease that has been present for many weeks or months. because no specific physical abnormalities occur with most liver diseases, it is important to remember that liver disease may be present when symptoms of illness are unexplained or nonspecific. sensitive and specific laboratory tests may easily detect such liver diseases. 5 second, most liver diseases cause similar signs and symptoms (table 61 -2). one of these is acholic feces, which occurs nearly exclusively in dogs with common bile duct obstruction. 6 owners b documented in humans, may occur in dogs and cats. a more likely to present with chronic rather than acute liver disease. the clinical signs listed in table 61 -2 are candidates for having a primary hepatopathy. in all such cases, further diagnostic studies should be performed to confirm or exclude liver disease ( figure 61 -5). breed, sex, age, and drugs may predispose dogs and cats to hepatopathies. the presence of numerous risk factors should be a stimulus for an extended diagnostic workup; other diseases should be investigated in the absence of such suspicions. caused by hypersensitivity to sulfonamides, destructive cholangiolitis is the most common drug-induced liver disease. 14 a recent history of therapy with sulfonamides or other potentially hepatotoxic drugs, combined with icterus makes this condition likely and should prompt immediate discontinuation of the medication. breed associations may occur when a disease is (in part) determined by genetic factors. breeders may, by chance, increase the incidence of hepatic diseases by familial selection. because dog breeds may represent more or less closed populations in a country, breed predispositions may vary among countries. therefore this section only mentions generally applicable predispositions; locally, other breed associations may be more pertinent. chronic hepatitis and cirrhosis, both of which are, as a rule, different stages of one disease, occur more frequently in certain breeds. 15 hepatitis may develop at any age, but typically not before 2 years of age. only lobular dissecting hepatitis tends to occur at a young age (i.e., often before 1 year of age). 16 breeds associated with hepatitis are doberman pinschers, bedlington terriers, west highland white terriers, american and english cocker spaniels, labrador retrievers, and many other breeds. recent copper excretion studies have shown that hepatitis is caused by copper retention and not vice versa in doberman pinschers. 17 the cause of copper retention remains unclear; many of the tested candidate genes (including murr 1, the affected gene in bedlington terriers) were excluded as monogenetic causes for copper-associated subclinical hepatitis in may note the light-gray appearance of stool, which in combination with icterus is virtually diagnostic for extrahepatic cholestasis. different combinations of clinical signs and symptoms may occur with any liver disease. statistically, one disease may be associated with a typical pattern of signs and symptoms in dogs and cats. however, overlapping patterns are so great that it is useless to try to identify the exact disease based on clinical signs and symptoms alone (table . clinical signs and symptoms associated with liver diseases of cats are similar to those in dogs, except for pu/pd, which is not clinically overt in most cats. certain liver diseases cause neurobehavioral signs associated with hepatic encephalopathy, 7 and these signs may wax and wane in their frequency and severity. any medication given may appear to be effective because of the natural fluctuation of signs. therefore signs of hepatic origin may be easily missed. seizures alone are never caused by hepatic encephalopathy; if they do occur, they occur in combination with other signs seen with this syndrome. 5 furthermore, very few, if any, medications to treat liver diseases have been tested in double-blind, placebocontrolled studies, making decisions about the best therapeutic regimens difficult. 8 it is usually not possible to differentiate between hepatic diseases and diseases of other organs based on symptoms and clinical signs. signs associated with hepatic diseases are nonspecific; similar signs may occur in diseases of many other organ systems, most notably the gi, neurologic, renal, and hematologic systems (see table 61 -2). 9 of the gi tract-related symptoms, nausea expressed as vomiting in acute cases or reduced, irregular appetite with occasional vomiting and weight loss over time, is very common in liver and biliary diseases of dogs and cats. for biliary diseases these are always the most prominent symptoms. diarrhea, however, is not a major symptom of liver disease, and in cases in which diarrhea is the leading symptom the liver is only rarely the causative organ (except for rare cases with complete common bile duct obstruction). a rare sign (not included in table 61 -2) is an ulcerative form of dermatosis, so-called superficial necrolytic dermatitis, hepatodermal, or hepatocutaneous syndrome. this syndrome occurs rarely in dogs with liver cirrhosis and nodular hyperplasia and whose pathogenesis is poorly understood. 10 this symptom and the more common symptoms of lethargy, inappetence, vomiting, diarrhea, weight loss, pu/pd, and neurobehavioral symptoms are frequently associated with diseases of other organs. therefore the history often discloses symptoms that may suggest hepatic disease, but may also be caused by other disorders. two main reasons account for the nonspecificity of liver-related symptoms. first, the liver is the central organ for many metabolic and detoxifying pathways; consequently, failing liver function may cause dysfunction of other organs. one example is hepatic encephalopathy; metabolic dysfunctions of the liver cause neurotransmitter dysfunctions of the brain, resulting in neurobehavioral signs. 11 second, toxic factors resulting from diseases of other organ systems (especially from the gi tract) often secondarily affect the liver. examples include hepatic lipidosis in diabetes mellitus, steroidinduced hepatopathy in cushing syndrome, reactive hepatitis in gi diseases, and centrolobular liver necrosis in acute, severe anemia. 5 therefore signs and symptoms of liver disease may be hidden within signs of other organ dysfunction, and vice versa. because clinical and physical examination findings may be compatible with hepatic disease, and because laboratory tests to detect hepatic disease are also abnormal with primary and secondary hepatopathies, it is often necessary to make a histologic diagnosis of the liver disorder to resolve this dilemma. 12, 13 lack of specific physical examination findings may prevent recognition of a primary liver disease. most dogs with illnesses causing pathetic innervation; therefore dilation (e.g., extrahepatic cholestasis), cholecystitis, or cholelithiasis should be suspected in vomiting dogs and cats. 5 vomiting is also common in upper gi disease. in many gi diseases, translocation of bacteria and endotoxins may cause secondary, nonspecific, reactive hepatitis. 30 this occurs frequently in dogs, but rarely in cats. reactive hepatitis is characterized by intrahepatic canalicular cholestasis, liver cell necrosis, and an exudative inflammatory reaction. clinical signs, symptoms, and diagnostic results associated with primary liver disease and reactive hepatitis are similar; therefore, a further diagnostic workup is important to reveal the primary cause. small bowel-type diarrhea occurs frequently with hepatic diseases (see table 61 -2). two primary mechanisms may account for clinical signs. first, cholestatic diseases (intrahepatic or extrahepatic caused by common bile duct obstruction) disrupt the normal enterohepatic cycle of bile acids; therefore less bile reaches the duodenum. 30, 31 decreased resorption of dietary fat may cause hyperosmotic intestinal contents and diarrhea. however, studies in rats show that cholestasis must be severe before steatorrhea as a result of disruption of the enterohepatic bile acid cycle occurs. another mechanism for diarrhea in liver disease is increased resistance to portal blood flow, resulting in portal hypertension and congestion of splanchnic organs. intestinal vasculature congestion reduces intestinal water resorption and increases intestinal volume content. this is the predominant mechanism underlying diarrhea in diseases such as chronic hepatitis, lobular dissecting hepatitis, portal vein thrombosis, and portal vein hypoplasia. 5 alternatively, when the primary cause of diarrhea is intestinal disease, the liver may be affected secondarily. in those cases, the hepatic macrophage system should remove the increased absorption of (endo)toxins or bacteria by the affected intestinal wall. increased exposure, however, can lead to secondary, nonspecific, reactive hepatitis. endotoxins also effectively inhibit bile formation and flow, leading to cholestasis. it is therefore common to find increased plasma liver enzyme activities and bile acid levels in cases of reactive hepatitis; clinical icterus may even be apparent. the cause of diarrhea can be determined only by further diagnostic methods, including histologic evaluation of liver biopsy specimens. reactive hepatitis resolves rapidly when the primary disease is treated successfully. in the authors' experience it is very rare to find diarrhea as single or the leading symptom in cases of liver disease. if present, it is usually one of the less prominent symptoms in the spectrum of other more prominent symptoms such as apathy, pu/pd, or vomiting. one may therefore elect to follow liver laboratory values after treatment of the intestinal disease and perform a liver biopsy if liver parameters fail to improve within a few weeks. hepatic encephalopathy is a complex of neurobehavioral signs resulting from portosystemic shunting of blood in combination with a reduction of functional liver mass. 11 it may occur in animals with cpss or in those with apscapsc because of portal hypertension. diseases associated with the latter form are chronic hepatitis, cirrhosis, portal vein hypoplasia, lobular dissecting hepatitis, and portal vein thrombosis. 32 cats, because of their dependence on some essential amino acids (e.g., arginine), may develop hepatic encephalopathy without portosystemic shunting, especially when they have a severe form of hepatic lipidosis. 33 doberman pinschers. 17 the hepatitis in doberman pinschers is sex linked, confined to females, and aggressive. 18 it is responsive to medication with penicillamine, 19 but may terminate in micronodular cirrhosis. this form of cirrhosis, predominantly seen in copper toxicosis, differs from other forms of chronic hepatitis, in which patients typically develop macronodular cirrhosis with large hyperplastic nodules. hepatitis is overrepresented in female doberman pinschers by a factor of 10; a study in finland showed that approximately 10% of doberman pinschers may be affected. 20 inherited copper toxicosis is also a well described entity in bedlington terriers worldwide. 21 both sexes may be affected. clinical signs usually develop after 4 years of age as a result of the gradual accumulation of copper. it is caused by a defect in the murr 1 gene, leading to a severely decreased excretion of copper by hepatocytes. other affected breeds are west highland white terriers (particularly in the united states), skye terriers, dalmatians, anatolian shepherd dogs, and labrador retrievers. [22] [23] [24] [25] [26] siamese cats may also be predisposed to copper-associated hepatopathies. 9 although essential for life, copper is usually ingested to excess and must be eliminated by the liver to prevent toxicity. the central role of the liver in copper homeostasis makes it vulnerable if elimination processes fail. 27 furthermore, increased copper levels add to the oxidative stress, which is an important component in chronic inflammatory and cholestatic diseases in dogs. 25 spaniels seem to have the form of chronic hepatitis unrelated to copper toxicosis and develop macronodular cirrhosis when left untreated. no sex predisposition exists, but there seems to be a worldwide overrepresentation of hepatitis in this breed. congenital portosystemic shunts (cpss) are seen in both sexes in various breeds. intrahepatic shunts predominate in large breeds, whereas extrahepatic shunts predominate in small and toy breeds. although cpss are most likely inherited in some fashion in all affected breeds, this has only been proven in irish wolfhounds 28 and cairn terriers. 29 worldwide predispositions occur in irish wolfhounds, australian cattle dogs, labrador retrievers, dachshunds, yorkshire terriers, cairn terriers, maltese terriers, and miniature schnauzers. 7, 9 in the united states, an increased prevalence of shunts has also been reported to occur in german shepherd dogs, doberman pinschers, and golden retrievers. cpss occur most often in mixed-breed cats; however, persian and himalayan cats are frequently overrepresented. clinical signs are usually seen in young dogs and cats (<1 year old) with congenital shunts. 5, 9 pathogenesis of common symptoms of primary liver diseases vomiting is one of the most common symptoms noted in dogs and cats with liver disease. vomiting may be caused by direct stimulation of the vomiting center via the chemoreceptor trigger zone in the fourth ventricle by (endo)toxins that are not cleared by the liver. 30 this typically occurs when toxins from the gi system bypass the liver and access other body systems. vomiting is common in all conditions that share portosystemic shunting and liver dysfunction (e.g., congenital shunts and acquired shunts because of hepatitis, fibrosis, cirrhosis, and portal vein hypoplasia or thrombosis). hepatic diseases that cause an abnormal liver shape may reposition the upper gi tract and induce nausea and vomiting by vagal stimulation. causes include hepatic tumors, especially liver cell (or hepatocellular) carcinomas, and unilateral collapse and contralateral hypertrophy, which may occur with thrombosis of a main branch of the portal vein. the gallbladder and larger bile ducts have a rich sym-form ammonium urate. affected dogs usually have clinical signs related to shunting and liver dysfunction, such as hepatic encephalopathy, pu/pd, or vomiting. in the other category, the enzyme uricase, which forms allantoin, is inactive because of an inborn error affecting only this function. ammonium urate urolithiasis occurs commonly in dalmatians but may occur in other breeds. 43 affected dogs only have signs related to urolithiasis (e.g., pollakiuria, stranguria, dysuria). an owner may note acholic feces, which can provide a direct clue to the underlying diagnosis. steatorrheic feces that do not contain normal bile pigment are seen only when bile flow into the intestinal tract is completely disrupted, usually as a consequence of extrahepatic obstruction of the common bile duct. 6 destructive cholangiolitis is the only intrahepatic process severe enough to seriously disrupt bile flow. the latter disease is caused by a hypersensitivity reaction to sulfonamide-containing drugs. the smaller bile ductules become necrotic and liver lobuli may be disconnected from the biliary tract. affected dogs have a history of recent medication with sulfonamides. acholic feces contain excess fat because resorption is impaired. the lack of normal black-brown fecal pigments occurs because their precursor, bilirubin, does not reach the duodenum. therefore, the feces from affected animals are gray-white and soft. animals with this condition often are icteric. the presence of icterus reduces the likelihood of exocrine pancreatic insufficiency as a diagnosis. abdominal distention may occur in dogs and cats with liver disease for several reasons. first, ascites is a frequent finding associated with liver disease in dogs as a result of portal hypertension, but is less common in cats. abdominal distention may also result from organ enlargement, which in the case of liver disease may include the liver and, in the spleen in the presence of portal hypertension. in contrast to dogs, cats often have hepatomegaly with liver disease. nonspecific symptoms, such as apathy, reduced appetite or anorexia, and weight loss, may occur in dogs and cats with liver disease. retarded growth is common in young animals. these problems reflect the central role of the liver in many metabolic and detoxifying functions. in addition, nausea, inappetence, vomiting, and diarrhea can result in a catabolic state, which, in turn, may aggravate hepatic encephalopathy. signs of early hepatic encephalopathy include depression and other nonspecific problems. anemia, another common finding in liver disease (see below), can cause general malaise. dogs with liver cell carcinoma often are hypoglycemic, 44 which may be the primary problem underlying apathy and weakness. production of insulin-like growth factors by the tumor may be responsible. as with historical findings, physical examination findings rarely provide enough information to pinpoint the liver as definitive cause of the presenting problems. possible findings include icterus, hepatomegaly, splenomegaly, ascites, and pale mucous membranes. petechiae of the skin or mucous membranes occur infrequently. of these possible findings, only icterus and hepatomegaly are more or less specific for liver diseases; other abnormalities on the physical examination occur more frequently with diseases of other organ systems. hepatic encephalopathy is caused by derangement of neurotransmitter systems caused by defective metabolic processes in the liver. 34 inadequate metabolism of ammonia and aromatic amino acids by the liver may reduce the excitatory glutamatergic and monoaminergic neurotransmitter system tones, respectively. 35 in addition, there is an increased tone of the inhibitory γ-aminobutyric acid (gaba) system. 36 these neurotransmitter derangements make anesthesia risky in some animals with liver disease. the liver inactivates many anesthetics and the unforeseen delay of recovery from anesthesia may suggest an underlying liver disease as a cause for nonspecific clinical signs. this occurs especially in dog and cats with portosystemic shunting, either congenital or acquired. in addition to reduced hepatic clearance, anesthetics exert their action via various neurotransmitter systems in the brain, which may already be functioning abnormally as a consequence of hepatic encephalopathy. 34 this is especially true of drugs that act via the gaba-benzodiazepine pathway. that pathway is already overstimulated and may provoke an exaggerated and prolonged anesthetic effect. pu/pd is one of the most frequent signs (50% of cases) in dogs with liver disease, but is less common in cats. pu/pd is most common in diseases associated with congenital or acquired portosystemic shunting and, therefore, with hepatic encephalopathy. in affected dogs, abnormal neurotransmitter disturbances lead to increased secretion of adrenocorticotropic hormone (acth) from the anterior and intermediate pituitary lobes. 37 chronically elevated acth stimulates increased levels of free cortisol. increased levels of free cortisol, in turn, affect the posterior lobe of the pituitary creating an increased threshold for the release of arginine vasopressin. 38, 39 thus, a higher plasma osmolality is required to stimulate antidiuresis through arginine vasopressin, and before reaching that threshold, affected dogs become thirsty and start drinking. 38 pu/pd is not only present in cases with hepatic encephalopathy, but also frequently in all other liver diseases. this may be caused by certain bile acids, which are often increased in plasma of animals with liver diseases. bile acids may inhibit the activity of 11β-hydroxyl steroid dehydrogenase. 40 this enzyme protects the aldosterone receptor from occupation by cortisol, by converting cortisol into cortisone, which cannot bind to the receptor. present in plasma in tenfold excess compared with aldosterone, cortisol can occupy and stimulate the aldosterone receptor thereby inducing pseudohyperaldosteronism and pu. 41 reduced hepatic formation of urea is another possible but undocumented mechanism that may play a role in the pathogenesis of pu/ pd. in urea deficiency states, the kidney does not have sufficient urea available to build up an osmotic gradient in the medulla. apart from this mechanism, pd also occurs in liver diseases not associated with hepatic encephalopathy (e.g., extrahepatic cholestasis and liver tumors). the mechanism is unclear; however, nausea with an impulse to drink and compensation of water loss by vomiting and diarrhea may play a role. 5 dysuria may occur as a result of insufficient liver function when nonmetabolized uric acid is excreted by the kidneys and precipitates to form uroliths. such calculi are seen in dogs but rarely in cats. 42 there are two main categories of liver dysfunction that cause ammonium urate urolithiasis. most frequently it is caused by congenital portosystemic shunting, whereby the liver is underdeveloped and fails to metabolize uric acid into allantoin. in urine, uric acid flocculates easily in the presence of high ammonia concentrations to a result of congestive heart disease can, in most cases, be recognized easily by physical examination of the circulatory system. measurement of central venous pressure is diagnostic. the exception is liver congestion caused by a thrombus in the caudal vena cava proximal to the liver, which is assessed by other methods. when the liver is overtly enlarged because of congestion, ascites is usually present. ascitic fluid has the typical slightly hemorrhagic appearance of congestive fluid. dogs with enlarged livers and no signs of congestive disease often have liver cancer, which may be primary, metastatic, or a form of malignant lymphoma. with most tumors, the liver is diffusely enlarged, but primary hepatocellular carcinomas or adenomas may cause enlargement of the affected lobe only. bile duct carcinomas disseminate easily over the biliary system and usually cause pronounced icterus and hepatomegaly. most cats with hepatic disease develop pronounced enlargement of the liver. in cats, liver enlargement occurs with cholangitis, hepatic lipidosis, amyloidosis, hepatic tumors (primarily malignant lymphoma), and congestive disease. when the liver is involved in feline infectious peritonitis, it may not be enlarged. cats with cpss have small livers. splenomegaly and ascites in association with liver disease are nonspecific findings. they occur especially with hepatic diseases causing portal hypertension. both findings are frequent in dogs but rare in cats. there is a positive undulation test with distinct ascites; slight ascites can be found with ultrasonography rather than physical examination. the liver may be enlarged with central causes of venous congestion. canine liver diseases associated with portal hypertension include chronic hepatitis and cirrhosis, portal vein hypoplasia, and lobular dissecting hepatitis. portal hypertension is sometimes seen with cirrhosis because of advanced cholangiohepatitis in cats. in these diseases, hepatic encephalopathy is also common. portal vein thrombosis is a prehepatic cause of portal hypertension that usually causes ascites. although as a rule the liver is small in these cases, unilateral obstruction of a main branch of the portal vein may cause hypertrophy of the rest of the liver, which may be palpable. diseases of the hepatic parenchyma, hepatic vasculature, and biliary tract are relatively common in dogs and cats. because the symptoms and signs accompanying liver disease are quite nonspecific, and the liver may be secondarily involved in diseases of other organs, liver disease can easily go undetected. therefore, after taking a thorough history and performing a physical examination, it is critical to perform additional biochemical tests with the highest possible diagnostic accuracy whenever liver disease is included in the differential diagnosis. if liver disease cannot be ruled out based on these examinations, additional testing is necessary to define the type of liver disease, most notably ultrasonography of the cranial abdominal quadrant and examination of a liver biopsy specimen. the algorithm in figure 61 -5 summarizes this approach. diagnosis usually depends on histopathologic examination of liver tissue, especially for parenchymal liver diseases, many biliary tract diseases, and tumors of the liver or biliary tract. although biopsy methods are beyond the scope of this chapter, excellent sources exist. 13 one note of caution: blood coagulation testing is vital before collecting a liver biopsy specimen for histopathology. most animals, for example, have one or more abnormal coagulation tests. [47] [48] [49] [50] [51] [52] factors involved may include vitamin k deficiency, reduced ascites and hepato-and splenomegaly may have been noted by the owner as abdominal enlargement. 5 biochemistry analyses are an integral part of the diagnostic process for liver diseases. most of these analyses are not a decisive factor in the diagnosis of liver disease, but serve to rule out liver disease from the differential diagnosis. 8 icterus is the most frequently encountered specific abnormality noted on the physical examination in dogs and cats with liver disease. however, only approximately 20% of dogs with hepatobiliary diseases and 30% to 40% of cats are icteric. icterus results from bilirubin accumulation in the blood and extravascular space as a result of increased production, reduced clearance, impaired conjugation by the liver, and/or impaired bile flow. in most cases, a combination of these factors is involved. cholestasis is predominant; therefore conjugated bilirubin is the fraction present in greatest quantity. hemolysis alone does not result in icterus with normal liver function. when hemolysis is severe, however, it may result in such a degree of portal hypoxia that the centrolobular zones of the liver lobules become necrotic. in those cases, icterus results from the combination of increased production and reduced liver function and cholestasis. 45 if hemolysis is the primary cause of icterus, it must be severe, and the mucous membranes will be extremely pale. primary liver diseases that may cause icterus are commonly accompanied by hemolysis. whereas the erythrocyte lifetime is reduced to 6 to 10 days in dogs with severe primary hemolytic disease; it is 20 to 60 days (normally 100 days) in hepatobiliary disease. increased production of bilirubin and liver dysfunction with cholestasis result in a combined conjugated and unconjugated hyperbilirubinemia in dogs and cats with primary hepatic or hemolytic disease. 46 icterus caused by hemolytic disease is characterized by pale mucous membranes, whereas the mucous membranes in animals with primary liver disease are normal or only slightly pale. the combined evaluation of icterus and the color of the mucous membranes immediately reveal the nature of the underlying process. as previously discussed, most hepatobiliary diseases are accompanied by increased degradation of red blood cells. the mechanisms behind hemolysis in liver disease are not completely clear. hypersplenism and reduced portal blood flow due to portal hypertension may drastically prolong the transit time of erythrocytes through the spleen, with a greater chance that they will be trapped when they are slightly abnormal. increased fragility of red cell membranes may be a result of the high bile acid levels in most liver diseases, whereas a reduced clearance of enteral endotoxins and bacteria by the liver may also induce immune-mediated hemolysis. apart from hemolysis, nonregenerative anemia also may occur as part of the syndrome of anemia of chronic disease as an expression of catabolism and slight deficiencies of iron and b vitamins. although common in liver diseases, 12 anemia, in contrast to icterus, is nonspecific. like icterus, hepatic enlargement is a distinct sign of an abnormal liver. in dogs, most liver diseases do not cause hepatomegaly. exceptions include tumors of the liver, liver congestion, and secondary liver involvement in metabolic diseases. examples of the latter conditions are glycogen accumulation in the liver in cushing disease, fatty liver with diabetes mellitus, and rare cases of amyloidosis of the liver. the more chronic liver diseases of dogs tend to reduce liver size, and acute diseases cause little change in size. liver enlargement as hormones and drugs, leading to an increase in their serum activities in patients without primary hepatic disease. additionally, serum hepatic enzyme activities can be increased as a consequence of secondary hepatopathies. the magnitude of hepatic enzyme activity increases may aid in the assessment of the severity or the extent of hepatic injury but should not be considered to be prognostic. the liver has a large capacity for regeneration, so even in cases of severe hepatic injury, with dramatically raised hepatic enzyme activities, a full recovery is possible. this is especially true when the injury is acute. conversely, in cases of chronic end-stage liver disease, such as cirrhosis, serum hepatic enzyme activities may not be markedly increased, or may even be within the reference interval as a result of the replacement of hepatocytes with fibrous tissue. consequently, serial evaluation of serum hepatic enzyme activities is more useful for assessing prognosis than measurement at a single point in time. consistent decreases of a previously increased activity are considered a favorable sign in acute liver injury, whereas a decreasing hepatic enzyme activity in a patient with chronic liver disease that is clinically deteriorating suggests loss of hepatocytes because of fibrosis. it is important to note that serum hepatic enzyme activities do not provide an assessment of liver function. alt is an enzyme found primarily in the cytosol of hepatocytes. alt is released into the serum when hepatocyte membrane permeability is increased, or if there is hepatocellular necrosis. alt is considered to be the most liver-specific enzyme. alt is also produced by cardiac muscle, skeletal muscle, and the kidneys. 1 apart from the hepatic form, only the muscle isoenzyme is clinically significant. although uncommon, severe muscle injury can result in an increased serum alt activity. hepatic microsomal induction in response to some drugs can also produce small increases in alt activity. some controversy exists regarding the serum half-life for alt in dogs. the mean serum half-life of alt was reported as being 149 minutes in one study 1 and 59 hours in another. 2 the serum half-life of alt is generally believed to be shorter in the cat than in the dog. a mean serum half-life of 207 minutes was reported in an experiment involving three healthy cats. 3 the shorter half-life in cats means that increases in serum alt activity are considered more clinically important in this species. as alt is metabolized in the liver its serum half-life may be longer in patients with liver disease. 4 increased cell membrane permeability in the absence of hepatocyte destruction can cause a rapid increase in serum alt activity. because of this, alt activity is a considered to be a highly sensitive marker of hepatocyte injury. this also means that an increased alt activity does not imply severe or irreversible hepatocellular injury. the highest increases in alt activity are seen during acute hepatic inflammation or necrosis, but because of the capacity for the liver to regenerate these do not indicate irreversible damage. consequently, a single measurement of alt activity does not provide an accurate prognosis. however, the degree of the alt activity increase is believed to have some correlation with the number of hepatocytes that have been injured. cholestasis can also result in an increased serum alt activity because of hepatocellular damage caused by the accumulation of bile acids. certain drugs can lead to increases in serum alt activity. these are usually minor, for example, phenobarbital used at therapeutic doses frequently leads to small increases in serum alt activity, in the absence of hepatic insufficiency. these increases are thought to occur as a result of subclinical hepatic injury rather than induction of hepatic microsomal enzymes. 5 toxic doses production of clotting factors, some degree of disseminated intravascular coagulation (dic), and severe protein deficiency. 13, 52 it should be noted that taking a fine-needle aspiration biopsy should be considered safe, even if abnormalities in coagulation are present. however, its diagnostic value is limited as the liver architecture is lost. diagnosis of circulatory hepatic diseases depends on information obtained from laboratory results, ultrasonography (most reliable diagnostic test to date), and histopathology. 13 a recent paper found that fasting ammonia concentration was superior to fasting bile acids for diagnosing portosystemic shunting in dogs. 53 ammonia is easily measured in practice with dry chemical methods, some of which provide reliable results. 54 another recent publication showed ultrasonography to be a reliable diagnostic method to noninvasively characterize the underlying hepatic disease in dogs with hyperammonemia. 55 diagnostic evaluation of the hepatobiliary system has several aims: (a) to determine if hepatobiliary disease is present, (b) to assess liver function, (c) to determine if liver disease is primary or secondary, (d) to definitively diagnose hepatobiliary disease, and (e) to monitor response to treatment. despite the apparent clarity of these aims, hepatobiliary disease can present a diagnostic challenge for a number of reasons. first, as clinical signs can be nonspecific, hepatobiliary disease should be a consideration when evaluating any patient with signs of systemic disease. dogs and cats with hepatobiliary disease may not have any clinical signs. furthermore, because of the liver's central role in metabolism and detoxification of endogenous toxins and xenobiotic agents, a number of extrahepatic diseases can secondarily affect the liver. it is important to distinguish these secondary hepatopathies from diseases that originate in the liver (primary hepatopathies). additionally, serum markers of hepatocellular damage, cholestasis, and hepatic function can be abnormal in the absence of hepatobiliary disease. finally, the liver's large reserve capacity means that detectable loss of liver function often occurs late in the course of disease. thus, when assessing a patient with suspected hepatobiliary disease, it is important to consider the clinical presentation, results of laboratory testing, diagnostic imaging findings, and the results of cytologic and/or histopathologic evaluation together. hepatic enzymes can be divided into markers of hepatocellular damage and markers of cholestasis. serum alanine aminotransferase (alt) and aspartate transaminase (ast) activities are the two most commonly measured markers of hepatocellular leakage, while serum alkaline phosphatase (alp) and γ-glutamyltransferase (ggt) activities are the two most commonly measured markers of cholestasis. although increased serum hepatic enzyme activities are considered to be sensitive, they are not specific for primary liver disease because they are produced by extrahepatic tissues. the relative importance of these extrahepatic isoenzymes varies, but their extrahepatic release can lead to increased serum activities. also, the production of some hepatic enzymes can be induced by certain alp (b-alp), and kidney alp (k-alp) are transcribed from the tissue nonspecific alp gene. the other gene encodes intestinal alp (i-alp) and probably glucocorticoid-induced alp (g-alp). 13 in dogs the serum half-lives of placental alp, k-alp, and i-alp are less than 6 minutes. 14 in cats the serum half-life of i-alp is less than 2 minutes. the half-lives of placental alp and k-alp are also assumed to be short in the cat as they have similar structures to i-alp. because of this, only l-alp, b-alp, and, in the dog but not the cat, g-alp, are believed to contribute significantly to serum alp activity. the serum half-life of l-alp is approximately 70 hours in the dog 14, 15 and 6 hours in the cat. 16 l-alp is bound to the membranes of the hepatocytes by glycosylphosphatidylinositol linkages. cleavage of these links by glycosylphosphatidylinositol-phospholipase allows the enzyme to be released into the bloodstream. 17 as bile acids have detergent-like properties; accumulation of bile acids during cholestasis facilitates this process. cholestasis can also result in the induction of synthesis of l-alp (and g-alp in the dog). consequently, serum alp activity is often severely increased in patients with cholestatic disorders. in the dog, alp is considered to be a sensitive marker for cholestasis with a sensitivity of 85%. 18 the short half-life of l-alp in cats means that increases in alp during cholestasis are not as high as in the dog. consequently, alp is a less-sensitive marker of cholestasis in the cat than in the dog, with a reported sensitivity of only 48%. 19 however, the shorter half-life in cats and the absence of g-alp means that any increase in alp activity should be considered clinically important in this species. an increased serum alp activity does not differentiate between intrahepatic or extrahepatic cholestasis. a wide variety of liver diseases can cause intrahepatic cholestasis. this is generally caused by hepatocyte swelling, causing obstruction of the small bile canaliculi. the increase of alp following a hepatic insult is delayed compared to rises in markers of hepatocellular leakage. the reason for this is that it takes time for the enzyme to be synthesized and released into the systemic circulation. alp often remains increased for some time after the resolution of liver injury. b-alp is released into the bloodstream as a result of the activity of osteoblasts. therefore any condition that results in increased bone formation can lead to increased serum alp activity. in animals that are skeletally immature, mild increases in serum alp activity are commonly observed. animals with increased osteoblast activity, such as those with hyperparathyroidism, neoplasia involving bones, and osteomyelitis, may have mild to moderate increases in alp activity. these causes of increased b-alp activity are unlikely to be confused with primary liver disease because the increases are smaller than would be expected with cholestasis, and because bone diseases are often clinically apparent. finally, an increased serum activity of b-alp was reported in a family of asymptomatic huskies. 20 in dogs, but not in cats, g-alp and tissue-nonspecific alp may be induced by corticosteroids. g-alp is believed to be an isoform of i-alp with a prolonged serum half-life that is produced by the liver. 13 posttranslational glycosylation of the g-alp is believed to be responsible the prolonged half-life. induction of g-alp may cause an increase in total serum alp activity after administration of exogenous corticosteroids. synthesis of this isoenzyme can also be induced by the administration of anticonvulsant drugs, such as phenobarbital. similarly, hypercortisolemia frequently causes an increased serum alp activity because of induction of g-alp. however, in dogs with excess serum concentrations of endogenous or exogenous corticosteroids, hepatocyte swelling caused by glycogen accumulation (vacuolar hepatopathy) may lead to intrahepatic cholestasis, another potential contributor to increased serum alp of phenobarbital can cause dramatic increases of serum alt activity and hepatic insufficiency. prednisone and other glucocorticoids can cause an induction of alt (and steroid hepatopathy) and consequently small increases in serum alt activity. serum alt activity can also be increased with any secondary hepatopathy. however, a persistently increased serum alt activity, even with an apparently normal liver function, is an indication for further diagnostic testing. serial evaluation of serum alt activity can be helpful to prognosticate but must be done while considering the patient's clinical signs and other laboratory values. in general, a declining serum alt activity after acute liver injury is considered a good sign. ast is another aminotransferase enzyme that is used as a marker of hepatocellular leakage. ast is found in skeletal muscle, the brain, liver, kidney, cardiac muscle, and to a lesser extent within other tissues. 6 the extrahepatic isoenzymes of ast are relatively more important than they are for alt. muscle disease can cause an increase in serum ast activity. because of this, ast is considered less liver specific than alt. however, by looking at serum ast activity in conjunction with the activities of other hepatic enzymes and muscle enzymes, it is usually possible to differentiate increases caused by muscle damage from increases caused by hepatic damage. again, there is controversy regarding the serum half-life of ast. in dogs one study 1 reported the half-life to be a mean of 263 minutes; another study reported a mean of 22 hours. 2 one study reported the mean half-life to be 78 minutes for cats. 7 unlike alt, a considerable proportion of ast (approximately 30%) is found within hepatocyte mitochondria rather than the cytosol. 8 the cytosolic fraction of ast is released into the serum from hepatocytes when cell membrane permeability is increased, or in case of hepatocellular necrosis. in contrast, the mitochondrial fraction is only released during hepatocellular necrosis. release of ast from hepatocytes into the serum parallels the release of alt. therefore, like serum alt activity, serum ast activity is considered a sensitive marker for hepatocyte injury. it has been suggested that increased ast activity may be more sensitive than increased alt activity for the detection of hepatocellular injury in cats. 9 corticosteroids and phenobarbital may cause mild increases in serum ast activity. because of the considerable overlap in the information provided by the measurement of serum alt and ast activities, measurement of serum ast activity may be redundant. alp is an enzyme bound to the membranes of the hepatocytes that comprise the bile canaliculi and the sinusoidal membranes. it is considered a sensitive marker for cholestasis, especially in the dog, but is not liver specific. cholestasis, canalicular cell necrosis, and increased hepatic synthesis may lead to the release of this enzyme into the circulation. synthesis of this enzyme can be induced by certain drugs, most notably corticosteroids. the possibility that an increase in serum alp activity could be caused by extrahepatic disease, or could be induced by glucocorticoids in the dog, can make the interpretation of this finding challenging. in the dog a wide variety of tissues exhibit alp activity, including intestinal mucosa, kidney (cortex), bone marrow, pancreas, testicle, brain, lung, kidney (medulla), lymph node, liver, skin, spleen, skeletal muscle, and cardiac muscle. 6 there is disagreement in the literature regarding the relative contributions of alp activity from each of these tissues in cats. [10] [11] [12] there are two genes encoding alp in the dog. different forms of alp arising from the same gene are called isoforms. differences among these isoforms arise because of differing posttranslational processing. liver alp (l-alp), bone globulins are produced in the liver, but not exclusively so. the liver produces α-globulins and β-globulins, whereas lymphoid cells produce immunoglobulins (γ-globulins). hepatic insufficiency rarely leads to a decrease in serum globulin concentration. conversely, inflammatory liver disease may be associated with hyperglobulinemia because the nonimmunoglobulin fraction produced by the liver includes several acute-phase proteins (c-reactive protein, haptoglobin, and serum amyloid a). the hepatic synthesis of these proteins is increased during systemic inflammation [26] [27] [28] [29] possibly leading to a rise in the total serum globulin concentration. additionally, immunoglobulin production may be increased in infectious, neoplastic, or autoimmune diseases. coagulation factors (except factor viii), anticoagulation factors (antithrombin and protein c), and the fibrinolytic protein plasminogen, are all synthesized by the liver. the liver is also the site of activation of the vitamin k-dependent clotting factors: ii, vii, ix, x, and protein c. furthermore, as bile acids are needed to emulsify fat and aid in the absorption of vitamin k from the intestine, vitamin k malabsorption may develop secondary to cholestasis. consequently, hepatobiliary disease can affect hemostasis in more than one way. in canine and feline liver disease, coagulation parameter abnormalities have been reported in specific clotting factor activities, prothrombin time, aptt, [30] [31] [32] proteins induced in the absence of vitamin k, 33, 34 fibrin degradation products, fibrinogen, and protein-c activity. 35 these abnormalities of hemostasis are not specific for liver disease but may support its presence. patients with liver disease may develop dic, which can be difficult to distinguish from coagulopathy because of reduced hepatic synthesis of clotting factors alone. although spontaneous bleeding seldom occurs in patients with liver disease, the assessment of the coagulation status of these patients is important, especially when an invasive procedure such as a liver biopsy is being considered. a recent study investigated the diagnostic value of serum protein c as a marker for hepatobiliary disease and portosystemic shunting in dogs. serum protein-c measurement was reported to aid in the differentiation of portal vein hypoplasia without portal hypertension (formerly called microvascular dysplasia) from portosystemic shunt (pss). dogs with portal vein hypoplasia without portal hypertension had a significantly higher serum protein-c concentration than those with portosystemic shunting. 35 urea is produced from ammonia in the liver, released into the systemic circulation, and subsequently excreted by the kidneys. serum urea nitrogen concentration may be close to or below the lower limit of the reference interval in patients with hepatic insufficiency, pss, 36 or urea cycle enzyme deficiencies. however, serum urea nitrogen concentration may also be decreased because of medullary solute washout caused by diuresis, malnutrition, or a protein-restricted diet, and is a normal finding in neonates. in a patient with liver disease, a high fasting serum urea nitrogen concentration relative to the serum creatinine concentration suggests gi hemorrhage. ammonia (nh 3 ) is produced in small intestinal enterocytes from the catabolism of glutamine and in the colon as a consequence of bacterial deamination. ammonia is a highly diffusible gas and passes readily through the bowel wall into the bloodstream. in the blood, at a ph of 7.4, most of the ammonia exists in the form of ammonium ions (nh 4 + ). the ammonium is transported in the blood from the intestines through the hepatic portal circulation to the liver. the extraction of ammonia from the portal circulation is highly efficient. endogenous ammonia is produced from the breakdown of activity. before diagnosing primary liver disease in a dog with an increased serum alp activity, induction of g-alp by endogenous or exogenous steroids should be ruled out. recently a group of scottish terriers were found to have increased serum alp activity with no identifiable underlying cause. 21 it is technically possible to selectively measure the activity of g-alp using techniques such as levamisole inhibition. measurement of g-alp activity was initially investigated as a way to differentiate increases in alp caused by corticosteroids from those caused by cholestasis. unfortunately, measuring g-alp is not clinically useful as g-alp activity may be increased in a variety of conditions, including hepatic disease, diabetes mellitus, hypothyroidism, and pancreatitis. ggt is a glycoprotein enzyme that is bound to the membranes of those hepatocytes that form the bile canaliculi and bile ducts and also periportal hepatocytes. in comparison to alp its distribution includes more distal areas of the biliary tract, but measurement of serum ggt activity is not useful to distinguish between intrahepatic and extrahepatic cholestasis. ggt is also produced by a number of extrahepatic tissues. most of the ggt activity in serum is thought to be a result of the hepatic isoenzyme. colostrum also contains ggt, which is responsible for the mild increases in serum ggt activity that are seen in suckling animals. 22 changes in serum ggt activity generally parallel those in serum alp activity, in that activity is often increased in patients with cholestasis. because ggt is also induced by glucocorticoids, its activity may be increased in patients with hyperadrenocorticism or those exposed to exogenous steroids. in dogs, an increased serum ggt activity is considered to be more specific, but less sensitive than alp activity for the presence of liver disease. 18 in cats, measurement of serum ggt activity is more sensitive but less specific for the detection of liver disease than alp. cats with hepatic lipidosis may be an exception to this as they often have a normal serum ggt activity but an increased serum alp activity. 19 the liver plays a central role in protein metabolism. it is responsible for the synthesis of plasma proteins, deamination of amino acids, conversion of ammonia to urea, amino acid synthesis, and interconversion of amino acids. 23 consequently, in patients with hepatic disease these functions may be compromised. albumin is an important plasma protein that is produced exclusively by the liver. the rate of albumin synthesis must equal the rate of albumin loss to maintain serum albumin concentrations. mild decreases in serum albumin concentration can occur from a variety of conditions. however, the differential diagnoses for severe hypoalbuminemia (<2 g/dl) are limited to hepatic insufficiency, severe exudative skin disease, protein-losing enteropathy, and proteinlosing nephropathy. it is possible to determine the cause of severe hypoalbuminemia from a combination of clinical findings, measurement of the serum globulin concentration, urinalysis (including protein creatinine ratio), tests of gi protein loss, and tests of liver function. as albumin contributes significantly to colloid oncotic pressure, 24 severe hypoalbuminemia can lead to ascites, pleural effusion, and/or subcutaneous edema. the liver has a large reserve capacity for the synthesis of albumin and albumin has a serum halflife of approximately 7 days in dogs. 25 consequently, hypoalbuminemia is a relatively insensitive marker for hepatic insufficiency and is only likely to be seen in patients with advanced chronic liver disease or portosystemic shunts (psss). for hepatobiliary disease in dogs or cats. in patients with severe hepatic insufficiency or pss 49 serum cholesterol, concentration may be decreased as a consequence of impaired hepatic synthesis. hypocholesterolemia might also occur as a result of inadequate dietary intake, maldigestion, malabsorption, or hypoadrenocorticism. the serum cholesterol concentration of patients with hepatobiliary disease may be within the reference interval. patients with cholestatic disease can become hypercholesterolemic. 50 fasting hypercholesterolemia also may be observed in patients with various endocrinopathies, obesity, protein-losing nephropathy, pancreatitis, or primary hyperlipidemias. serum triglyceride concentration may be increased or normal in patients with liver disease. however, an increased fasting serum triglyceride concentration is not a sensitive or specific marker for hepatobiliary disease in dogs or cats. a mild increase in serum triglyceride concentration may develop in patients with cholestasis. there is some evidence that hypertriglyceridemia is associated with gallbladder mucocele formation. 51 hypertriglyceridemia is associated with increased serum hepatic enzyme activities in miniature schnauzers. 52 increased fasting serum triglyceride concentrations are also observed in patients with endocrinopathies, obesity, pancreatitis, and primary hyperlipidemias. the liver plays a central role in carbohydrate metabolism and is responsible glycogen storage, conversion of galactose and fructose into glucose, gluconeogenesis, and the synthesis of many compounds from carbohydrates. 23 blood glucose measurement is not a sensitive or specific marker for liver disease. the liver has a large reserve capacity for glucose production. consequently, hepatic insufficiency must be severe for hypoglycemia to occur. hypoglycemia occurs in a proportion of patients with congenital pss. 53 hepatic neoplasia can also lead to hypoglycemia. this is thought to be caused by release of insulin-like substances. 54 a variety of extrahepatic conditions can also lead to hypoglycemia. bilirubin is a yellow pigment produced from the breakdown of heme-containing compounds. measurement of serum bilirubin concentration can be used to assess liver function. hyperbilirubinemia can be the result of hepatobiliary or extrahepatic disease. icterus is the yellowish pigmentation caused by the retention of bilirubin in the soft tissues. laboratory assessment is the most sensitive way to detect increased serum bilirubin concentrations. hyperbilirubinemia is classified as prehepatic, hepatic, or posthepatic in origin. bilirubin may be artifactually increased by in vitro hemolysis or by the administration of synthetic hemoglobin polymers. when assessing a hyperbilirubinemic patient it is critical to localize the underlying cause. bilirubin is the major product of the degradation of hemecontaining compounds by cells of the mononuclear phagocyte system. bilirubin is released from the mononuclear phagocyte system and is transported in the plasma. the bilirubin is reversibly bound to albumin as it is water insoluble. the unconjugated bilirubin is absorbed through the hepatocyte cell membranes and is bound to glucuronic acid (conjugation). conjugated bilirubin is water soluble and is actively excreted from the hepatocytes into the bile canaliculi, eventually being excreted into the intestines. once in the intestine some of the bilirubin is converted into urobilinogen by bacteria. some of the urobilinogen is reabsorbed from the intestines, but most of this is immediately reexcreted by the liver. when nitrogenous substances in the body, especially glutamine. in the liver the ammonium is converted to urea by the enzymes of the urea cycle, or is used during the conversion of glutamate to glutamine. 37 urea enters the circulation and is excreted by the kidneys. ammonium that is not removed by the liver enters the systemic circulation. the liver has a large reserve capacity for the conversion of ammonia into urea. because of this, plasma ammonia measurement is a relatively insensitive marker for hepatic insufficiency. however, measurement of blood ammonia concentration is a sensitive test for congenital psss and apsc shunts (also known as acquired psss). this is because when portosystemic shunting occurs, the ammonia absorbed from the intestines bypasses the liver and reaches the systemic circulation directly. the sensitivity of plasma ammonia measurement for the detection of pss is reported to be between 81% and 100% in dogs [38] [39] [40] [41] and 83% in cats. 41 the measurement of postprandial venous ammonia is more sensitive than the measurement of fasting ammonia (sensitivities of 91% and 81%, respectively) for the detection of congenital pss. 42 however, the sensitivity for detecting dogs with hepatocellular disease is only 36%. generally, hyperammonemia is considered specific for hepatic insufficiency or pss. however, although they are uncommon, urea-cycle enzyme deficiencies may also cause an increased blood ammonia concentration. these enzyme deficiencies can be hereditary as a result of the absence of a particular enzyme 43 or secondary to cobalamin or arginine deficiency. 44, 45 arginine deficiency is especially relevant in cats with hepatic lipidosis. ammonia is one of the substances that cause hepatic encephalopathy (he). therefore blood ammonia measurement is a useful marker for he. however, other substances can also cause he and the plasma ammonia concentration of a patient with he may be within the reference interval. ammonium ions are labile in plasma, so sample handling is critical when measuring plasma ammonia concentration. samples should be collected, placed immediately on ice, and the plasma separated from the red blood cells as soon as possible. the plasma must be kept cooled and should be analyzed within 30 minutes of collection. these handling requirements have meant that ammonia measurement has been mainly confined to practices with immediate access to a commercial laboratory. measurement of plasma ammonia is available on an in-house dry chemistry analyzer (vettest, idexx laboratories, westbrook, me) although this method was only considered to reliably agree with a reference method for serum ammonia concentrations greater than 150 µm. 46 a recent study found that a point of care blood ammonia analyzer (pocketchem ba, menarini diagnostics, florence, italy) may be suitable for the measurement of blood ammonia concentrations in dogs and cats. 47 ammonia tolerance tests (atts) have been investigated in an attempt to increase the sensitivity of ammonia measurement for detecting hepatic insufficiency and pss. however, the oral administration of ammonium salts can cause vomiting and potentially worsen he signs. ammonium chloride or sulfate can also be given rectally, which is less likely to produce adverse. this method is sensitive for the detection of pss in dogs. 48 the liver plays a central role in lipid metabolism and is responsible for oxidation of fatty acids, synthesis of cholesterol, synthesis of lipoproteins, and synthesis of fatty acids from proteins and carbohydrates. 23 serum cholesterol concentrations may be increased, normal, or decreased in patients with liver disease. increased or decreased fasting serum cholesterol concentrations are not sensitive or specific (fasting or postprandial) suggest hepatic dysfunction, pss, or cholestasis, but they are not specific for any particular liver disease. bile acids are exclusively synthesized in the liver from cholesterol. nearly all of the bile acids that are produced by the hepatocytes are conjugated to an amino acid. in both dogs and cats conjugation is primarily to taurine, but dogs may also convert to a conjugation with glycine. 61 in contrast, even taurine depleted cats conjugate their bile acids almost exclusively to taurine. 62 the conjugated bile acids produced by the liver are called primary bile acids. these are secreted in the bile, and then stored in the gallbladder. cholecystokinin is released from endocrine cells in the small intestine. this hormone stimulates gallbladder contraction and the flow of bile into the duodenum. when the gallbladder contracts the bile acids are released into the intestines. spontaneous gallbladder contraction also occurs during the interdigestive phase. 63 bile acids act as ionic detergents, aiding the emulsification of dietary lipids and their subsequent intestinal absorption in micelles. 64 bile acids are recycled in a process known as enterohepatic circulation ( figure 61-6 ). primary bile acids are lipid insoluble and thus are only absorbed from the intestines when they bind to specific high affinity ileal mucosal receptors. 64 this ileal reabsorption is very efficient. the reabsorbed bile acids enter the portal circulation and upon reaching the liver they are efficiently extracted from the plasma and subsequently reexcreted. the total bile acids pool can be recirculated several times in a day. consequently, the rate of hepatic bile acid synthesis and the fasting serum bile acids concentration in dogs and cats with normal hepatic function is low. because of the increased release of stored bile acids during the postprandial period, small increases in total sba concentration occur in animals with normal hepatic function. primary conjugated bile acids can undergo bacterial deconjugation in the intestinal lumen. the resulting unconjugated bile acids are called secondary bile acids. these are readily absorbed from the colon by passive diffusion. first-pass extraction and reexcretion of secondary bile acids is less efficient than that for primary bile acids. consequently, secondary (unconjugated) bile acids are often present in postprandial serum samples. 65 hepatobiliary disease can cause increased sba concentrations by interfering with hepatocellular function, by causing decreased bile flow (cholestasis), or by altering the hepatoportal blood flow. the main clinical use of sba measurement is to assess hepatic function in patients suspected to have hepatic disease, with serum exposed to air the urobilinogen remaining in the intestines is altered and oxidized into the brown pigment sterocobilin. 23 prehepatic hyperbilirubinemia is caused by increased production of bilirubin as a result of hemolysis. the liver has a large reserve capacity for bilirubin excretion so, for hemolysis to cause hyperbilirubinemia, hepatic bilirubin clearance must be decreased. 55 this occurs if the hemolytic anemia results in hepatocyte dysfunction because of hypoxia. if hepatic hypoxia occurs serum hepatic enzymes activities are often increased. prehepatic hyperbilirubinemia is mainly distinguished from other causes of hyperbilirubinemia by the presence of severe anemia. other supportive evidence includes the presence of a regenerative erythroid response, characteristic changes in red blood cell morphology, and possibly the detection of red blood cell bound antibodies. 56 hepatic hyperbilirubinemia is caused by a decreased rate of hepatocyte bilirubin uptake, conjugation, or excretion (as a result of intrahepatic cholestasis). usually, hepatocyte dysfunction and intrahepatic cholestasis occur concurrently. hepatic enzyme activities (both hepatocellular leakage markers and cholestatic markers) are often increased, although they can also be increased with both prehepatic and posthepatic hyperbilirubinemia. because of the hepatic reserve capacity, hepatic disease must be severe in order to cause hyperbilirubinemia. a range of primary and secondary hepatopathies can cause hepatic hyperbilirubinemia. hepatic hyperbilirubinemia can usually be distinguished from prehepatic hyperbilirubinemia by assessment of the patient's hematocrit, and from posthepatic hyperbilirubinemia by abdominal ultrasound. other markers of hepatic insufficiency, when present, provide additional support for the presence of hepatic hyperbilirubinemia. posthepatic hyperbilirubinemia is a result of extrahepatic bile duct obstruction. this is often caused by pancreatic inflammation or, much less commonly, neoplasia. the main diagnostic tool for documenting extrahepatic bile duct obstruction is abdominal ultrasound. typically, extrahepatic bile duct obstruction leads to dramatic increases in serum cholestatic enzyme activities (compared to hepatocellular leakage enzyme activities) and hypercholesterolemia. when the bile duct is completely obstructed, acholic (pale-colored) feces may be noted. rupture of the biliary tract frequently leads to hyperbilirubinemia as bilirubin accumulates in the abdomen. it is possible to measure the concentration of serum conjugated bilirubin. however, this test is not considered to be clinically useful for distinguishing between prehepatic, hepatic, or posthepatic hyperbilirubinemia, and is thus rarely performed. bilirubin can covalently (nonreversibly) bind to albumin. this biliprotein cannot be cleared by the liver and thus persists in the plasma. biliprotein has a serum half-life comparable to albumin. this is of clinical importance because it means hyperbilirubinemia (and icterus) may persist for several weeks after the resolution of its cause. 57 bile acids (or bile salts when they are deionized) are formed from cholesterol in the liver and are the major constituent of bile. serum bile acids (sbas) measurement is a useful test of liver function in dogs and cats. sbas are either measured as a fasting sample (after withholding food for 12 hours) or by collecting paired fasting and 2-hour postprandial samples. 58 both of these tests are simple to perform and safe. enzymatic measurement of the concentration of total bile acids in serum has become widely available and has replaced other techniques such as radioimmunoassays. 59, 60 once collected, the samples of serum can be stored at room temperature, making it possible to send them to an outside laboratory for evaluation. lipemia and hemolysis of the blood samples should be avoided as both can interfere with the assay. increased sba concentrations recent study did not support the use of a 13 c-labeled galactose breath test for assessment of liver function in dogs. 80 the metabolism of endogenous substances has been investigated to find possible markers of hepatic cellular metabolism. dogs with hepatic disease (hepatitis and neoplasia) had significantly higher serum l-phenylalanine concentrations than did healthy dogs and those with nonhepatic diseases. 81 further investigations are needed to determine the utility of these tests for assessing liver function in veterinary patients. urine specific gravity can be decreased in patients with hepatic insufficiency or pss. this can be caused by an inability to fully concentrate urine, resulting in pu, or from primary pd. bilirubin is commonly measured semiquantitatively in canine and feline urine using urine dipsticks. bilirubinuria (<2+ on a dipstick) can be a normal finding in dogs (especially males). 82 bilirubinuria in dogs without hemolytic or hepatobiliary disease can occur as a consequence of the loss of unconjugated bilirubin that is bound to albumin in proteinuric patients and renal filtration of small amounts of conjugated bilirubin that has leaked from the liver. additionally, the renal tubular cells of male dogs have the enzymes needed to produce and conjugate bilirubin. as cats have a higher renal threshold for bilirubin than dogs, bilirubinuria should always be considered abnormal in cats. bilirubinuria in cats and excessive bilirubinuria in dogs implies hemolytic or hepatobiliary disease. because dogs have a relatively low renal threshold for bilirubin, bilirubinuria is often detected before bilirubinemia or jaundice. ammonium biurate crystals are detected in the urine sediment by light microscopy. uric acid is a product of purine catabolism and is converted to allantoic acid by hepatic urate oxidase. in cases with severe hepatic insufficiency or pss, the serum uric acid concentration may be higher than the renal threshold. this combined with hyperammonemia may lead to ammonium biurate precipitation in the urine. urate urolithiasis seems to be more common in patients with pss than those with other types of hepatic dysfunction. between 40% and 70% of dogs with pss were found to have urate crystalluria. 83 however, it should be noted that urate crystalluria is not specific for hepatobiliary disease. the erythrocyte series may be affected by hepatobiliary disease, resulting in erythrocyte dysmorphias and anemia. these abnormalities are suggestive of, but are not specific for, hepatobiliary disease. patients with hepatobiliary disease can be anemic as a result of blood loss, in which case signs of a regenerative response are normally present within 3 days of hemorrhage. acute, severe hemorrhage may occur in patients with hepatobiliary disease following invasive procedures such as liver biopsy or as a consequence of hemorrhage from a hepatic neoplasm or hepatic rupture. less-severe anemia may occur as a result of gi bleeding. 84 chronic gi blood loss may eventually lead to iron-deficiency anemia. this is characterized by microcytic hypochromic erythrocytes and a variable regenerative response. additionally, hepatobiliary disease may lead to anemia of chronic disease, which is typically nonregenerative with normocytic normochromic erythrocytes. red blood cell morphologic changes are sometimes observed in dogs with hepatobiliary disease. poikilocytosis, characterized by the presence of acanthocytes and target cells, may be seen in patients with chronic hepatic disease. this is thought to be a result of altered phospholipid metabolism. patients with pss can have microcytic red blood cells. this is more common in dogs than in bilirubin concentrations that are within the reference interval. measurement of postprandial sba concentration does not seem to have an advantage over fasting sba concentrations or vice versa. sensitivity can be increased by collecting paired preprandial and twohour postprandial samples. 66 numerous studies show that sba measurement is a useful test for diagnosing hepatobiliary disease, including pss in dogs and cats. 41, [66] [67] [68] [69] [70] a recent study found the sensitivity of fasting sba measurement for diagnosing pss (using a cutoff value of 20 µmol/l) to be 93% for dogs and 100% for cats. the reported specificities were 67% for dogs and 71% for cats. 41 however, the sensitivity of sba measurement for detecting hepatic insufficiency is lower than that for detecting pss. measurements of sba concentrations have several limitations. first, this test does not allow differentiation between various types of hepatobiliary disease. also, measurement of serum bile acids in a patient with proven cholestasis is of no clinical benefit. additionally, there is limited utility in measuring sbas concentrations in patients with hyperbilirubinemia, although potentially sba measurement could be useful in distinguishing prehepatic from hepatic or posthepatic causes of hyperbilirubinemia. with prehepatic causes of hyperbilirubinemia, the bile acid concentrations should be within the reference interval. however, in most cases prehepatic hyperbilirubinemia is easily distinguished by the presence of severe anemia. it should also be noted that the magnitude of increases of sba concentration are not correlated with prognosis or disease severity. it is important to note that fasting sba concentrations may be higher than the upper limit of the reference interval or higher than the postprandial value because of spontaneous contraction of the gallbladder in the fasting state, or because of delayed gastric emptying. this could result in an increased fasting sba concentration in the absence of hepatobiliary disease. increased fasting and postprandial serum bile acids concentrations can be the result of increased bacterial deconjugation of primary bile acids into secondary bile acids. 71 false-negative results may occur if enterohepatic circulation of bile acids does not occur from a lack of gallbladder contraction. this could be a problem if a patient is anorectic, does not eat enough food, consumes a diet with insufficient protein or fat, vomits the test meal, or has delayed gastric emptying. ceruletide is an injectable cholecystokinin analogue that has been used to stimulate gallbladder contraction when using sba measurement to diagnose hepatobiliary disease. 72, 73 this test circumvents many of the factors that influence postprandial sba concentrations. urine bile acids measurement has been described in dogs and cats. the diagnostic performance was similar to that of sba measurement in both species. this test does not offer any advantages over sba measurement. [74] [75] [76] [77] excretion of exogenous tracers, such as the anionic cholephilic dyes, bromsulphalein, and indocyanine green have been used historically to assess hepatic function in veterinary patients. however, these tests are considered unreliable and have been replaced by the measurement of sba concentrations. the metabolism of exogenous substances can be used to assess liver function. a variety of substances have been investigated as markers for hepatic metabolism in human medicine. assessment of the metabolism of aminopyrine has been investigated in dogs and to a lesser extent in cats. the 13 c-labeled aminopyrine demethylation blood test involves intravenous administration of 13 c-labeled aminopyrine to the subject. the aminopyrine is metabolized by the liver, resulting in the production of 13 co 2 . this is measured in the blood by fractional mass spectroscopy. 78, 79 further investigation of the utility of this test for assessment of liver function is needed. a in dogs with liver disease than in dogs with extrahepatic disease, and were higher in dogs with cirrhotic liver disease than in dogs with noncirrhotic liver disease. blood ha concentration may prove be a useful marker for hepatic fibrosis in dogs but further studies are necessary to evaluate its clinical utility. histopathologic analysis of liver biopsies or identification of a shunting blood vessel is often required to definitively diagnose hepatic disease. however, the pattern of laboratory test abnormalities, particularly when interpreted in conjunction with the patient's clinical presentation, and the results of diagnostic imaging, can increase or decrease a clinician's index of suspicion for specific liver diseases (table 61-3) . it is important to note that there is considerable overlap between the patterns for different diseases. to avoid misinterpretation and misdiagnosis when evaluating a patient for liver disease, it is essential to consider the limitations of the laboratory tests discussed above. diagnostic imaging is an important part of the investigation of hepatobiliary disease in dogs and cats. diagnostic imaging may help to determine whether or not hepatobiliary disease is present, identify the cause of a secondary hepatopathy, aid in the diagnosis of specific hepatobiliary diseases, and provide prognostic information. however, with the exception of diagnosis of a pss, imaging seldom yields a definitive diagnosis. radiography and abdominal ultrasound are the most frequently used imaging modalities for assessment of the hepatobiliary system in dogs and cats, but alternative imaging techniques are now being used more frequently. abdominal radiographs allow assessment of hepatic size, shape, opacity, and location in most patients. 91 radiography may also allow identification of extrahepatic abnormalities that affect the liver. however, radiographs provide limited information about the hepatic parenchyma. it is important to note that patients with hepatobiliary disease often have normal abdominal radiographs. radiography allows subjective assessment of liver size. cranial displacement of the gastric axis may be observed on lateral abdominal radiographs when microhepatia is present. however, subtle microhepatia is unlikely to be appreciated radiographically. psss and hepatic cirrhosis are the most common conditions causing microhepatia. mild bilateral renomegaly may also be cats. 83 microcytosis also occasionally occurs in patients with hepatocellular disease. altered iron metabolism is thought to lead to a delay in red blood cell precursors gaining a sufficient amount of hemoglobin to be released into the circulation. this delay leads to the precursors undergoing an extra cell division in the bone marrow, resulting in microcytosis. 85 microangiopathy can occur as a result of hepatic neoplasia or dic, and may lead to the formation of schistocytes. the leukocyte series may be affected by hepatobiliary disease in a variety of ways. the resultant abnormalities are inconsistent and are not specific for hepatobiliary disease. leukocytosis, leukopenia, and sometimes an inflammatory leukogram may be present when infectious and, less commonly, inflammatory or neoplastic processes affect the hepatobiliary system. a leukocytosis was found to be present in 44% of dogs with chronic hepatitis. 86 the thrombocyte series is occasionally affected by hepatobiliary disease, but changes are both inconsistent and nonspecific. mild to moderate thrombocytopenia may occur in patients with severe liver disease. 86 this may be the result of a decreased production of thrombopoietin by the liver. disseminated intravascular coagulopathy associated with liver disease also may lead to thrombocytopenia. additionally, infectious diseases affecting the liver, such as leptospirosis may result in thrombocytopenia. 87 genetic testing for copper hepatotoxicosis has been developed in bedlington terriers. affected bedlington terriers have an autosomal recessive defect of their commd1 gene. dogs with a homozygous affected genotype develop copper hepatopathy as a result of impaired biliary excretion of copper. initially, a microsatellite marker that is in linkage disequilibrium with the mutation was discovered and used to identify affected dogs and select dogs homozygous unaffected dogs for breeding. 88 subsequently, a mutation of the commd1 gene (a deletion of exon 2) was identified as the cause of the condition in the majority of bedlington terriers. 89 a genetic test for this disease has become commercially available (vetgen, ann arbor, mi). this test is run alongside the linked marker as a small proportion of bedlington terriers do not have the deletion but are nevertheless affected by the disease. these dogs are likely to have a rare second mutation of their commd1 gene which the linkage markers may track. hyaluronic acid is a major constituent of the ecm and hyaluronic acid (ha) concentration in blood has been used as a marker for hepatic fibrosis in humans. a recent study investigated the use of ha concentration as a marker for hepatic disease in dogs. 90 this study found that blood ha concentrations were significantly higher the size of the liver can be subjectively assessed by abdominal ultrasonography. the findings of a small liver and cranial displacement of the stomach suggest microhepatia. hepatomegaly is another subjective finding and can be generalized or focal. the finding of rounded liver lobe margins suggests hepatomegaly. hepatic parenchymal changes can be classified as being diffuse, multifocal, or focal. a wide variety of disease processes can cause diffuse changes to the hepatic parenchyma. these changes can be isoechoic, hypoechoic, hyperechoic, or of mixed echogenicity. in some cases the architecture of the liver will not be altered, but in other cases changes will occur. examples of diseases in which the echogenicity of the hepatic parenchyma is diffusely changed but no changes in architecture occur include cholangitis, neoplasia, hepatic lipidosis, other vacuolar hepatopathies, toxic hepatopathy, and early micronodular hyperplasia with various degrees of fibrosis. 100 hyperechogenicity of the liver compared to the falciform fat, poor visualization of the intrahepatic blood vessels, and increased attenuation of the ultrasound beam have been used as criteria for the sonographic diagnosis of feline hepatic lipidosis. 101 diseases where the hepatic architecture is altered are easier to detect sonographically. these include neoplasia, micronodular hyperplasia, and chronic hepatitis with fibrosis. cystic structures, abscesses, hematomas, and granulomas are examples of focal parenchymal liver disease. these lesions are usually easily detected sonographically. sonography seldom allows a definitive diagnosis of hepatic parenchymal disease to be made. in one study the overall accuracy of ultrasound for discrimination among different categories of diffuse liver disease was 36.5% for dogs and 54.6% for cats. hepatic lipidosis in cats could be diagnosed slightly more accurately than other diffuse hepatic diseases. 102 cytologic or histologic evaluation of a hepatic tissue sample is usually needed to make a definitive diagnosis. hepatic neoplasia, whether primary or metastatic, can be diffuse, multifocal, or focal in its distribution. round cell tumors are the most likely tumor type to diffusely infiltrate the liver. these tumors can cause hypoechoic, hyperechoic, or mixed-echoic changes, or appreciated radiographically for patients with pss. urate uroliths can be radiolucent so they might not be visible on plain abdominal radiographs. hepatomegaly can be generalized or focal. generalized hepatomegaly can be caused by a number of conditions including neoplasia, vacuolar hepatopathies, congestion, or amyloidosis. focal hepatomegaly can be caused by neoplasia, abscesses, granulomas, or a liver lobe torsion. radiographic signs associated with hepatomegaly are rounded hepatic borders, caudal displacement of the gastric axis, and extension of the hepatic silhouette beyond the costal arch. radiography does not allow appreciation of mild hepatomegaly. additionally, it can be normal for the hepatic silhouette to extend beyond the costal arch in brachycephalic breeds, chondrodystrophic breeds, neonatal animals, or geriatric animals. 91 the liver is normally appreciated as an area of homogenous softtissue opacity on radiographs. radiolucent areas within the liver indicate accumulation of gas within the hepatic parenchyma, biliary tract, or portal vasculature. gas in the parenchyma of the liver can be associated with an hepatic abscess. 92 gas in or around the gallbladder has been reported in dogs with emphysematous cholecystitis. 93 although uncommon in dogs and cats, if choleliths or choledocholiths contain enough calcium, they may be appreciated as mineral opacities within the hepatic silhouette. 94 mineralization of the gallbladder wall can be associated with a biliary adenocarcinoma in the dog. 95 parenchymal mineralization can be associated with granulomas, abscesses, 96 hematomas, neoplasia, or hepatic necrosis. 91 angiography allows the visualization of the hepatoportal vasculature, including abnormal vessels. this often provides a definitive diagnosis of pss and is indicated in patients that are suspected of having a pss, where the shunt cannot be adequately evaluated by abdominal ultrasound. anatomical characterization of congenital pss is important when planning attenuation, and angiographic procedures often allow this. there are several techniques for mesenteric portography. operative mesenteric portography is commonly performed immediately prior to surgical attenuation of a congenital pss and involves catheterization of a mesenteric vein and injection of a contrast agent. operative contrast portography allows evaluation of the portal vasculature before and after shunt attenuation. this technique has the disadvantage of being relatively invasive. cranial mesenteric portography can be accomplished less invasively by using ultrasound guidance to percutaneously catheterize the splenic vein. 97 however, this can be technically demanding and may not be possible in smaller patients. transvenous retrograde portography has been described and involves the catheterization of the jugular vein. 98 this technique allows selective catheterization of the shunting vessel and measurement of portal pressures (figure 61-7) . transvenous retrograde portography has been applied during percutaneous transjugular coil embolization of intrahepatic shunts. 99 percutaneous splenoportography involves percutaneous injection of contrast media into the spleen. this technique is simple to perform, but there is a risk of complications such as splenic infarction or hemorrhage. abdominal ultrasonography is the most commonly used imaging modality for evaluating small animal patients with suspected hepatobiliary disease. ultrasonography allows assessment of the hepatic parenchyma and biliary tract. evidence of extrahepatic disease causing a secondary hepatopathy may also be detected. additionally, ultrasound guidance is often used when collecting samples for cytologic and histologic evaluation of the liver. gallbladder mucoceles can also lead to biliary obstruction, which might also be appreciated sonographically. the sensitivity of ultrasound for the detection of a gallbladder wall rupture in dogs with gallbladder mucoceles is reported to be 85.7%. 109 abdominal ultrasound can be used to assess the liver for vascular disease. congenital pss is classified as being intrahepatic or extrahepatic. although angiographic techniques are considered to be the gold standard for the detection and characterization of pss, abdominal ultrasound is being used increasingly for this purpose. sonographic assessment of the portal vasculature is time-consuming and highly operator dependent. because of this there should be a high index of suspicion for pss before performing these studies. secondary findings consistent with pss include mild bilateral renomegaly, urolithiasis (because of urate crystalluria), and microhepatia. ascites and hepatic parenchymal changes are not consistent with congenital pss. extrahepatic shunts typically occur in small-breed dogs and arise from the splenic vein or the right gastric vein while intrahepatic shunts typically occur in larger breeds of dogs and arise from the right or left portal branch. an intrahepatic pss is usually easier to detect sonographically than an extrahepatic pss in dogs. cats typically have single extrahepatic shunts with a wider degree of anatomical variation than in the dog. ultrasonography has been reported to have a sensitivity of 92% and a specificity of 98% for detecting pss in dogs. 110 portal hypertension can develop as a result of chronic hepatitis with fibrosis, hepatic arterioportal fistulas, portal vein thrombosis, primary portal vein hypoplasia, extraluminal compression of the portal vein, or after ligation of a congenital pss. the finding of hepatofugal or reduced velocity hepatopetal blood flow using doppler ultrasound is consistent with portal hypertension. 111 however, not all patients with portal hypertension will have these changes. ascites frequently, but not always, develops secondary to portal hypertension and this can be readily detected on abdominal ultrasound examination. acquired portosystemic collaterals (also known as acquired pss) may develop when sustained prehepatic or hepatic portal hypertension is present. 112 sonography may allow detection of portal hypertension and apscapsc although apsc vessels are more difficult to identify than congenital pss. posthepatic portal hypertension does not result in the development of may not affect the echogenicity of the liver at all. neoplasia can also lead to the appearance of nodules within the hepatic parenchyma. malignant liver nodules have a variable appearance and size and can be difficult to distinguish from nonmalignant conditions such as cysts, hematomas, benign hyperplastic nodules, granulomas, or abscesses. the finding of one or more target lesions in the liver or spleen had a positive predictive value of 74% for detecting malignancy, and thus should not be considered a specific finding. 103 cytologic or histologic evaluation of a tissue sample is needed to differentiate between malignant and benign liver nodules. tumors, such as hepatoma or hepatocellular carcinoma, can also focally infiltrate the liver. contrast-enhanced harmonic ultrasound allows assessment of tissue perfusion patterns. gas-filled microbubbles are administered intravenously to the patient. the microbubbles are relatively echogenic. when they reach the tissue of interest, they produce a more potent harmonic signal than the surrounding tissue. this technique allows enhanced differentiation between tissues with varying perfusion patterns. in one study the sensitivity of contrast enhanced ultrasound for differentiation between benign and malignant liver nodules in dogs was reported to be 100% and the specificity was reported to be 94.1%. 104 sonography is also a valuable tool for the evaluation of the biliary system. biliary disease can be classified as being obstructive or nonobstructive. the term cholangitis refers to a group of nonobstructive biliary diseases, which are more common in cats than in dogs. typical ultrasound findings in cats include a hypoechoic hepatic parenchyma and prominent portal vasculature. 105 additional findings can include evidence of pancreatic inflammation, thickening of the gallbladder wall, and dilation of the intrahepatic and extrahepatic biliary system. it is important to note these changes are not always present. cytologic or histologic confirmation and bacterial culture are needed to confirm this diagnosis. generalized gallbladder wall thickening can occur as a result of cholecystitis, cholangitis, or hepatitis. however, the gallbladder wall can also appear to be thickened when peritoneal effusion or hypoproteinemia are present. gall bladder wall masses can be identified sonographically as a focal thickening of the gallbladder wall. sonography has also been used to assess gallbladder motility in dogs. 106 it should be noted that gravity dependent gallbladder sludge can be found in dogs without hepatobiliary disease, so this finding should be considered incidental. 107 abdominal ultrasound is the most commonly used imaging modality for the detection of biliary obstruction in dogs and cats. findings consistent with biliary obstruction include common bile duct distention, intra-and extrahepatic bile duct distention, and/or gallbladder dilation. a retrospective study showed that common bile duct dilation greater than 4 mm was 97% sensitive for the detection of biliary obstruction in cats. 108 sonography can also aid in identifying the cause of biliary obstruction. biliary obstruction can be classified as being luminal or extraluminal. extraluminal causes include nonneoplastic pancreatic disease, abdominal adhesions, and, rarely, pancreatic neoplasia. luminal causes include gallbladder mucocele, biliary neoplasia, inflammation, and cholecystolithiasis. biliary tract obstruction can progress to biliary rupture and bile peritonitis. sonographic signs of biliary rupture include loss of gallbladder wall continuity, free peritoneal fluid, and signs of localized peritonitis. gallbladder mucoceles occur in the dog, but have not been described in cats. mucoceles have a variable sonographic appearance; typical findings include a stellate or finely striated bile pattern with a hypoechoic rim, which is not gravity dependent, and gb lt specificity of 100%. 119 the disadvantages of ct and mri include their limited availability, cost, and the need for anesthesia. although cytologic evaluation of the liver provides a definitive diagnosis, often histologic examination is also required. there are a variety of techniques to collect cytologic samples of the hepatobiliary system. abdominal effusion, when present, can be collected percutaneously. fine-needle aspirates (fna) of the liver can be collected percutaneously under ultrasound guidance. cholecystocentesis can also be performed percutaneously with ultrasound apsc vessels, but can lead to a distention of hepatic veins and ascites. nuclear scintigraphy involves administering a radioactive tracer substance (radiopharmaceutical) to the patient, which localizes to a specific organ or tissue. the radioactive decay of this substance is detected by a gamma camera and used to form images. scintigraphy has been used to detect pss and to assess gallbladder emptying in small animals. however, specialized equipment and a license for the use of radioisotopes are required. consequently, availability of this imaging modality is currently limited to academic institutions and specialty referral hospitals. technetium-99m pertechnetate is the most commonly used radiopharmaceutical for assessing the portal circulation of small animal patients. two techniques have been described: per-rectal portal scintigraphy and transsplenic portal scintigraphy. by analyzing the radiation emitted from regions of interest drawn over the patient's liver and heart, pss can be detected and a shunt fraction can be calculated. this allows for the minimally invasive diagnosis of pss, differentiation of pss from portal vein hypoplasia without portal hypotension (previously known as microvascular dysplasia), and comparison of the degree of shunting before and after shunt attenuation. transsplenic portal scintigraphy is preferred over perrectal portal scintigraphy as it is simpler to perform, uses lower doses of the radiopharmaceutical, and is more sensitive (figure 61-9 ). transsplenic portal scintigraphy is 100% sensitive and specific for the diagnosis of congenital pss, and significantly more likely than per-rectal portal scintigraphy to detect shunt number and termination in dogs. 113 nuclear scintigraphy has been used to quantify liver function and to assess biliary tract patency in dogs. in a retrospective study hepatobiliary scintigraphy was found to be 83% sensitive and 94% specific for the detection of extrahepatic biliary obstruction in dogs and cats. 114 computed tomography (ct) (figure 61 -10) and magnetic resonance imaging (mri) have been used to detect hepatic parenchymal neoplasia in humans. compared to abdominal ultrasound these techniques have an improved accuracy for the diagnosis of hepatic neoplasia in humans. however, there is limited data in the veterinary literature evaluating their diagnostic performance. in one study the diagnostic accuracy of ct for detecting hepatic masses was not found to be significantly different from that of abdominal ultrasound in dogs. 115 in another study mri was found to have a sensitivity of 100% and a specificity of 86% for the differentiation between benign and malignant liver lesions in dogs. 116 ct angiography is being used increasingly in dogs for the diagnosis of congenital pss and other hepatic vascular diseases. it offers the advantage of being less invasive than operative angiography, allows for improved assessment of the portal vasculature, and allows the creation of a three-dimensional reconstruction. the vasculature detail afforded by ct angiography is particularly useful when planning attenuation of a congenital pss. the diagnostic utility of ct angiography for detecting and characterizing pss in dogs was shown to compare favorably to that of other techniques, including surgical exploration. 117 transsplenic ct portography has been described in dogs without pss. this technique offers more intense enhancement of the splenic and portal veins than ct angiography. 118 mri angiography diagnoses pss in dogs with a sensitivity of 80% and a the findings above may aid in making a diagnosis of liver disease but are not a substitute for histopathologic analysis, as cytologic specimens do not allow assessment of the hepatic architecture. furthermore, only a tiny proportion of the liver is sampled when cytologic samples are examined. these limitations are reflected by the results of a retrospective study that found the overall agreement between the histopathologic and cytologic diagnosis of liver disease to be 30.3% for dogs and 51.2% for cats. 123 cytologic evaluation of bile can also be useful for the diagnosis of biliary disorders, particularly in cats. world small animal veterinary association (wsava) standards for the clinical and histological diagnosis of canine and feline liver disease suggest that the cytologic evaluation of bile forms part of the minimum diagnostic requirement for cats with extrahepatic cholestasis and for dogs guidance. these techniques are minimally invasive and the risk of complications is relatively low but caution should be exercised in patients with bleeding disorders. liver disease can cause abdominal effusion by several mechanisms. in cases with hepatic insufficiency, severe hypoalbuminemia (<1.5 g/dl) can occur. this can lead to the formation of a pure transudate as the result of a reduced plasma colloid oncotic pressure. increased capillary hydrostatic pressure because of portal hypertension may lead to formation of a pure or modified transudate. hepatic neoplasia can also lead to a formation of a modified transudate. biliary tract rupture can lead to bile peritonitis and abdominal effusion. an exudate with a bilirubin concentration greater than twice that of the plasma is suggestive of bile peritonitis. 120 cytologic evaluation of hepatic fna can aid in making a diagnosis of liver disease. suppurative, mixed inflammatory, lymphocytic and, more rarely, eosinophilic patterns of inflammation can be appreciated cytologically. each pattern of inflammation suggests a group of possible diagnoses. the finding of dark green or black bile casts suggests cholestasis. infectious diseases such as histoplasmosis can be definitively diagnosed based on the cytologic finding of the infectious agent ( figure 61-11 ). hepatocellular vacuolation can be classified as being caused by lipid or not. lipid vacuolation of hepatocytes is characterized by colorless cytoplasmic vacuoles. severe lipid vacuolation is suggestive of hepatic lipidosis in cats ( figure 61 -12). however, feline hepatic lipidosis often occurs secondary to another disease process. a group of cats with cytologic findings suggestive of hepatic lipidosis were reported to have underlying infiltrative liver disease. 121 nonlipid vacuolation is characterized by generalized hepatocyte swelling and lacy vacuolation ( figure 61-13 ). vacuolar hepatopathy occurs secondary to a wide variety of extrahepatic disease processes in dogs. 122 metastatic tumors and round cell tumors, such as lymphoma ( figure 61-14) , affecting the liver can often be diagnosed cytologically. additionally, cytologic evaluation can aid in distinguishing liver nodules because of extramedullary hematopoiesis from those caused by neoplasia. however, it is not possible to distinguish hepatic nodular hyperplasia from hepatic adenoma or well-differentiated carcinoma cytologically. some cases of hepatocellular carcinoma can be diagnosed cytologically if criteria for malignancy are present. and cats suspected to have cholangitis. 124 the finding of neutrophils and bacteria on bile cytology supports a diagnosis of feline neutrophilic cholangitis. cytology is essential for the diagnosis of this disease, as cats with neutrophilic cholangitis may not have typical hepatic histopathologic changes and it can be difficult to distinguish these cats from those with lymphocytic cholangitis. bile should also be submitted for aerobic and anaerobic bacteriologic culture. histopathologic evaluation is required to make a definitive diagnosis of most liver diseases. histopathologic evaluation of the liver allows a morphologic and sometimes an etiologic diagnosis to be made (see chapter 29) . in addition to routine staining with hematoxylin and eosin, a variety of other staining techniques can be employed to demonstrate hepatic pathology. to optimize the value of histopathologic evaluation of the liver, particular attention should be paid to specimen collection, specimen handling, and communication between the clinician and the pathologist. although liver biopsy is considered to be relatively safe, the patient should be assessed for bleeding disorders before this procedure. this assessment should include a platelet count, coagulation times, and a buccal mucosal bleeding time. liver biopsies can be collected in a number of ways. each method has advantages and disadvantages, and there is controversy in the veterinary literature as to which technique is optimal. laparotomy allows collection of relatively large wedge biopsies, with direct visualization. this technique does not require specialized equipment or training, and excessive bleeding can be readily identified. however, laparotomy requires general anesthesia and is the most invasive biopsy technique. percutaneous needle biopsy techniques have been described. these techniques may be possible under heavy sedation and are the leastinvasive method for collecting liver biopsies. ultrasound guidance is often used, allowing biopsy of focal lesions. it is also possible to biopsy tissue that is deeper within the hepatic parenchyma than is possible with other techniques. however, the specimens that are collected are relatively small and may be inadequate for accurate assessment in some patients. a prospective study showed that there was agreement between the histomorphologic diagnoses made upon examination of needle biopsies and those made on wedge biopsies collected during laparotomy or necropsy for only 48% of dogs and cats. 125 excessive hemorrhage after biopsy may not be identified immediately. laparoscopy allows collection of biopsies using forceps with laparoscopic guidance. this technique requires general anesthesia, but is less invasive than laparotomy. the biopsies collected are larger than needle biopsies and excessive bleeding can be visualized. however, laparoscopy requires specialized equipment and training. the use of biopsy forceps may result in crushing artifact and the tissue collected may be too superficial to identify lesions that lie deeper within the hepatic parenchyma. 126 regardless of the technique used, a tiny proportion of the organ is sampled and, because liver disease can affect the hepatic parenchyma in a heterogeneous manner, sampling error is possible. to reduce the effect of sampling error, several biopsies from different areas of the liver should be collected and focal lesions should be specifically biopsied. the clinician should provide the pathologist with all the pertinent information from the patient's history, physical examination findings, the results of laboratory testing, and the findings from diagnostic imaging. in turn the histomorphologic diagnosis that the pathologist makes should be interpreted by the clinician along with the other clinical data. when the histopathologic diagnosis does not fit the clinical picture, the pathologist should be consulted and when necessary a second opinion should be requested. variation in the assessment of hepatic pathology between pathologists was highlighted by a study that found agreement between examiners for only 44% of needle biopsies and 65% of wedge biopsies examined. 125 hopefully, the adoption of wsava standards for the clinical and histological diagnosis of canine and feline liver diseases since the aforementioned study will reduce this interobserver variation. quantification of hepatic metal concentrations requires submission of tissue for flame atomic absorption spectroscopy. although zinc has a role as an antioxidant, hepatic copper and iron retention can lead to oxidative liver injury. copper is the most frequently course of the evaluation. in patients that are asymptomatic and have abnormal biochemical testing, repeat evaluation is sometimes warranted. a general guideline is to obtain a liver biopsy in asymptomatic patients if there are moderate to severe elevations in serum hepatic enzyme activities that persist for at least 3 months, or if there are mild to moderate elevations in serum hepatic enzyme activities that persist for at least 6 months. if clinical signs of hepatic disease develop, then biopsy should not be unreasonably delayed. if there are concurrent elevations in serum bile acids, biopsy should also not be delayed. other indications for hepatic biopsy are ultrasound imaging abnormalities. if there are focal hepatic masses or diffuse echotextural changes, a biopsy may be warranted, depending on results of laboratory testing. in one study, abdominal ultrasound findings alone were not reliable for obtaining a diagnosis of infiltrative hepatic disease with diffuse changes in echogenicity (either hypoechoic or hyperechoic, uniform or mottled). 1 in another study, sonographic detection of a hepatic mass greater than or equal to 3 cm, ascites, abnormal hepatic lymph node(s), and abnormal spleen were predictive of liver neoplasia based on cytology. 2 conversely, sonographic detection of hepatic nodules less than 3 cm was predictive of vacuolar hepatopathy on cytology. thus several sonographic findings, alone or combined, may be predictive of liver ultrasound-guided fine-needle aspiration cytology results. in light of the fact that ultrasound-guided fine-needle aspiration cytology of the liver has limitations , the results of ultrasound and cytology should be adjuncts to other findings. another indication for hepatic biopsy is the need to assess response to therapy. in cases of chronic hepatitis in dogs, it is often difficult to determine if there is ongoing inflammation and resolution/ progression of fibrosis during long-term therapy. this is particularly true when the patient is receiving glucocorticoid therapy as these medications cause variable increases in serum alp and transaminase activities independent of the underlying disease. repeat or serial hepatic biopsy analysis is often helpful to guide therapeutic decisions in these cases. among the most serious complications of liver biopsies are hemorrhage, infections, and injury to the adjacent viscera. consequently the clinician must take into account the clinical question, the appropriate invasive biopsy method, and methods of managing postbiopsy complications. postbiopsy hemorrhage is often the first concern, although it is unclear as to what the best predictor of hemorrhage is in patients about to undergo hepatic sampling. in one study of 200 human patients in which bleeding was evaluated laparoscopically, there was no correlation between any in vitro coagulation test and "liver bleeding time." 3 other studies in man have used laparoscopy and ultrasonography to assess hepatic bleeding time following needle biopsy, and most have shown similar poor correlation between coagulopathies and hepatic bleeding times. similar studies have not been reported in veterinary medicine. there also have been studies in human and veterinary medicine evaluating risk factors for bleeding complications (as opposed to "hepatic bleeding times"). 4,5 bigge et al. correlated coagulation profile findings and bleeding complications after ultrasound-guided biopsies in 310 dogs and 124 cats. 5 there was no apparent correlation between coagulation parameters and major complications following liver biopsy. studies show that clotting times assessing proteins induced by vitamin k antagonism are more sensitive in detecting coagulopathies in patients with hepatic disease. 6, 7 the measured of these metals and quantification is essential for the diagnosis of hepatic copper retention. these measurements are usually performed on freeze-dried pieces of liver. specimens for metal measurement should not be stored in saline and should be kept in metal-free containers. recently, it was shown that measurements of the concentration of copper and iron, but not zinc, can be ascertained from deparaffinized-archived liver tissue. 127 hepatobiliary diseases can be challenging to diagnose. although diagnostic tests that employ biochemical, molecular biologic, serologic, functional, as well as imaging techniques are capable of establishing the etiology of some chronic or acute liver diseases, in most instances the gold standard for definitive diagnosis and the assessment of stage and severity of liver diseases is the histologic evaluation of a liver sample. recent advances in imaging technology, the use of multiple imaging modalities, and newer biopsy methods have resulted in improvement in the ability to safely procure hepatic tissue for evaluation. there are several means of obtaining hepatic samples including fine-needle aspiration, ultrasound-guided biopsy, laparoscopy, and laparotomy. all techniques have both advantages and disadvantages, which should be carefully considered before choosing the appropriate sampling method. many biochemical tests are available to evaluate the anabolic and/ or catabolic function of the liver and the hepatic circulation. these include measurement of concentrations of bile acids, ammonia, bilirubin, and the ability to excrete organic dyes. other tests of hepatic function include measurement of serum albumin, glucose, urea nitrogen, and clotting factor analysis. hepatic function can be markedly abnormal despite maintenance of the hepatocellular membrane and therefore normal serum activities of hepatic enzymes. examples include psss, terminal cirrhosis, and metastatic hepatic neoplasia. likewise, the liver can continue normal anabolic or catabolic function despite severe hepatocyte leakage of intracellular enzymes because of its marked reserve capacity. this can occur, for example, in certain cases of hepatocellular necrosis, blunt abdominal trauma, or primary hepatic neoplasia. thus, the limitations of serum hepatic enzyme activities must be taken into consideration. hepatocellular leakage enzyme activities include alt and ast. enzyme activities that increase with biliary tract obstruction include serum alp and ggt. no laboratory test identifies a specific problem, helps determine specific therapeutic management, or predicts an outcome. this is because different diseases produce similar alterations in hepatic function or in laboratory tests. once biochemical tests identify the presence of hepatic disease, the diagnosis must be pursued further. in some instances, diagnostic imaging can reveal specific abnormalities (e.g., psss and extrahepatic bile duct obstruction). when results of imaging do not give a specific etiology, the next step is often to pursue a morphologic diagnosis obtained by analysis of a biopsy specimen. often it is a judgment call as to when to pursue hepatic biopsy. in cases with severe clinical signs and/or severe biochemical abnormalities, biopsy is usually warranted early in the fine-needle aspiration has several advantages. little to no sedation is usually required. because the size of the needle is so small, there is little risk of hemorrhage. therefore multiple sites can easily be sampled. the procedure is rapid and can usually be performed on an outpatient basis. there is also less cost to the client. the primary disadvantage of fine-needle aspiration is its questionable accuracy. the sample size often limits the number of available cells to obtain an accurate diagnosis, and hemodilution makes it difficult to assess whether inflammatory cells were present in the liver or peripheral blood. there are several important elements used to interpret pathologic information including lobular architecture, presence and location of inflammation within a lobule, presence and severity of fibrosis, metal accumulation, vascular abnormalities, and lobule heterogeneity. these criteria cannot be accurately determined using a cytologic sample obtained using fna. several studies have compared fine-needle aspiration cytology with biopsy with histopathology. [9] [10] [11] [12] in one study with a total of 34 cases, there was good correlation in 35% of cases, partial correlation in 35% of cases, and no correlation in 30% of cases. 9 poor correlation was found with a variety of histologic changes, including vacuolar change, lipidosis, cholestasis, inflammation, and neoplasia. in a similar study with 97 cases, complete agreement between fineneedle aspiration and histopathology was seen in only 30% of cases in dogs: 25% agreement with inflammation, 14% agreement with neoplasia (mainly carcinoma), and 64% agreement with vacuolar hepatopathy. 10 in cats, there was overall agreement in 51% of cases: 27% agreement with inflammation, 33% agreement with neoplasia (lymphoma), and 64% agreement with vacuolar hepatopathy. although vacuolar hepatopathy was the most sensitive diagnosis, it was also the most common misdiagnosis using cytology. in another study, the best correlation between hepatic cytology and biopsy was seen with lipidosis, lymphoma, and carcinoma, whereas the worst performance was seen with inflammatory and fibrotic disorders. 11 another study found high sensitivity and specificity with fine-needle aspiration in detecting inflammatory hepatic disease in dogs. 12 however, further information was not provided such as the severity of the inflammation or other histopathologic features. additionally, for noninflammatory hepatic disease, cytology was inaccurate in 76% of cases. proteins-induced-by-vitamin-k-antagonism test is more than twice as sensitive in dogs and more than three times as sensitive in cats in detecting coagulopathies compared with prothrombin time (pt) and aptt. 6, 7 however, in a pilot study performed by me, hepatic bleeding times assessed via laparoscopy did not correlate with proteins induced by vitamin k antagonism times. thus it appears that indices of coagulation in the peripheral blood are generally unreliable guides of the risk of bleeding after liver biopsy, and hence, are of limited value in determining contraindications to this procedure. this lack of correlation may be explained by the high concentration of clotting factors in the hepatic parenchyma and by mechanical compression of the needle tract by the elastic tissue within the liver. in most cases of significant hemorrhage, technical errors such as damaging a large vessel are the cause rather than persistent oozing from a needle biopsy site. controlled studies in veterinary patients will be necessary to make final conclusions regarding postbiopsy hemorrhage in the patient with a coagulopathy. in one study of normal dogs, biopsies taken from the left lateral hepatic lobe using a biopsy punch, biopsy needle, ligature method, laparoscopic biopsy forceps, and ultrasonically activated scalpel resulted in minimal hemorrhage (<2 ml). 8 however, this investigation did not assess the risk of hemorrhage in dogs or cats with hepatic disease. these risks will be discussed later under each sampling method. with the exception of fine-needle aspirations, each patient should have a prebiopsy packed cell volume and 3 and 6 hours postbiopsy packed cell volume for close monitoring of potential hemorrhage. fine-needle aspiration involves obtaining a small amount of hepatic tissue for cytologic analysis, and is typically performed in conjunction with, and guided by, ultrasound. ultrasound imaging helps determine if there is a diffuse abnormality (e.g., increased or decreased echogenicity, diffuse mottling) or if there are focal abnormalities (e.g., discrete nodules, cysts, masses, or focal areas of heterogenous mottling). an appropriate site to be sampled is chosen. often multiple sites are chosen to represent different lobes, and in the case of focal lesions, to sample more than one area of abnormal tissue and sample seemingly normal tissue. the sites are also chosen based on accessibility. for example, a solitary nodule in the dorsocranial aspect of the liver in a large deep-chested dog would be impossible to reach with a 1.5-inch needle. a lesion adjacent to the gallbladder or caudal vena cava would involve considerable risk. the clinician would need to decide whether the relative risk of sampling such lesions is the appropriate decision, or whether other methods of sampling would be more appropriate such as laparoscopy or laparotomy. figure 61 -15 depicts a typical setup for fine-needle aspiration. usually a 22-gauge, 1.5-inch needle is used. for most patients, the procedure is performed without sedation or local anesthetic. if it is determined that the animal is moving too much during the initial ultrasound examination, a sedative may be necessary (or an anesthetic in extreme cases). the needle is inserted without a syringe using ultrasound guidance. the needle is rapidly agitated in and out (sometimes referred to as mimicking the action of a sewing machine) and simultaneously twisted multiple times for a few seconds to obtain a sample. this method relies on capillary action rather than suction to get tissue into the needle, resulting in less hemodilution. after removing the needle from the liver, a syringe is attached and cells are expelled onto a glass slide for cytologic examination. often three to five separate attempts are made to increase the sample size and diversity. examination is performed prior to biopsy. this allows planning of the procedure based on echo pattern, lesion size, proximity to other organs, proximity to blood vessels, determination of cystic or solid tissue, and optimal approach of the needle path. care must be taken prior to taking samples to ensure that vessels and other organs are not within the path of the needle. for diffuse lesions, the transducer is typically placed caudal and to the left of the xiphoid, and aimed at the left medial or lateral lobes. in patients with a small liver, it may be difficult to adequately visualize the needle without gastric gas interference. placing these animals in a 45-degree right lateral oblique position can reduce this interference. if the animal is under general anesthesia, an assistant can compress a rebreathing bag to hold the animal in deep inspiration, which serves to move the diaphragm and liver caudally to improve visualization. the area is surgically prepared. the ultrasound transducer is covered with sterile wrap and sterile lubricant is used to enhance skin contact. a small stab incision is made in the skin at the desired needle insertion site. while one hand maneuvers the transducer, the other hand advances the needle into the liver under direct ultrasound visualization. the image should be optimized to maximize the chance of recognizing the needle within the liver. to allow distinction of the needle from other echogenic structures, the needle can be gently moved in and out with minimal movements (attempting to move the liver within the abdominal cavity rather than the needle within the liver). occasionally the needle cannot be seen, and indirect evidence of organ penetration must be used such as movement of the liver or visualization of movement at the liver border. the needle is then directed so the trajectory will avoid other structures when it is fired. the needle is then fired, and immediately removed. for most cases, four to five samples are obtained, and are submitted for aerobic/anaerobic culture, histopathologic evaluation, and metal (copper, zinc, and iron) quantification. in one study, liver tissues with high metal concentrations had significantly lower copper and iron in needle-core versus wedge biopsy specimens. 13 consequently the value of needle-core biopsy specimens for measurement of metal concentrations is questionable. careful examination for post-biopsy hemorrhage is then performed. external digital pressure may be used to help control hemorrhage in smaller patients. usually an abdominal compression wrap is ineffective for controlling hemorrhage. ultrasound-guided biopsy has many of the advantages of fine-needle aspiration, including the need for minimal sedation in some patients, the ability to sample multiple sites, and low to moderate cost to the client. additionally, tissue is obtained for histopathology. one disadvantage of ultrasound-guided biopsy is the risk of bleeding (especially when multiple sites are sampled and largergauge needles are used). in one study, 96 percutaneous transabdominal hepatic needle biopsy samples were obtained with no adverse consequences noted 14 ; however, this study was performed in normal dogs, and still carries high risk. additional disadvantages of ultrasound-guided biopsy include the needing sedation or anesthesia in some patients, difficulty of imaging small livers, difficulty of obtaining liver tissue in patients with fibrosis, and, most importantly, the obtaining of samples that have a questionable representation of the underlying hepatic pathology. the diagnostic accuracy of needle biopsy has been questioned by many clinicians, observing that results of needle biopsy analysis often do not adequately reflect the clinical and laboratory features of the patient. this questionable accuracy is in most part a result of potential for sampling error. this method still results in a relatively small sample size, possible although fine-needle aspiration is easy to perform, involves little risk, and little to no sedation, the information is of little value if it is inaccurate as often as it is accurate. there is institutional bias regarding its accuracy, which may relate to the experience and expertise of the cytologists. given its clear limitations, fine-needle aspiration is best used as an adjunctive diagnostic modality in conjunction with other techniques or clinical findings, and does not replace histopathology. the clinician must be aware of its inherent inaccuracy before undertaking fine-needle aspiration and relying on the cytologic findings. ultrasound-guided hepatic biopsy uses a cutting-type needle as a sampling tool. automated needles are preferred and should be either completely automated or semiautomated. these are spring-loaded needles similar in style to the manual tru-cut needle. completely automated needles thrust the inner obturator (containing the biopsy tray or specimen notch) followed by the outer cutting sheath into the liver in a fraction of a second. these needles can be operated with one hand while the other hand operates an ultrasound transducer to allow precise placement of the biopsy instrument. there is minimal displacement of the liver, a shorter intraparenchymal phase, and a more reliable yield of tissue. this allows a smaller diameter needle to be used and a lighter degree of sedation in some cases. using the rapid cutting action, the hepatic tissue tends to be less fragmented. semiautomated needles require manual placement of the internal obturator into the liver, followed by an automatic thrusting of the outer cutting sheath by a spring-loaded mechanism. these needles have the additional advantage of control over the final needle position, as the tip of the needle can be precisely localized before the outer cutting sheath is deployed. i generally use a 16-gauge needle for ultrasound-guided hepatic biopsy. figure 61 -16 depicts a typical setup for ultrasound-guided biopsy. in most dogs, the liver can be biopsied using local anesthesia and minimal sedation. most cats require general anesthesia to safely obtain tissue. it must be emphasized that the degree of sedation must be tailored to each individual patient. a careful ultrasound peritoneal space through a stab incision using a number 11 scalpel blade. after ensuring no obstruction and negative pressure using the infusion and aspiration of saline, the abdomen is insufflated with carbon dioxide gas and maintained at a pressure of approximately 12 mm hg. a scope port (cannula) is then placed 4 cm right lateral to the veress needle. the veress needle is then removed and replaced with an instrument port. hepatic sampling is achieved using a "spoon" or oval cup biopsy forceps. multiple samples are obtained under direct visualization, and samples are submitted for aerobic/anaerobic culture, histopathologic evaluation, and metal (copper, zinc, and iron) quantification. following procurement of all the biopsy specimens, the sites are inspected for hemorrhage. the abdomen is then decompressed, and lidocaine and bupivacaine are infused into the peritoneal cavity through either port. both the instrument and scope ports are removed, and the port site incisions are closed using either a cruciate or simple interrupted pattern in the body wall, subcutaneous tissue, and skin. this technique enables gross evaluation of the entire liver, extrahepatic biliary system, and surrounding structures while obtaining multiple large specimens of liver. the ability to obtain multiple samples decreases the risk for sampling artifact in cases of regional diversity within the liver. additionally, by directly visualizing the hepatic parenchyma, the clinician can correlate the histopathologic findings and clinical data with the gross appearance of the liver to render the most accurate diagnosis. this method also enables the visualization of smaller masses and irregularities that may not be evident with ultrasonographic imaging. these masses can also be individually sampled. laparoscopy also gives the clinician an excellent view of the liver regardless of the hepatic size or conformation of the patient, making it an easy method to sample the liver in patients that are difficult to image with ultrasound. there is generally minimal bleeding during this procedure, even in patients with in vitro coagulopathies. using a "spoon" or oval-cup biopsy forceps typically results in a marked decrease in the amount of hemorrhage when compared with needle biopsies. any hemorrhage can be directly visualized for adequate clot formation. if hemorrhage persists, direct pressure using a blunt probe for 5 minutes can be used. if the site continues to bleed, electrocautery can be applied to the biopsy site or a topical hemostatic agent (gelfoam) can be placed directly on the biopsy site using laparoscopic forceps. disadvantages of laparoscopy include the need for expensive equipment, the need for extensive training, the need for general anesthesia in most cases, and higher cost to the client. laparoscopy gives the clinician the advantages of a laparotomy (large sample size, ability to best direct sampling, and ability to take multiple samples, thus resulting in the highest diagnostic accuracy), though with a relatively minimally invasive procedure. the complication rate (especially hemorrhage) is far less than with ultrasoundguided biopsy in my practice. for these reasons, it is my method of choice for obtaining hepatic biopsy specimens in most cases. wedge biopsy via laparotomy is another potential method for obtaining hepatic biopsies. if a random liver biopsy is needed and a section of liver is protruding, a guillotine suture can be used. a preformed encircling ligature of 4-0 monofilament absorbable suture material is placed around the protruding section of liver. fragmentation of fibrous tissue, and may not enable sampling of abnormalities located in other lobes (the left medial or lateral lobes are generally sampled because of their ease of imaging). in one study, percutaneous hepatic sampling using core biopsies resulted in 92% diagnostic quality samples, however these were not compared with large wedge biopsy to assess the accuracy of this method. 15 in another study, the diagnostic accuracy of the tru-cut-type needle biopsy was compared with the gold standard of surgical wedge biopsy of the liver in 124 patients. 16 the overall discordance between the two methods was 53% in dogs and 50% in cats, with a greater than 60% discrepancy occurring with chronic hepatitis or cirrhosis, cholangitis/ cholangiohepatitis, portosystemic vascular anomalies, microvascular dysplasia, fibrosis, and miscellaneous disorders. these disorders are the most commonly seen among dogs and cats with hepatobiliary disease. the greatest accuracy was with neoplasia (80% concordance). figure 61 -17 is an example of a mass amenable to an ultrasound-guided needle biopsy. use of a 14-gauge versus 18-gauge needle may reduce this discordance as it raises the number of portal triads sampled from an approximate mean of four to seven, though larger needles carry the risk of increased hemorrhage. in summary, ultrasound-guided hepatic biopsy is relatively easy to perform, but involves more risk to the patient (primarily bleeding). like fine-needle aspiration, ultrasound-guided biopsy has questionable accuracy. the accuracy may be increased by using a larger-gauge needle, but this carries a greater risk of postbiopsy hemorrhage. if the patient is suspected of having inflammatory disease, vascular abnormalities, or significant fibrosis, or is at risk for hemorrhage, laparoscopy or laparotomy should be considered. chapter 28 provides a detailed description of laparoscopic liver biopsy. briefly, laparoscopy is performed under general anesthesia with the patient in dorsal recumbency tilted 45 degrees to the left. a veress needle is placed at the level of the umbilicus into the acute hepatitis and necrosis are common morphologic hepatic lesions in dogs and cats presenting with acute liver disease caused by infectious, toxic, metabolic, and ischemic disorders (box 61-1). however, acute liver disease can also be associated with other pathologic processes such as severe hepatic lipidosis (cats), granulomatous hepatitis (fungal infections), intrahepatic cholestasis (bacterial cholangitis, leptospirosis), and malignant infiltration (lymphoma, malignant histiocytosis). canine adenovirus i, canine and feline herpesvirus in the neonate, clostridium piliforme, and toxoplasma gondii are specific examples of infectious agents that cause acute hepatic necrosis (with variable inflammation), often as part of a multisystemic disorder. 2 although leptospirosis is a well-recognized infectious cause of acute liver disease in dogs, hepatic necrosis is an uncommon histologic feature and hepatic lesions are typically characterized by cholestasis, liver cell dissociation, and nonspecific reactive hepatitis. 3 despite the large number of potential causes of acute hepatitis and necrosis, a specific etiology is often not determined. 4, 5 in a recent case series of 101 dogs with primary hepatitis (acute and chronic hepatitis) that were presented to a referral clinic, 21 dogs were diagnosed with morphologic features of acute hepatitis. 4 a cause could not be determined in the majority of these cases, although increased hepatic copper was detected in five dogs with acute hepatitis, suggesting that copper accumulation could be a significant contributing factor. 4 despite numerous potential causes of hepatocyte death, two general mechanisms are recognized: apoptosis and necrosis. 2 these two mechanisms have traditionally been considered to be distinct events. however, it now appears that apoptosis and necrosis are alternate outcomes of the same initiating causes and signaling pathways. 1 apoptosis is adenosine triphosphate-dependent (caspasedependent) programmed cell death that causes shrinkage of the cell (apoptotic bodies or acidophil bodies) with orderly resorption of cellular contents, minimal leakage of cellular components, and minimal secondary inflammation. 1,2 necrosis occurs when depletion of adenosine triphosphate results in cellular swelling, loss of integrity of the cell membrane and cell lysis, with release of cell contents and secondary inflammation. 1, 2 diffuse hepatic necrosis is the most consistent histological lesion detected in dogs and cats with acute liver failure. 5 acute liver failure (alf) is a rare clinical syndrome (usually fatal) that occurs when a sudden severe insult to the liver compromises at least 70% of functional hepatic mass. liver cell death exceeds hepatic regenerative capacity, resulting in clinical signs of liver failure. 5 the clinical and laboratory features of alf are not specific for the inciting cause but reflect disruption of one or more major hepatic functions. once hepatocellular injury has occurred (and assuming the patient survives), the morphologic hepatic response to injury may include parenchymal regeneration, fibrosis, and ductular proliferation. 2 nearly complete hepatic regeneration is possible if hepatocyte injury is limited and the reticulin network remains intact. 2 with severe parenchymal destruction or extensive loss of hepatocytes, periportal ductular proliferation, hepatic fibrosis, postnecrotic scarring, and regenerative hepatic nodules are more likely. 2 dogs with acute hepatitis may also progress to chronic hepatitis. 4 the clinical presentation of dogs and cats with acute hepatitis and necrosis varies with the underlying cause and the extent and severity of the hepatic lesions. the spectrum of hepatic involvement may the ligature is then tightened until it has crushed the hepatic parenchyma. after completing several throws in the knot, the sample is excised 1 to 2 mm distal to the ligature using metzenbaum scissors or a scalpel blade. if a specific area of liver is needed, a sample can be obtained using the transfixation method or a biopsy punch. the transfixation method entails placing a ligature through the liver lobe approximately 8 to 10 mm from its edge. the ligature is tightened to crush through the hepatic parenchyma along one border of the desired biopsy specimen. an additional throw is made at a right angle to the first ligature, and this throw is tightened to crush the parenchyma of the second border of the specimen. the sample is removed 1 to 2 mm distal to the crushed area using a scalpel blade or metzenbaum scissors. if the desired area does not lie near the edge of a liver lobe, a 6-mm biopsy punch can be used. the biopsy punch should be inserted into the hepatic parenchyma ensuring not to penetrate the opposite surface. if the biopsy site is close to the hilus, extra caution must be used so that no more than half of the thickness of the liver is penetrated. the biopsy sample is removed from the liver using scissors. hemorrhage can be controlled by filling the defect with a topical hemostatic agent (gel foam) and applying digital pressure for 3 to 5 minutes, or by suturing the hepatic capsule with fine, absorbable monofilament suture in a cruciate pattern. akin to laparoscopy, this method has similar advantages and disadvantages as listed previously. although it is more invasive, it allows easier biopsy of other abdominal organs (such as intestine and mesenteric lymph node) and the ability to perform therapeutic maneuvers (such as hepatic mass removal or biliary diversion). etiology hepatocyte death (necrosis and apoptosis) in dogs and cats occurs secondary to a broad variety of insults, including infectious agents, drugs and toxins, hypoxia, immunologic events, and metabolic disorders. hepatic necrosis and acute inflammation often occur together and the relationship between these two processes is complex. acute inflammation may be the primary event, or necrosis of hepatocytes can be followed by a substantial inflammatory response, the "hallmark" of necrotic cell death. 1 the term acute hepatitis traditionally has been used when infectious agents cause hepatocellular necrosis, even though in the early stages, hepatic inflammation can be minimal or absent. 2 controversy exists among veterinary pathologists regarding the preferred terminology (acute hepatitis versus acute hepatic necrosis), when necrosis predominates and is caused by noninfectious insults such as toxins or ischemia. 2 for the purposes of this discussion, lesions of acute hepatitis and acute hepatic necrosis are discussed together, recognizing that the primary contributions of each lesion may be variable, depending on the cause, host response, and passage of time. acute hepatitis, a form of primary hepatitis, should be differentiated from "nonspecific reactive hepatitis," a response of the liver to a variety of extrahepatic disorders that is characterized by focal inflammation without necrosis. 2 nonspecific reactive hepatitis is discussed in a later section of this chapter. synthesis. 7 increased activity of the cholestatic liver enzymes, alkaline phosphatase, and ggt, also commonly occur with acute hepatitis and necrosis, but the magnitude of the increase is much less than for the alt and ast. abnormalities in biochemical tests such as hyperbilirubinemia, increased sbas, hypoglycemia, and hyperammonemia indicate compromised hepatic function. hyperbilirubinemia and bilirubinuria support more significant hepatic injury once prehepatic (hemolytic) causes have been discounted. primary biliary tract disorders including posthepatic mechanisms of hyperbilirubinemia should also be considered in the differential diagnosis. other considerations for hypoglycemia in conjunction with acute liver disease include xylitol toxicity (excess insulin release) and sepsis. hypoalbuminemia usually suggests chronic rather than acute liver disease, because of the long serum half-life of albumin. if azotemia is detected, dehydration, gi blood loss, and concurrent renal damage (e.g., leptospirosis, nonsteroidal antiinflammatory drugs [nsaids]) should be considered. interpretation of azotemia is facilitated by concurrent urinalysis. renal injury is supported by findings of cellular or granular casts, glucosuria, isosthenuria, and proteinuria. the complete blood cell count may reveal an inflammatory response suggesting underlying infectious or inflammatory disorders, and it is also useful for ruling out hemolytic anemia as cause of jaundice. documentation of a coagulopathy is required for the clinical diagnosis of alf. laboratory findings indicative of a coagulopathy include prolonged pt and activated partial thromboplastin time (aptt), decreased fibrinogen, increased fibrin degradation products, and thrombocytopenia. abdominal radiographs are often unremarkable in dogs and cats with acute hepatitis and necrosis. the liver may appear normal or increased in size. on abdominal ultrasound, the liver may appear normal or hypoechoic. thoracic and abdominal imaging may be helpful to evaluate for other causes of acute hepatic disease, and biliary tract disorders. because dogs and cats with acute hepatitis and necrosis present with nonspecific signs of acute liver disease, the clinician should maintain a broad perspective regarding the many potential diseases and processes that can acutely affect the liver. prior to obtaining a liver biopsy, ancillary testing (cytology or biopsy of more accessible lesions, infectious disease titers or molecular tests, diagnostic imaging) should be performed to evaluate for systemic disorders with secondary hepatic effects or multisystemic infections, thus providing a diagnosis of other causes of acute liver disease in a less-invasive manner. when acute hepatitis or hepatic necrosis is suspected (or confirmed by liver biopsy), a thorough history is essential to identify exposure to potential hepatotoxins and infectious agents. the owner should be questioned regarding recent medications, including prescription and over-the-counter drugs, and alternative medicines such as herbal and dietary supplements. the potential for exposure to chemicals or hepatotoxins (amanita mushrooms, blue-green algae, sago palms, aflatoxins, or xylitol) should be assessed (for more details, see "drug and toxin-induced liver injury" section). other pertinent historical questions include current vaccination history (canine adenovirus, leptospirosis), travel history (fungal infections or tick-borne diseases), and exposure to other animals (infectious causes). liver biopsy is required to document the presence of acute hepatitis and necrosis; evaluate for specific causes; and differentiate acute from chronic disease. in patients with mild (or absent) clinical signs and liver enzyme elevations that correspond to recent medication administration, a liver biopsy may be postponed, the medication discontinued, and clinical signs and liver enzymes monitored for include (a) subclinical (biochemical abnormalities only), (b) clinical signs of acute liver disease, or (c) the clinical syndrome of alf. when liver injury is mild (focal necrosis and inflammation), clinical signs may be absent, mild, or related to an underlying cause in another organ system. in this setting, hepatic involvement may not be recognized until biochemical evaluation reveals increased liver enzyme activity or mild hyperbilirubinemia. it has been suggested that many dogs with acute hepatitis are not recognized clinically, because signs are mild and self-limiting, and dogs recover spontaneously regardless of treatment. 6 clinical signs of acute hepatitis include acute onset of lethargy, anorexia, vomiting, diarrhea, pu, and pd, in a previously healthy animal. these are nonspecific findings of acute liver disease, which overlap those of other systemic disorders. the finding of icterus on the physical examination is a more specific indicator of hepatobiliary disease, especially in the absence of anemia. dogs and cats with acute diffuse hepatic necrosis often present with alf. 5 in addition to the signs of acute liver disease described above, animals in alf show signs of he (depression, behavioral changes, dementia, ataxia, pacing, circling, blindness, hypersalivation, seizures, and coma) and clinical evidence of a bleeding tendency (melena, hematemesis, or cutaneous and mucosal hemorrhages), which suggest severe hepatic dysfunction. 5 signs of alf are rapidly progressive (over hours to days) and this clinical syndrome is often fatal, with reported mortality varying from 25% to 100%. 5 with acute hepatic disease, the history typically reveals acute onset of signs in a previously healthy animal. however, liver failure that is recently recognized may not necessarily be recent in onset. with occult chronic liver disease, clinical signs may be vague and go unrecognized by the owner until a final phase of hepatic decompensation. the owner should be questioned about any subtle signs of chronic illness that would suggest the underlying liver disease may be chronic rather than acute, and that the current illness may be an exacerbation or decompensation of chronic liver disease. dogs and cats with alf are generally in good nutritional status compared with those with chronic hepatic disease. findings of cachexia, emaciation, ascites, or edema suggest a more protracted illness and are characteristic of chronic rather than acute liver disease. it is important to make a distinction between acute and chronic liver disease as the intensive supportive care indicated in alf might not be warranted in chronic end-stage liver disease. the long-term prognosis is better for acute hepatitis than chronic hepatitis. 4 an initial database consisting of complete blood cell count, serum chemistry, and urinalysis should be obtained in dogs and cats with acute liver disease. liver enzyme elevations are a common finding in dogs and cats with acute hepatitis and necrosis. with mild hepatic injury or focal hepatic necrosis, increased alt activity may be the only finding on an otherwise unremarkable biochemical profile. alt and ast activities are moderately to markedly increased, because of enzyme leakage from damaged hepatocytes. 7 although alt activity increases with many hepatic diseases, the largest magnitude of increase is seen with acute hepatic necrosis and roughly correlates with the number of involved cells. 7 alt activity may be increased as much as 100 times the upper range of normal, with increases in ast activity that parallel but are generally lower (30 times the upper limit of normal) than the alt. it should be noted that some recognized hepatotoxins (aflatoxin and microcystin in blue-green algae) are not associated with severe or protracted increases in alt activity because of toxin-suppressed transaminase improvement over a 2-to 3-week period. for patients with alf and coagulopathy, the clinician must carefully weigh the benefits of histologic characterization versus the risk of excessive bleeding from the procedure. acute hepatitis is characterized histologically by a mononuclear or mixed inflammatory pattern, accompanied by hepatocellular apoptosis or necrosis. 2 necrosis should be further characterized by the pathologist as to the morphologic pattern of injury (focal, multifocal, confluent, bridging, massive, or piecemeal) because the pattern of necrosis may provide insight into the pathogenesis of the lesion. 2 for example, because centrilobular hepatocytes have an abundance of cytochrome p450 enzymes, these hepatocytes are preferentially affected in drug-induced hepatotoxicity, when cytochrome p450 metabolism of the parent drug results in toxic metabolites. 8 quantitative copper analysis and histochemical staining for copper are recommended, as copper accumulation may be an underappreciated cause of acute hepatitis in dogs. 4 infectious causes of acute hepatitis may be diagnosed on liver biopsy, or by additional tests performed on liver tissue (culture, immunohistochemistry, polymerase chain reaction [pcr], virus isolation; table 61 -4). unfortunately, in most cases, routine liver biopsy is unlikely to reveal a specific cause of acute hepatitis. 2, 4 findings of inflammation and necrosis/apoptosis accompanied by nodular regeneration and fibrosis suggests chronic rather than acute hepatitis. the long-term prognosis is better for acute hepatitis than for chronic hepatitis. 4 if a probable cause of acute hepatitis and hepatic necrosis can be determined, then specific treatment is directed at the primary etiology (e.g., discontinuing potentially hepatotoxic medications, treating for leptospirosis with doxycycline, or chelating hepatic copper with penicillamine). in most cases specific therapy is unavailable and treatment is directed at more general supportive and symptomatic treatment of liver disease. glucocorticoid therapy is not typically indicated in the treatment of acute hepatitis. 4 empirical treatment with antioxidants such as s-adenosylmethionine (same; 20 mg/ kg po q24h), milk thistle (siliphos; 3 to 6 mg/kg po q24h), or vitamin e (10 to 15 iu/kg q24h) may be warranted, as oxidative stress is believed to play a role in drug (carprofen, potentiated sulfonamides, diazepam, methimazole, lomustine, others), and toxin (aflatoxin, organic solvents, and heavy metal toxicity) induced hepatic injury. 9 same and milk thistle have additional cytoprotective properties that could be beneficial in necroinflammatory hepatopathies and hepatotoxicity. antioxidants and cytoprotective agents are discussed in more detail in chapters 40 and 46, respectively. liver biochemistries should be monitored to assess patient response to therapy. repeat liver biopsy performed 6 to 8 weeks after the initial diagnosis has been recommended, to confirm that acute hepatitis has improved or resolved, or to document a progression toward chronic hepatitis. 4, 6 it has been suggested that most dogs with mild idiopathic acute hepatitis (not in alf) recover after several days, regardless of treatment. 6 for patients with alf, aggressive supportive treatment is required. goals of therapy are to treat the underlying cause when possible, allow adequate time for hepatic regeneration and repair, and prevent or control complications of liver failure, such as hypoglycemia, coagulopathy and anemia, he, gi ulcers, and septicemia. intravenous n-acetylcysteine (nac), a glutathione source/ antioxidant, is the antidote of choice for treatment of acetaminophen toxicity. nac also appears to have additional potential benefits (improved systemic hemodynamics and tissue oxygen delivery), and should be considered for use in any dog or cat with alf. 9 the optimal dose regimen when nac is used for this purpose has not been determined. treatment of complications of liver failure are discussed in "complications of liver disease" section. the prognosis for recovery in dogs with acute hepatitis is good, as most dogs recover uneventfully. 4 however, there is a potential for dogs with acute hepatitis to develop chronic disease. 4 if animals present with signs of advanced liver failure (e.g., he, coagulopathy, hypoglycemia), the prognosis is guarded. if the animal survives, hepatic lesions such as periportal ductular proliferation, hepatic fibrosis, postnecrotic scarring, and regenerative hepatic nodules are likely. 2 if a hepatic drug reaction is suspected, reexposure of the patient to the suspect drug should be avoided. etiology hepatic abscesses from bacterial infection of the liver occur uncommonly in dogs and cats. [10] [11] [12] [13] abscesses may form as solitary or multiple macroscopic masses or microabscesses. in newborn animals, gram-positive and gram-negative bacteria cause hepatic abscesses, presumably related to postpartum umbilical infections. 14 in adult animals, gram-negative enteric bacteria (especially escherichia coli) and anaerobes (especially clostridia spp.) are most commonly identified; multiagent infections are frequent. 10, 12 other organisms such as yersinia spp., actinomyces spp., nocardia asteroides can also cause hepatic abscesses as part of a systemic infection. 14 the pathogenesis of hepatic abscesses in dogs and cats is unclear. hepatic abscesses are usually associated with extrahepatic infections or regional hepatic parenchymal damage. small numbers of bacteria, including clostridium spp., can be cultured from liver tissue of healthy dogs. hypoxia of hepatic tissue caused by hepatic neoplasia, liver lobe torsion, or trauma may predispose to abscess formation, because small numbers of existing anaerobes (e.g., clostridium spp.) can proliferate under these conditions. other potential sources of bacteria include hematogenous spread (via the umbilical vein, hepatic artery, or translocation of intestinal bacteria into the portal blood), ascension via bile ducts, penetrating abdominal and caudal thoracic wounds, and direct extension from local suppurative diseases. concurrent diseases or potential predisposing factors in dogs include systemic infections (pneumonia, pyelonephritis, prostatitis, pyometra, endocarditis), gallbladder rupture, pancreatitis, diabetes mellitus, liver lobe torsion, coexisting hepatic disease such as hepatic neoplasia (infected necrosis), longterm phenobarbital administration, long-term corticosteroid administration, and previous surgical biopsy. 10, 11 concurrent diseases in cats include cholecystitis, pyothorax, and hepatic neoplasia. 12 solitary abscesses are more common in dogs, whereas cats are more likely to be septic and have multiple hepatic abscesses. 11, 12 no association with feline leukemia virus or feline immunodeficiency virus infection has been made. 12 solitary liver abscesses are more likely to involve the right liver lobe in cats and the left liver lobe in dogs. 11, 12 clinical examination when adult dogs and cats are diagnosed with hepatic abscesses, they are usually older than 8 years of age. [10] [11] [12] clinical signs are nonspecific and can be attributed to sepsis, inflammation, and hepatic dysfunction. the most common signs are anorexia, lethargy, vomiting, and diarrhea. 10, 11 clinical signs of hepatic involvement may be overshadowed by signs of the associated disease process (e.g., neoplasia, pyelonephritis, pancreatitis). dogs with hepatic abscess may have a history of failure to respond to antibiotics or improvement that relapsed when antibiotics are discontinued. 10 physical examination findings are often vague and include depression, dehydration, fever, abdominal pain, hepatomegaly, abdominal mass, and abdominal effusion. [10] [11] [12] hypothermia is a more common finding than fever in cats with hepatic abscesses. 12 because the clinical findings are vague and nonspecific, hepatic abscesses often go undetected until an abdominal ultrasound is performed or they rupture and are discovered during laparotomy. rupture of a hepatic abscess leads rapidly to peritonitis, septic shock, and death. clinicopathologic abnormalities are consistent with an inflammatory hepatic disease. potential findings on the complete blood count include neutrophilia with a left shift (or neutropenia and degenerative left shift if rupture occurs), mild anemia, and thrombocytopenia. 10, 12 increased alt and alp activity are common findings although the alt may be in the normal range. 10 liver enzyme elevations are a less-consistent finding in cats with hepatic abscesses and 79% in cats. 12 the survival rate appears to be better when solitary abscesses are detected. 12, 13 granulomatous hepatitis is characterized histologically by focal or multifocal aggregates of activated macrophages with an epithelioid appearance, usually accompanied by lymphocytes and plasma cells. 14 this inflammatory response is distinct from that encountered in canine chronic hepatitis. systemic infectious diseases are an important cause of granulomatous hepatitis and this lesion has been described with fungal infections (histoplasmosis, coccidioidomycosis, many others), bacterial infections (mycobacteria, bartonella, nocardia, actinomyces, rhodococcus), protozoal diseases (cytauxzoonosis, leishmaniasis); parasitic diseases (visceral larval migrans, schistosomiasis, alveolar echinococcus, hepatozoon americana), and disseminated protothecosis (see table 61 -4). 15, 16 in cats, feline infectious peritonitis (coronavirus) is an important cause of multisystemic granulomatous or pyogranulomatous inflammation. other causes of granulomatous inflammation include a local response to foreign material (crystalline material, sutures, plant material) or a drug reaction. in humans, granulomatous liver lesions have been associated with administration of diltiazem, sulfonamides, quinidine, allopurinol, interferon-α, and phenytoin. 17, 18 however, drug therapy as a cause of granulomatous hepatitis in dogs and cats has not been specifically reported. granulomatous lesions in the liver has been described in a small number of dogs with lymphangiectasia, lymphosarcoma, and histiocytosis. 19 many cases of granulomatous hepatitis are idiopathic. 16 hepatic lipogranulomas ("fatty cysts"), which are often found in dogs with congenital portosystemic shunt, are aggregates of pigment-laden foamy macrophages and should not be confused with granulomatous hepatitis. clinical findings with granulomatous hepatitis are highly variable, depending on the underlying cause. when granulomatous hepatitis is identified on liver biopsy, special stains for fungal and mycobacterial organisms should be performed. other diagnostics to either identify an organism (cytology, culture, fecal exam, pcr) or detect antibodies against the organism (serology) vary widely with the underlying agent (see table 61 -4). if a cause cannot be found after a thorough diagnostic evaluation, consideration should be given to presumptive treatment for undiscovered infectious agents such as atypical mycobacteria, bartonella spp., or systemic fungal infection. corticosteroids or other immunosuppressant agents should only be used when diagnostic testing and empirical treatment have been unsuccessful, as steroid-induced immunosuppression may exacerbate an underlying infection. 19 eosinophilic hepatitis occurs rarely in dogs and cats. 2 potential causes include visceral larval migrans (toxocara), schistosomiasis, liver fluke infections, sarcocystis canis, and possibly, fungal infections (see table 61 -4). 2 with parasitic causes, eosinophils are often located at or near the site of the parasitic lesion in the liver. dogs and cats with systemic allergic, parasitic (heartworms), or hypereosinophilic syndromes, may also have scattered eosinophils in the liver, a variant of nonspecific reactive hepatitis. 2 hepatic druginduced liver injury should also be considered. phenytoin and minocycline are associated with eosinophilic infiltrates in humans with drug-induced liver injury. 18, 20 potentiated sulfonamides have been suggested to cause drug-induced eosinophilic hepatitis in dogs, 2 although a more typical pattern is acute hepatocellular necrosis or a cholestatic hepatopathy. 21 when eosinophilic infiltrates are identified, efforts should be directed at diagnosing parasitic causes (fecal, (increased alt and alp activity occurred in less than 50% of cats). 12 other potential biochemical findings include hyperglobulinemia, mild hyperbilirubinemia, and hypoglycemia (sepsis). laboratory abnormalities may also reflect the associated disease processes (e.g., hyperglycemia with diabetes mellitus, increased pancreatic lipase immunoreactivity with acute pancreatitis). if an abscess ruptures, cytology of the abdominal infusion reveals septic suppurative inflammation. abdominal radiographs may be normal or reveal hepatomegaly, hepatic mass lesion, or decreased abdominal detail or effusion associated with secondary peritonitis. with proliferation of gas-producing organisms, radiolucent areas may be seen in the liver. ultrasonographic examination permits earlier detection of hepatic abscesses. 11 ultrasonographically, a liver abscess appears as a hypoechoic or anechoic structure with irregular, hyperechoic margins. 11, 13 the ultrasonographic pattern is similar to that seen with hepatic hematomas, cysts, neoplasia, and biliary cystadenoma. gas may be seen within the abscess. 11 if abscess rupture has occurred, concurrent abdominal effusion may be detected. additional ultrasonographic findings may reflect associated disorders such as pancreatitis, cholecystitis, or pyelonephritis. ultrasound-guided fine-needle aspiration of a suspected liver abscess can be safely performed to obtain samples for cytology and culture to confirm the diagnosis. 11 if ultrasonography is not available, the diagnosis of hepatic abscesses is usually established during exploratory laparotomy (or at necropsy). an attempt should be made to isolate and identify the organism(s) associated with abscessation so that appropriate antibiotic therapy can be instituted based on sensitivity testing. aerobic and anaerobic cultures can be performed on abscess contents (by fine-needle aspiration), abdominal exudate, blood or hepatic tissues. treatment of hepatic abscesses consists of surgical resection or drainage of focal lesions, administration of appropriate antibiotics, correction of associated fluid, electrolyte, and acid-base imbalances, and identification and treatment of any underlying disease process. treatment of large unifocal hepatic abscesses has typically involved surgical resection of affected tissue, which may necessitate partial or full lobectomy. 10, 12 if perforation and peritonitis are present, surgical abdominal drainage and lavage are indicated. ultrasound-guided percutaneous drainage of a solitary abscess may resolve the abscess or allow stabilization until surgical resection can be performed. 11 the successful management of focal hepatic abscesses (up to 8 cm in diameter) by ultrasound-guided percutaneous drainage and alcoholization has been described in five dogs and one cat. 13 broad-spectrum combination antibiotic therapy (directed toward both aerobic and anaerobic bacteria) should be initiated as soon as cultures have been obtained. results of a gram stain on the exudate may provide preliminary information as to type of organism and guide the empirical choice of potentially effective antibiotics. recommendations for broad-spectrum antimicrobial coverage of hepatobiliary infections include either a fluoroquinolone combined with amoxicillin/clavulanate or a fluoroquinolone combined with penicillin and metronidazole, until culture results are available. the dose of metronidazole should be adjusted in animals with hepatic dysfunction (7.5 mg/kg po q8-12h). antibiotic therapy should be continued for at least 6 to 8 weeks. response to treatment can be monitored with serial ultrasound examinations and repeated blood work. historically, hepatic abscesses have carried a grave prognosis, with an overall reported mortality rate of approximately 50% in dogs 10 case series (see chapter 62) . breeds of dogs at increased risk for chronic hepatitis include the bedlington terrier, 22, 23 west highland white terrier, 24, 25 doberman pinscher, [26] [27] [28] american and english cocker spaniel, [29] [30] [31] [32] [33] skye terrier, 34 dalmatian, 35 labrador retriever, [36] [37] [38] and english springer spaniel. 39 unfortunately, with the exception of hereditary copper-associated liver disease in bedlington terriers, information is lacking for most of the breed-related disorders. female dogs appear to be at increased risk in some studies, 4,40 while others report that male and female dogs are equally affected. 41, 42 within particular breeds, sex differences have been noted (female doberman pinschers, labrador retrievers, and english springer spaniels; male cocker spaniels). 26, 29, 36, 38, 39 dogs diagnosed with chronic hepatitis are generally 4 to 7 years of age, but adult dogs of any age (or breed) can be affected. 4, 29, 41 etiology and pathogenesis ideally, canine chronic hepatitis should be classified on an etiologic basis. however, with the exception of copper-associated liver disease in bedlington terriers, the cause, pathogenesis, natural history, optimal treatment, and prognosis of these disorders are unknown (table 61 -5) . idiopathic chronic hepatitis is the most common clinical diagnosis. 4, 32, 40, 41 infectious causes. viral infections are a common cause of chronic hepatitis in humans, but are not currently recognized as an important etiology in dogs. in humans, viruses have the potential heartworm test; see table 61 -4), systemic eosinophilia, and hypersensitivity reactions. if no specific cause can be determined, empirical treatment with fenbendazole should be considered, followed by corticosteroid therapy as described for idiopathic chronic hepatitis. the term nonspecific reactive hepatitis is used to describe the slight to moderate widespread inflammatory infiltrates of the liver that occur secondary to a spectrum of extrahepatic disease processes. 2 lesions of nonspecific reactive hepatitis are associated with febrile and inflammatory disorders, especially those involving the gi tract and pancreas, or they may represent residual evidence of a previous intrahepatic inflammatory disorder. 2 inflammation occurs in portal or parenchymal areas and necrosis is absent. neutrophils predominate with acute extrahepatic disorders, whereas mononuclear inflammation occurs with chronic extrahepatic disorders or residual hepatic inflammation. the liver may be secondarily affected by systemic disorders because of changes in liver blood flow, portal blood delivery of bacteria, drugs, hormones, cytokines, or other substances from the gi tract, or activation of intrahepatic kupffer cells (monocyte-macrophage system) involved in the hepatic immune response. it may be challenging to differentiate nonspecific reactive hepatitis from resolving acute hepatitis or mild chronic hepatitis, without supportive clinical information. clinical signs in dogs and cats with nonspecific reactive hepatitis are usually referable to the extrahepatic disorder. liver enzyme elevations (alt-two times the upper limit of normal; alpthree-to fourfold increases) are common, thus mimicking primary hepatic disease. however, tests that reflect liver function, including serum bile acids, are usually normal. it is important to consider extrahepatic disorders that can secondary affect the liver, prior to focusing on primary hepatic disease. treatment is directed at the underlying extrahepatic disorder. chronic hepatitis, a heterogeneous group of inflammatorynecrotizing diseases of the liver, occurs commonly in dogs, but is rare in cats. cholangitis, which is inflammatory liver disease that targets the biliary tract, rather than hepatocytes, is more common in cats but also occurs in dogs.the term chronic hepatitis, rather than chronic active hepatitis or chronic persistent hepatitis, is recommended. 2, 15 if the etiology is known, it should be included as an adjective, such as "drug-induced chronic hepatitis," or "copper-associated chronic hepatitis"; otherwise, it is considered "idiopathic chronic hepatitis." chronic hepatitis in dogs is defined based on histopathologic features of hepatocellular necrosis or apoptosis associated with inflammation and evidence of regeneration and fibrosis. 2 lymphoplasmacytic inflammation is characteristic, but a neutrophilic component may be present. 2 the histopathologic features of chronic hepatitis are similar, regardless of the underlying cause. chronic hepatitis has the potential to progress to cirrhosis. 15, 16 recommendations have been made to include a clinical component to the definition of chronic hepatitis, such as documenting an increase in alt activity along with histologic evidence of hepatic inflammation for a minimum of 4 months. however, many dogs with chronic liver disease are not clinically apparent until the advanced stages, so duration can be difficult to evaluate. the early stages may not be recognized unless biochemistries are monitored for hepatic injury. a familial predisposition to develop chronic hepatitis has been suggested by demographic studies, pathologic surveys, and clinical hepatitis (acute and chronic) accounted for one-third of all dogs with primary hepatitis. 4 hepatic copper accumulation and hepatopathy have been described in cats but appears to be rare. 58, 59 the severity of hepatic injury correlates with the amount of hepatic copper, but subcellular localization of molecules and the molecular association also plays a role. 54 serum copper levels do not accurately reflect hepatic copper content and quantitative analysis of copper in the liver is required. 56 hepatic copper concentration in normal dogs is between 150 and 400 µg/g dry weight (parts per million). 28, 57 inflammatory hepatic injury does not consistently occur until copper concentrations exceed 2000 µg/g dry weight. 60, 61 however, there may be breed variations; for example, in doberman pinschers hepatic inflammation is present with copper concentrations of less than 2000 µg/g. 27, 57 transient acquired fanconi syndrome has been described in dogs with excess hepatic copper accumulation. 62, 63 copper granules were demonstrated on renal biopsy in some but not all dogs. potential mechanisms for hepatic copper accumulation include primary metabolic defects in hepatic copper metabolism, cholestasis causing impaired biliary excretion of copper, and excess copper absorption. 54, 56 a primary defect in hepatic copper metabolism occurs in bedlington terriers with a genetic mutation in the gene encoding the copper transport protein, commd1 (formerly murr1), resulting in a defect in biliary copper excretion. 64, 65 in to cause hepatitis either because of a persistent hepatic infection or as a transient infection that triggers an immune response because of a cross-reaction between the virus and liver antigens. 43 in an attempt to identify infectious causes of canine hepatitis, pcr screening of liver tissue was performed in 98 dogs with various stages of hepatitis to look for canine adenovirus type 1, hepadnaviridae, hepatitis a virus, hepatitis c virus, hepatitis e virus, helicobacter spp., leptospira spp., and borrelia spp. 44 based on negative results, the authors concluded that canine hepatitis is not typically caused by these infectious agents. 44 however, dogs that are experimentally infected with canine adenovirus type i, but are partially immune, can develop chronic hepatitis that progresses to cirrhosis. 45 the virus could not be detected beyond the first week postinfection, although the disease progressed over a period of months. 45 canine adenovirus antigen has been demonstrated by immunohistochemical techniques in formalin-fixed liver sections from five of 53 dogs with various hepatic inflammatory lesions, suggesting that canine adenovirus 1 (cav-1) may play a role in spontaneous chronic hepatitis. 46 in contrast, pcr and immunohistochemistry failed to detect canine adenovirus in liver tissue of 45 dogs with chronic liver disease. 47 whether cav-1 is a significant cause of chronic hepatitis under natural conditions is unknown. another proposed viral cause of chronic hepatitis and cirrhosis is the "canine acidophil cell hepatitis virus," reported from great britain in the 1980s. 48, 49 this transmissible agent, most likely a virus, is distinct from cav-1. it was transmitted experimentally by subcutaneous injection of serum or liver extracts from affected dogs, resulting in experimentally induced acute and chronic hepatitis. no further studies have been published to clarify the nature of this infectious agent or the associated hepatitis. canine leptospirosis is typically associated with acute cholestatic hepatic disease and acute renal failure. however, persistent infection can cause chronic hepatitis in the absence of azotemia. 50, 51 leptospira serovar grippotyphosa was incriminated as a cause of chronic hepatitis in a kennel of american foxhounds, based on serologic evidence and demonstration of spirochetes in the liver. 50 leptospira serogroup australis (serovars australis, bratislava, and muenchen) infection was suspected to cause chronic hepatitis in 16 young beagle dogs in a breeding colony routinely vaccinated against leptospirosis serogroups canicola and icterohaemorrhagica. 51 canine leishmaniasis has been associated with histologic evidence of chronic hepatitis, 52 but clinical features suggestive of hepatic involvement (hepatomegaly, ascites, or icterus) were absent. histologic findings revealed granulomatous hepatitis in most dogs, but some dogs had marked portal infiltration with lymphocytes and plasma cells, and mild portal fibrosis. leishmania amastigotes were routinely identified in macrophages in liver or other affected tissues. bartonella clarridgeiae dna was amplified from a liver biopsy of a doberman pinscher with copper-associated chronic hepatitis, although the significance of this finding is unclear. 53 copper accumulation. copper is an essential trace element in diets and is required for a number of physiologically important enzymes. cells have highly specialized and complex systems for maintaining intracellular copper concentrations. at toxic concentrations, free intracellular copper initiates oxidative damage causing hepatocellular necrosis and inflammation. 54, 55 normal copper metabolism has been reviewed in detail elsewhere. 54, 56 copper accumulation in the liver can be associated with significant hepatic injury resulting in acute hepatitis, chronic hepatitis, and cirrhosis ( figure 61-18) . 4, 54, 57 it is one of the few well-documented causes of canine chronic hepatitis. in one study, copper-associated a b liver of 37 dogs but was not identified in any control samples from healthy livers. none of the dogs had decreased serum levels of α 1antitrypsin. positive α 1 -antitrypsin staining was a more consistent finding in english and american cocker spaniels with chronic liver disease, than in other breeds. the authors concluded that accumulation of α 1 -antitrypsin might play a role, but it could not be determined if it was the cause or a result of chronic liver disease. 31 drugs and toxins. drug or toxin exposure is a potential cause of canine chronic hepatic disease. drugs that have been incriminated include anticonvulsants (phenobarbital, primidone, phenytoin), oxibendazole-diethylcarbamazine, lomustine, and possibly carprofen. [70] [71] [72] [73] chronic hepatitis and cirrhosis from long-term phenobarbital therapy is most widely recognized. 70, 71 exposure to aflatoxin from contaminated commercial dog food is usually associated with alf, but low-level long-term exposure in dogs can result in chronic hepatic injury (biliary hyperplasia, fibrosis, nodular regeneration). a breeding colony of german shepherd dogs developed chronic hepatitis and cirrhosis that was suspected (but never confirmed) to be a result of exposure to a porphyrinogenic substance, based on the finding of aggregates of crystalline pigments with orange birefringence with polarized light. 74 early recognition of drug-or toxin-induced chronic hepatic injury requires biochemical monitoring of liver enzymes, as dogs are clinically asymptomatic in the early stages. autoimmune/immune mechanisms. autoimmune hepatitis has not been documented in dogs. however, some dogs with chronic hepatitis appear to respond to corticosteroid therapy and thus may correspond to autoimmune hepatitis in humans. 41 autoimmune hepatitis in humans is a progressive chronic hepatitis of unknown cause that is believed to occur when an environmental agent (viruses, medications) triggers a cascade of t-cell-mediated events directed at liver antigens, in a genetically predisposed individual. 43 women are more commonly affected than men. hyperglobulinemia is a common finding. an infectious cause is difficult to document, as exposure may have occurred many years prior to the overt autoimmune disease. 43 certain drugs may induce or unmask an autoimmune hepatitis, or simply cause hepatocellular injury that mimics autoimmune hepatitis. 43 an autoimmune component to doberman pinscher hepatitis has been speculated, because of the breed's predisposition, high female predominance, and the finding that expression of mhc class ii antigens on hepatocytes of affected dogs correlates with degree of inflammation and decreases after treatment with prednisolone. 75 dogs with chronic hepatitis may have concurrent disorders associated with immune aberrations (immune hemolytic anemia, hypothyroidism, atopy, glomerulonephritis), but whether this is coincidental or indicative of the presence of multiple immune disorders as seen with autoimmune hepatitis in humans is unknown. 37, 76, 77 autoimmune hepatitis in humans is diagnosed when other causes of acute or chronic hepatitis have been excluded and serum autoantibodies (antinuclear, antismooth muscle, antibody to liver/ kidney microsomes type 1, antibody to liver cytosol type 1) are detected. 43 a number of studies have evaluated the role of liverassociated antibodies and cell-mediated response in dogs with chronic hepatitis, but none answers the question of whether the immune response is the primary cause of the hepatitis or a secondary phenomenon. twenty-four dogs with chronic hepatitis were evaluated for circulating autoantibodies (against cell nuclei, smooth muscle, liver membrane, and mitochondria) by indirect immunofluorescence. 77 antibodies to cell nuclei and liver membranes were the early stages, copper is sequestered in hepatic lysosomes and hepatic damage is minimal. however, with progressive accumulation of copper, hepatic injury becomes significant. the average copper concentration in bedlington terriers with chronic hepatitis is approximately 6000 µg/g dry weight and values up to 12,000 µg/g dry weight have been reported. 23, 57 inherited copper-associated liver disease is also described in the west highland white terrier, skye terrier, doberman pinscher, dalmatian, and labrador retriever, but with the possible exception of dalmatians, the hepatic copper levels are much lower than in bedlington terriers. [25] [26] [27] 34, 35, 38 the pathogenesis of copper accumulation and the relationship to chronic liver disease in these breeds is poorly understood. it seems likely that these breeds have a hereditary disorder of copper handling, but it is unlikely to be the same as described for the bedlington terrier. hepatic copper accumulation in the liver may also be a consequence rather than the cause of chronic hepatitis. because copper is normally excreted in the bile, chronic cholestasis and impaired bile flow can result in secondary copper accumulation. 57, 66 secondary copper accumulation is predominantly periportal and is usually less than 2000 µg/g dry weight. 57, 66 the effect of cholestasis on hepatic copper content was evaluated in three groups of dogs: bedlington terriers with copper toxicity, dogs with extrahepatic biliary obstruction (the prototype example of a cholestatic disorder) and chronic hepatitis in breeds not known to be at risk for copper-associated liver disease. 66 hepatic copper content was evaluated by a semiquantitative method based on copper staining of liver tissue with rubeanic acid, using a scale of 0 (no copper) to 5. 67 copper staining revealed absent to mild increases (scores of 0 to 2+) in dogs with biliary obstruction and chronic hepatitis when compared with bedlington terriers (scores of 5+). it was concluded that copper scores of 3+ or higher were suggestive of a primary copper storage disease. 66 unfortunately, quantitative copper analysis was not evaluated. markers of oxidative injury and altered defense mechanisms were similar in the three groups, consistent with the concept that copper, inflammation, and cholestasis can all contribute to oxidative injury. 66 high dietary copper intake appears to be an unlikely explanation for hepatic copper accumulation and liver disease in dogs. 56 however, the copper content of commercial dog foods ranges from 12 to 16 mg/kg dry matter, which is relatively high compared with recommended minimum daily copper requirements in dogs. 56 there is speculation that the recent increase in pathologically elevated hepatic copper concentrations (specifically evaluated in labrador retrievers), may coincide with a pet food industry recommendation to replace cupric/cuprous oxide in feed formulations because of its low bioavailability. 68 many dogs with copper-associated chronic hepatitis also have increased hepatic iron concentrations. 69 hepatic iron accumulation usually correlates with degree of inflammation. 40, 69 whether iron, as an oxidant, interacts with copper to contribute to lesions seen in copper-associated hepatitis remains to be determined. α 1 -antitrypsin deficiency. inherited α 1 -antitrypsin deficiency is a well-recognized cause of chronic hepatitis and cirrhosis in humans, and may play a role in the pathogenesis of chronic hepatitis in some dogs. 31 α 1 -antitrypsin is a circulating protease inhibitor that is synthesized and secreted by the liver. α 1 -antitrypsin deficiency in affected humans results in defective formation and impaired hepatic secretion of α 1 -antitrypsin, resulting in hepatic accumulation of α 1 -antitrypsin and hepatic injury. serum levels of α 1 -antitrypsin are typically low. in a study of 57 dogs with chronic liver disease, α 1antitrypsin was detected by immunohistochemical staining in the early stages of chronic hepatitis, ultrasonography of the liver may be normal or reveal nonspecific changes in echogenicity. when chronic hepatitis has advanced to cirrhosis, potential ultrasonographic findings include microhepatia, irregular hepatic margins, focal lesions representing regenerative nodules, increased parenchymal echogenicity associated with increased fibrous tissue, and ascites. splenomegaly and acquired psss may also be detected. a liver biopsy is essential for the diagnosis of chronic hepatitis. wedge biopsies are preferred over needle biopsies because they provide more tissue and are more likely to represent pathologic process(es) in the liver. when cirrhosis is present, laparotomy or laparoscopy often provide a better appreciation for the gross nodularity of the liver than can be ascertained from blind percutaneous needle biopsy (figure 61-19 ). chronic hepatitis is characterized histologically by moderate to severe inflammation (usually combinations of lymphocytes and plasma cells) associated with piecemeal necrosis. piecemeal necrosis, also referred to as interface hepatitis, is necrosis involving the layer of hepatocytes adjacent to the portal tract or "limiting plate." 2 the term bridging necrosis is used when necrosis and inflammation dissect across the hepatic lobule from portal areas to central veins or to adjacent hepatic lobules and suggests a severe form of chronic hepatitis. 2 histopathologic evaluation of the liver should not only consider etiology, but the pathologist should also comment on the activity (amount of inflammation, extent of apoptosis and necrosis) and the stage of disease (extent and pattern of fibrosis; architectural distortion suggestive of cirrhosis). 2 biopsies from dogs with chronic hepatitis should routinely be evaluated for copper accumulation. on hematoxylin and eosin staining, excess copper appears as golden brown refractile granules. 28 histochemical stains, such as rhodanine or rubeanic acid, can be used to semiquantitatively evaluate for copper in the liver (see figure 61 -19) . these stains consistently detect copper when amounts exceed 400 µg/g dry weight. 60 values obtained by quantitative copper analysis have a strong correlation with the number and size of granules seen with histochemical stains within the range of 400 to 1000 µg/g of liver tissue. 60 zonal distribution of copper detected, but were also found in dogs with other types of hepatic disease, suggesting a nonspecific secondary response. patterns of circulating autoantibodies found in dogs differed significantly from those found in humans with chronic liver disease. 77 in another study, serum anti-liver-membrane-protein antibody-positive dogs (1 : 40 to >1 : 1600) had higher alt activity, total bilirubin concentration, and more severe hepatic lesions than did anti-liver-membraneprotein antibody-negative dogs, but it was not determined whether autoantibodies were primary or secondary. 78 cd3+ lymphocytes are the most common hepatic lymphoid cells in dogs with chronic hepatitis and are associated with hepatic necrosis, 79-81 but also account for 54% of hepatic lymphocytes in normal dogs. 82 historical and physical examination findings in dogs with chronic hepatitis are indicative of chronic hepatic disease, and are similar regardless of the underlying cause. signs are often initially vague and nonspecific, such as anorexia, lethargy, vomiting, diarrhea, weight loss, pu, and pd. 4, 32 with increased severity of hepatic dysfunction, signs of overt liver failure develop, such as ascites, jaundice, and he. the presence of ascites and he suggest that chronic hepatitis has progressed to cirrhosis, and ascites is a negative prognostic indicator. 4, 42 melena associated with gastroduodenal ulceration or coagulopathy is also more likely with advanced liver disease. 32 because of the large functional reserve capacity of the liver, the onset of signs may appear very recent, initially suggesting an acute rather than chronic hepatic disorder. clinicopathologic features that support chronicity include poor body condition, ascites, microhepatia, hypoalbuminemia, and histologic evidence of fibrosis. in the early (subclinical) stages, dogs are asymptomatic and only identified by biochemical screening for liver enzyme elevations. increased serum alt activity, reflecting ongoing hepatic injury, is reported in 75% to 95% of dogs with chronic hepatitis. 4,7 serum alt activity may exceed 10 times the upper normal limit. 7 periods of normal alt activity may reflect cyclic disease activity and the varying severity of necrosis. 7 serum alp activity is also commonly increased, but the magnitude of the increase is generally lower than seen with alt activity. when chronic hepatitis advances to cirrhosis, liver enzyme activity may be normal, indicating decreased viable parenchymal mass. 7 abnormalities in biochemical tests such as hyperbilirubinemia, hypoalbuminemia, decreased blood urea nitrogen (bun), hypoglycemia, and increased sba indicate hepatic dysfunction and a more advanced stage of disease. 32 hyperglobulinemia can be seen in dogs with cirrhosis, but it remains to be determined whether this corresponds with increased autoantibodies as occurs in humans with autoimmune hepatitis, or whether it reflects nonspecific systemic antibody production in response to antigens from the portal blood which bypass the liver through acquired psss. 83 mild nonregenerative anemia may be a reflection of chronic disease. regenerative anemia can occur from blood loss secondary to a coagulopathy or bleeding gi ulcers. copper-associated hemolytic anemia has only been documented in bedlington terriers. abnormal hemostatic parameters (prolonged aptt and pt) are indicative of severe hepatic dysfunction or dic. a prolonged pt and thrombocytopenia may be negative prognostic indicators. 16, 37 analysis of ascitic fluid reveals a transudate or modified transudate. 32, 42 abdominal radiographs are unremarkable except when advanced stages of disease are accompanied by microhepatia or ascites. in the kg every 48 hours is most often recommended for treatment of canine chronic hepatitis. complications of corticosteroid therapy include gi bleeding (which may precipitate he), secondary infections, iatrogenic cushing disease, and worsening of ascites. dexamethasone (0.2 mg/kg po q24h) may be preferred in dogs with ascites or edema, because it lacks mineralocorticoid activity, which could exacerbate these signs. prednisone is often used in combination with azathioprine, especially if side effects of prednisone become objectionable. azathioprine is an antimetabolite with antiinflammatory and immunemodulating effects, and is commonly used in combination with prednisone in humans with autoimmune hepatitis. 43 the dose of azathioprine in dogs is 1 to 2 mg/kg/day po every 24 hours for 1 to 2 weeks, then tapered to every 48 hours for maintenance therapy. prednisone (0.5 to 1.0 mg/kg/day) is given on the alternate days. because azathioprine may cause bone marrow suppression and acute hepatotoxicity, the complete blood count and biochemical profile should be monitored. antiinflammatory agents and immunosuppressive drugs are discussed in more detail in chapters 38 and 49, respectively. because glucocorticoids increase liver enzyme activity (especially serum alp activity), response to therapy is best evaluated by a followup liver biopsy performed 3 to 6 months after starting therapy. if glucocorticoid therapy is eventually discontinued, clinical and biochemical parameters should be periodically monitored to detect a relapse. dogs with hepatic copper concentrations greater than 1500 µg/g, should be treated with the copper chelator penicillamine at a dose of 10 to 15 mg/kg po every 12 hours. 57 treatment usually requires months to years to produce significant decreases in hepatic copper. a mean decrease in copper of approximately 1500 µg/g was achieved in bedlington terriers treated for 6 months. 86 dogs with secondary copper accumulation appear to respond more rapidly, possibly because hepatic copper content is lower in these breeds. 86 doberman pinschers with subclinical hepatitis treated with penicillamine for 4 months had a mean decrease in copper from 1036 µg/g to 407 µg/g. 87 penicillamine has additional effects beyond copper chelation, which may be beneficial in dogs with chronic hepatitis, including inhibition of collagen deposition, stimulation of collagenase activity, immunosuppression, and immunomodulation. 54 common side effects of penicillamine therapy include anorexia, nausea, and vomiting, which can be minimized by giving the medication with a small amount of food. the copper chelator, trientine (10 to 15 mg/kg po q12h), is also effective for reducing hepatic copper concentrations. 86 it has fewer side effects than penicillamine and is effective in dogs with hemolytic anemia caused by copper release from necrotic hepatocytes. iatrogenic copper deficiency (microcytosis and hepatic dysfunction) has been described in a dog treated with long-term copper chelation therapy (trientine) and a copper-restricted diet. 88 decisions on duration of chelator therapy are based on followup liver biopsies with periodic monitoring of quantitative hepatic copper content. oral zinc salts can be used for maintenance therapy after copper chelation, or as initial therapy in dogs with hepatic copper concentrations between 400 µg/g dry weight and 1500 µg/g dry weight. zinc supplementation is typically used in conjunction with dietary copper restriction. zinc decreases intestinal copper absorption by inducing the intestinal copper-binding protein, metallothionein, within intestinal epithelial cells, which preferentially binds dietary copper and prevents its absorption. zinc acetate is given at a dose of 100 mg po bid for 2 to 3 months, then at a maintenance dose of 50 mg po bid. 89 a minimum of 3 months of zinc therapy is required before copper uptake from the intestinal tract is blocked. 89 accumulation should be noted, as copper accumulation starting in the centrilobular area is more likely with a primary metabolic defect in copper metabolism. 54 copper granules can also be detected on cytology of hepatic aspirates or impression smears stained with rhodanine or rubeanic acid. quantitative analysis for copper, by atomic absorption analysis on fresh hepatic tissue, is the definitive method to document increased hepatic copper content. needle core biopsy specimens may not be reliable for metal analysis, as copper and iron values are consistently lower in needle core versus wedge biopsy samples. 84 formalin-fixed tissues should be avoided, because formalin may contain copper or leach copper from the tissue. 57 hepatic copper can be reliably determined retrospectively on deparaffinizedarchived liver biopsy specimens. 84 once chronic hepatitis has been confirmed, a careful consideration of known causes of chronic hepatitis is essential (see table 61 -5) . findings that would support a primary metabolic defect in copper metabolism include a previously recognized breed predisposition, copper accumulation that precedes cholestasis or inflammation, centrilobular (zone 3) distribution of copper, histochemical score for copper of 3+ or greater, or quantitative copper measurements that exceed 2000 µg/g dry weight. 54, 57, 66 special stains of the liver should be requested to evaluate for infectious agents such as leptospirosis; serum antibody titers for leptospirosis may be indicated. a history of chronic drug therapy should be sought, especially long-term anticonvulsant therapy or other drugs listed in table 61 -5. recommendations for treatment of chronic hepatitis are empirical at best, because of the lack of controlled therapeutic studies on a well-defined population of dogs with this disorder. if a probable cause or category of injury can be determined, then specific treatment is directed at the primary etiology, for example, discontinuation of phenobarbital, treatment of leptospirosis, or chelation of hepatic copper with penicillamine. in most cases, specific therapy will be unavailable. treatment of chronic hepatitis in dogs has traditionally centered on the use of corticosteroids, presuming that, as in humans with the autoimmune form of hepatitis, immunologic mechanisms (inflammatory cells and mediators, local cytokines), contribute to hepatic inflammation and progression to cirrhosis. corticosteroids have antiinflammatory, immune-modulating, and antifibrotic effects, which may be beneficial in chronic hepatitis. a large retrospective study suggested that corticosteroid therapy at initial immunosuppressive doses (2.2 mg/kg/day; eventually tapered to 0.6 mg/kg/day) improved survival in dogs with chronic hepatitis. 41 however, many concurrent drugs were given and, undoubtedly, a heterogeneous group of disorders were included under the diagnosis of "chronic hepatitis." corticosteroid therapy appears warranted in dogs with histologic features of active inflammation and persistent increases in serum liver enzyme activity, for which known causes of chronic hepatitis (including infectious causes) have been excluded. 41, 85 glucocorticoid therapy is not indicated for treatment of chronic hepatitis caused by drug therapy, infectious agents, or primary hepatic copper accumulation. the optimal dose and duration of corticosteroid therapy for treatment of canine chronic hepatitis is unknown, including whether immunosuppressive doses are required, or whether lower, anti-inflammatory levels would suffice. 76 even in humans with autoimmune hepatitis, immunosuppressive doses of corticosteroids may not be required. 43 prednisone (or prednisolone) at an initial dose of 1 to 2 mg/kg/day po and then gradually tapered to 0.5 to 1.0 mg/ is correlated with the amount of hepatic copper. hepatic injury is believed to occur when progressive copper accumulation exceeds the storage capacity of the lysosomes; copper is released to the cytoplasm, damaging mitochondria, initiating lipid membrane peroxidation, and eventually causing cell death. affected dogs can be asymptomatic (in the early stages) or show signs of acute hepatic necrosis, chronic hepatitis, or cirrhosis. 54 in young dogs, copper accumulates in centrilobular (zone 3) hepatocytes and is sequestered in hepatic lysosomes. during this first stage, copper concentrations are between 400 and 1500 µg/g, dogs are asymptomatic, biochemical testing is within normal limits, and liver biopsy findings are unremarkable. in the second stage, when hepatic copper concentrations are between 1500 and 2000 µg/g, copper granules are also found in midzonal (zone 2) and periportal (zone 1) hepatocytes. although dogs are still asymptomatic, focal hepatic inflammation (centrilobular mixed cell foci, with necrotic hepatocytes, lymphoplasmacytic inflammation, and copper-laden macrophages) is seen on biopsy, and increased serum alt activity reflects hepatocellular injury. in the most advanced stage, when hepatic copper concentration exceeds 2000 µg/g, morphologic changes reveal chronic hepatitis that may progress to cirrhosis, and clinical and biochemical evidence of liver disease become apparent. clinical signs include anorexia, lethargy vomiting, and weight loss. with progression to cirrhosis, findings of jaundice, ascites, and he may develop. biochemical findings vary with the stage of disease. increased serum alt activity is the most sensitive laboratory indicator, although findings will be normal in young dogs in stage i, because of the lack of hepatic inflammation. other serum biochemical abnormalities typical of chronic hepatic dysfunction eventually develop. in some cases, acute hepatic necrosis and alf occur. hepatocellular necrosis may be associated with release of copper from necrotic hepatocytes, resulting in hemolytic anemia. during episodes of hemolysis, plasma copper levels are increased; other findings include low packed cell volume, hemoglobinemia, and hemoglobinuria. liver biopsy and quantitative analysis of hepatic copper concentrations is required for definitive diagnosis and staging of the disease. serum copper or ceruloplasmin concentrations are not helpful to make a diagnosis. 57 liver biopsies should be performed in all bedlington terriers considered for breeding, in order to identify and remove affected dogs from breeding programs. screening of asymptomatic dogs with a liver biopsy at 6 months and 15 months of age can determine if an affected dog is homozygous or heterozygous (a carrier). 15 affected dogs (both homozygous and heterozygous) typically have increased hepatic copper by 6 months of age. however, copper concentrations in dogs who are carriers (heterozygous) return to normal by 1 year of age, whereas copper concentrations in homozygous dogs continues to increase. 57 selective breeding programs in the netherlands has decreased the prevalence of bedlington terrier copper-associated liver disease from 46% (1976) (1977) (1978) (1979) (1980) (1981) (1982) (1983) (1984) (1985) (1986) to 11% (1990-1997) . 92 dna testing of bedlington terriers is available from vetgen (www.vetgen.com). this assay evaluates a linkage-based dna marker (co4107, allele 2) that is located in the chromosome close to the gene for copper toxicity. 93 the test can identify normal, affected, and carrier dogs with 90% accuracy. however, the marker can only be relied on for diagnosis of the genetic status of an individual dog when supported by a pedigree study. 93, 94 significant discrepancies were reported in 22 bedlington terriers, when comparing results of liver biopsy and the dna marker. 94 this may be attributed to different subpopulations of bedlington terriers with variations in the disease-causing mutation of the commd1 gene or a second mutant copper gene could play a role. 94 liver biopsy for quantitative zinc administration should be separated from meals by at least 1 hour and should theoretically not be prescribed at the same time as a copper chelator. 54 serum zinc concentrations should be monitored to achieve a level of 200 to 400 µg/dl. zinc concentrations greater than 500 µg/dl may be toxic (hemolytic anemia). low-copper diets are most beneficial for managing early (subclinical) copper accumulation in dogs affected with primary metabolic defects in hepatic copper metabolism. feeding a low-copper diet decreases hepatic copper content in labrador retrievers with subclinical copper-associated liver disease. 90 additional treatment with zinc does not appear to increase the copper-lowering effect of dietary management. 90 foods containing large amounts of copper (liver, other organ meats, shellfish, eggs, bean/legumes, chocolate, nuts, cereals, and copper-containing vitamin supplements) should be avoided. because oxidative stress is a significant mechanism for hepatic damage associated with copper accumulation and necroinflammatory hepatic disorders, 66,91 antioxidant therapy with vitamin e (10 to 15 iu/kg/day), or same (20 mg/kg/day) has been advocated. 9 other cytoprotective agents such as silymarin (milk thistle) and ursodeoxycholic acid may also be beneficial. 9 chapters 40 and 46 discuss cytoprotective agents used in the treatment of hepatobiliary disease in detail. when end-stage cirrhosis is diagnosed, treatment is mainly supportive, as cirrhosis itself is essentially irreversible. measures should also be instituted to control the complications of chronic liver failure, such as ascites, he, gastroduodenal ulcers, and coagulopathy, which are discussed in more detail in "complications of liver disease" section. the response to treatment of chronic hepatitis is variable, which is not unexpected as it is likely a heterogeneous group of diseases. some dogs can eventually be taken off medication and remain in remission, but more often, therapy must be continued indefinitely. other dogs fail to respond, especially those that have advanced disease with cirrhosis. 4, 32 in one study, the estimated median survival time in 42 dogs with idiopathic chronic hepatitis was 18 months (range: 0 to 49 months) and in 23 dogs with copper-associated chronic hepatitis it was 17 months (range: 7 to 27 months). 4 mean survival time in 20 dogs with cirrhosis was 1 week. 32 bedlington terriers develop chronic hepatitis and cirrhosis from copper toxicity, as a consequence of an inherited metabolic defect resulting in impaired biliary copper excretion. 23, 57, 76 the disorder is transmitted by autosomal recessive inheritance. the gene responsible for this metabolic disorder is commd1, which is different than that described for copper toxicity (wilson disease) in humans, in which the gene involved is atp7b. 64 there is no gender predilection. at one time, it was speculated that as many as 60% of the breed might be affected. 57 hepatic copper concentration in normal bedlington terriers ranges from 91 to 358 µg/g with a mean of 206 ± 56 µg/g dry weight. 23 bedlington terrier copper-associated liver disease is associated with progressive, hepatic copper accumulation (copper levels of up to 12,000 µg/g) unless treatment is instituted. the lowest hepatic concentrations of copper are found in the youngest dogs and concentrations increase with age, peaking at around 6 years. copper content usually declines thereafter in affected dogs, but not to normal. this decline may be a result of replacement of copper-containing hepatocytes by fibrous tissue or regenerative nodules that do not contain copper. the severity of hepatic disease recent efforts have focused on identification of affected doberman pinschers prior to advanced hepatic disease. in finland, a survey of 626 randomly selected, clinically healthy doberman pinschers, revealed that 8.8% of dogs had increased alt activity, and 3.4% had hepatitis (parenchymal and portal mononuclear inflammation and positive stains for copper). 99, 100 the mean age of dogs with subclinical hepatitis was 3.8 years, compared with clinically affected dogs (5.5 years). the asymptomatic period lasted an average of 19 months. the prevalence of subclinical doberman hepatitis was investigated in 106 randomly selected 3 year old doberman pinschers in the netherlands. 27 subclinical hepatitis was identified in 22 dogs (19 females and three males); hepatic copper concentration was higher in dogs with hepatitis (419 ± 414 µg/g dry weight) than those without liver disease (197 ±113 µg/g). 27 serial liver biopsies over at least a 2-year period, revealed that hepatitis persisted only in dogs with copper levels greater than 400 µg/g dry weight, and copper levels continued to increase in these dogs (939 ± 299 µg/g), supporting a relationship between copper, inflammation and hepatitis. 27 it has also been proposed that hepatic copper is incidental to chronic hepatitis in this breed, based on the findings that five of 35 doberman pinschers with chronic hepatitis had normal copper levels, and histologic changes were similar regardless of copper status. 81, 26, 75 an immune-mediated mechanism has been suggested, based on the finding that expression of mhc class ii antigens on hepatocytes of dogs with doberman hepatitis was correlated with degree of inflammation. 75 aberrant mhc class ii molecule expression on nonlymphoid cells could be a result of toxins, drugs, viral infection, or autoimmunity, and hepatocytes with mhc class ii expression might become a target as an antigen-presenting cell for cd4+ t cells. 75 dogs treated with low-dose prednisolone (0.1 to 0.5 mg/kg/day) for 4 to 5 months had significantly decreased expression of mhc class ii antigens. 75 chronic hepatitis should be suspected in any doberman pinscher (especially females) with clinical or biochemical evidence of hepatic disease. definitive diagnosis requires liver biopsy. other causes of chronic hepatitis should also be considered, since doberman pinschers appear to be at risk for drug-induced hepatitis. 71 early detection of chronic hepatitis provides the best opportunity for treatment. it has been recommended that all doberman pinschers older than 1 year of age be screened for alt activity. 99 persistent increases in alt activity suggest further evaluation including liver biopsy is warranted. the magnitude of increased alt activity is not different between subclinical and clinically affected dogs. hyperbilirubinemia is suggestive of more advanced disease. 28, 99 effective treatment for doberman pinschers with chronic hepatitis has not been established. however, a preliminary study showed that if diagnosed in the subclinical stage, treatment with penicillamine (200 mg total dose po bid for 4 months) lowered hepatic copper content and improved hepatic histopathology. 87 traditionally, antiinflammatory or immunosuppressive drugs such as prednisone with or without azathioprine have been instituted. the efficacy of this treatment remains to be determined but generally, the response is poor if dogs are presented in advanced stages of liver failure. the use of ursodeoxycholic acid (15 mg/kg po bid) deserves special consideration in this chronic cholestatic disorder, but has not yet been objectively evaluated. treatment of copperassociated hepatitis in doberman pinschers with advanced disease is usually unsuccessful. most dogs die within weeks to months. the prognosis appears more favorable if the disease is detected in the early stages, but the optimal therapeutic regimen remains to be determined. copper and morphologic examination remain the best option for diagnosis in the individual dog. 94 a database for certification of bedlington terriers is maintained on the web site (www.caninehealthinfo.org) of the canine health information center, which is sponsored by the akc/canine health foundation and the orthopedic foundation for animals. affected bedlington terriers who are asymptomatic (copper >400 µg/g dry weight but less than 1500 µg/g dry weight) should have dietary copper restriction and zinc supplementation. bedlington terriers with copper accumulation (copper >1500 µg/g dry weight) and chronic hepatitis should be treated with a copper chelator such as penicillamine or trientine. 57 early diagnosis and treatment with either zinc or copper chelators will allow most dogs to lead a normal life. 57 treatment of hemolytic anemia may require a blood transfusion. trientine dihydrochloride (but not penicillamine) may be effective in chelating circulating copper during a hemolytic episode. chapter 43 discusses copper-chelating agents in more detail. doberman pinschers are at increased risk for the development of severe chronic hepatitis and cirrhosis. 26, 28, 81, 95 doberman hepatitis accounted for 4% of all deaths in a dutch population of 340 dobermans. 96 middle-aged (4 to 7 years) female dogs are at increased risk, but males also may be affected. although a hereditary mechanism is suspected, the pathogenesis of this disorder is unclear. 28 copper accumulation appears to be associated with hepatic damage, but the pathogenesis is different from the bedlington terrier disorder. 27 immune mechanisms may also play a role. 75, 81 many doberman pinschers are diagnosed in the advanced stages of hepatic failure. 26 evidence of excessive bleeding (gingival bleeding, epistaxis, and melena) are common. signs of he often predominate in the terminal stages. common physical examination findings include ascites, jaundice, and weight loss. splenomegaly (associated with portal hypertension) is common. laboratory findings included increased alt and alp activity, hyperbilirubinemia, hypoalbuminemia, hyperammonemia, coagulopathy, and thrombocytopenia. 26 typical histologic lesions include portal inflammation (lymphocytes, plasma cells, and macrophages), piecemeal necrosis, bridging necrosis, bile duct proliferation, and portal fibrosis. hepatic copper concentrations are increased in most affected dogs and are typically between 1000 and 2000 µg/g dry weight, although values as high as 4700 µg/g have been reported. 28, 81 the significance of the increased hepatic copper concentration in this breed remains controversial. copper accumulation was originally attributed to secondary mechanisms, as doberman pinschers with advanced disease (chronic hepatitis and cirrhosis) have biochemical and histologic evidence of cholestasis. however, evaluation of affected dogs in the early (subclinical) stage, reveals that copper accumulation precedes cholestasis, 27, 95 and decreased biliary excretion of radiolabeled copper has been documented. 97 the hepatic distribution of copper and location of inflammation varies with the stage of disease. in the early stages, the copper (and focal inflammation) is centrilobular. 27, 81, 95 as the disorder progresses, copper accumulation and inflammation are more pronounced in periportal regions and areas of bridging necrosis. 95 although copper appears to be related to the hepatic inflammatory reaction, copper levels are typically less than 2000 µg/g dry weight, the minimum amount of copper that is believed to cause hepatocellular injury in bedlington terriers and west highland white terriers. 60,61 a primary copper retention disorder has been proposed, 28 but the genes associated with bedlington copper toxicity (commd1) and wilson disease in humans (atp7b) have been excluded. 98 same, milk thistle), and symptomatic therapies that are not designed to lower hepatic copper. 37 feeding a low-copper diet to 20 labrador retrievers with hepatic copper accumulation (seven of 20 dogs had varying degrees of hepatitis) was effective in decreasing hepatic copper concentrations, but severity of inflammation remained unchanged. 90 additional treatment with zinc did not appear to increase the copper-lowering effect of dietary management. 90 long-term survival appears variable. 36, 37 dogs who died within 2 months of diagnosis were more likely to have a prolonged pt and thrombocytopenia. 37 dalmatians are reported to have acute hepatic necrosis, chronic hepatitis, and cirrhosis associated with increased hepatic copper concentrations. 35, 102 cholestasis is not a prominent biochemical or histologic feature until later in the disease, suggesting that hepatic copper accumulation is more likely to be caused by a familial metabolic disorder rather than secondary to altered hepatic biliary copper excretion. most dogs presented initially with acute gi signs (anorexia, vomiting, and diarrhea). biochemical findings revealed markedly increased alt activity with lesser increases in alp activity. hyperbilirubinemia and hypoalbuminemia were seen with advanced disease. glucosuria (in the absence of hyperglycemia) and proteinuria were identified in some dogs. ultrasound findings were usually unremarkable. liver biopsy revealed piecemeal necrosis, bridging fibrosis, and inflammation (predominantly lymphocytes or neutrophils). the mean hepatic copper level was 3197 µg/g dry weight (normal <400 µg/g dry weight) with a range of 754 to 8390 µg/g dry weight. in five of nine dogs, copper exceeded 2000 µg/g. rapid progression of the disease was characteristic. copper chelation therapy may be beneficial if diagnosed before advanced liver disease occurs. chronic hepatitis and cirrhosis associated with hepatic copper accumulation (800 to 2200 µg/g dry weight) in genetically related skye terriers has been described. 34 in the early stages, copper accumulation is absent, and biopsy findings indicate hepatocellular degeneration with cholestasis and mild inflammation. chronic lesions are associated with intracanalicular cholestasis, chronic hepatitis, and cirrhosis. skye terrier hepatitis is speculated to be a disorder of disturbed bile secretion with subsequent accumulation of copper. american and english cocker spaniels have an increased incidence of chronic hepatitis and cirrhosis. 29, 30 the cause is unknown. hepatic copper accumulation does not appear to be a consistent feature. it is unclear whether accumulation of α 1 -antitrypsin in hepatocytes, a well-recognized cause of cirrhosis in humans, is important in the pathogenesis. 31 male cocker spaniels (average age: 5 years) are at increased risk. 29, 30 despite the chronicity and severity of the underlying hepatic lesions, most affected dogs have a short duration of clinical illness, usually less than 2 weeks. ascites is the most consistent presenting complaint. profound hypoalbuminemia (mean: 1.7 g/dl) is a consistent laboratory finding. total serum bilirubin concentration is normal or only mildly increased, supporting that cholestasis is not a key feature of the disorder. ascitic fluid analysis is consistent with a transudate or modified transudate. on liver biopsy, hepatic lesions are consistent with chronic hepatitis and cirrhosis. treatment of cocker spaniels with chronic hepatitis consists of general supportive therapy for the complications of liver failure. corticosteroid therapy prior to progression to cirrhosis may west highland white terrier west highland white terriers are at increased risk to develop chronic hepatitis and cirrhosis. 25, 76, 101 males and females are equally affected. the mode of inheritance for the familial copper-associated disorder has not been established. 24 decreased biliary excretion of radiolabeled copper occurs in affected dogs. 89 centrilobular (zone 3) copper accumulation occurs during the first year of life, but rarely exceeds 2000 µg/g dry weight. 24 in contrast to the bedlington terrier, west highland white terriers do not continuously accumulate copper over their lifetime; in fact, copper content may actually decrease with time. 56 in one report of 395 clinically normal west highland white terriers, most dogs had hepatic copper levels between 100 and 1500 µg/g dry weight with normal liver biopsies. 25 in west highland white terriers with chronic hepatitis, histologic lesions include multifocal hepatitis, subacute bridging necrosis, massive necrosis, and cirrhosis. 25 the relationship of hepatic copper to chronic hepatitis in the west highland white terriers is unclear. there appears to be at least two types of chronic hepatitis. 25 some dogs have copper-associated hepatitis with elevated copper content (>2000 µg/g) and multifocal centrilobular hepatitis. copper concentrations do not usually exceed 3500 µg/g dry weight. lesions of chronic hepatitis can also be seen in the absence of substantial copper accumulation, and have been described as "idiopathic chronic hepatitis." 25 quantitative copper analysis is necessary to determine if copper accumulation is a significant (>2000 µg/g dry weight) contributing factor. if chronic hepatitis and cirrhosis are associated with increased hepatic copper content (>2000 µg/g dry weight), treatment for hepatic copper accumulation should be instituted. mature west highland white terriers with chronic hepatitis and less than 2000 µg/g dry weight of copper may not require chelation therapy, as hepatic copper accumulation is not continuous throughout life. other therapeutic options for treatment of idiopathic chronic hepatitis, such as glucocorticoids, should be considered in these dogs. labrador retrievers are at increased risk for chronic hepatitis. 29, 37 age at presentation ranges from 2.5 to 14 years, with an average age of 7 to 9 years. [36] [37] [38] a female predisposition was noted in two studies, 36, 38 whereas in another study, males and females were equally affected. 37 most affected dogs have increased hepatic copper, which has been described as centrilobular (zone 3) or diffuse. [36] [37] [38] copper concentrations exceeded 2000 µg/g dry weight in 10 of 12 dogs (mean copper: 3369 µg/g; range: 2375 to 4972; reference interval: 120 to 400 µg/g). 38 most dogs also had elevated iron levels with a mean of 4117 µg/g (reference interval: 350 to 1750). 38 a genetic basis is suspected, based on the finding of increased copper concentrations in asymptomatic related dogs, but the genetic defect remains to be determined. 36 a retrospective survey of hepatic copper content in labrador retrievers during two time periods (1980-1997 and 1998-2008) , revealed significantly higher copper concentrations in the more recent period both in dogs with chronic hepatitis and in control dogs; no difference in age or gender was noted. 68 it was speculated that increased hepatic copper might reflect increased dietary copper bioavailability, because of pet food industry recommendations to replace cupric/cuprous oxide in feed formulations. 68 treatment with penicillamine (15 mg/kg po q12h) appears to be effective in decreasing hepatic copper content and inflammation. 36, 90 however, some dogs appear to respond to immunosuppressive (prednisone, azathioprine), supportive (ursodeoxycholic acid, clinical features of cirrhosis in dogs include ascites (portal hypertension, hypoalbuminemia), he (intrahepatic and extrahepatic portosystemic shunting of blood), and evidence of decreased hepatic function (hypoalbuminemia, increased sba, coagulopathy, hyperbilirubinemia). findings on hepatic ultrasonography (small nodular liver, splenomegaly, and acquired psss) are suggestive for cirrhosis, but liver biopsy is required for confirmation. because cirrhosis is essentially irreversible, treatment is mainly supportive, emphasizing measures that control complications of severe generalized liver failure, such as ascites, encephalopathy, gastric ulcers, coagulopathy, and infection (see "complications of liver disease" section). if clinical signs of liver failure are already present, the prognosis is poor. prevention of fibrosis, an important long-term goal, is best achieved by early specific treatment directed at the probable cause of injury (e.g., discontinuing a suspect drug, penicillamine for copper-associated liver disease, antiinflammatory drugs for idiopathic chronic hepatitis, surgical relief of extrahepatic biliary obstruction). many therapeutic agents used for treatment of liver disease, such as penicillamine, prednisone, azathioprine, milk thistle, ursodeoxycholic acid, and zinc, have potential antifibrotic properties, 9 and are discussed in more detail in other chapters. colchicine, a microtubule assembly inhibitor which increases collagenase activity, has been recommended for treatment of hepatic fibrosis, but its effectiveness in dogs has not been critically evaluated. the recommended dose in dogs is 0.025 to 0.03 mg/kg/day po. reported side effects include nausea, vomiting, and diarrhea. bone marrow toxicity and myoneuropathy have been reported in humans. lobular dissecting hepatitis is a specific histologic form of cirrhosis seen in neonatal or young adult dogs. 2, 4, [110] [111] [112] it is suggested to be a nonspecific response to a variety of hepatic insults. 111 the age at presentation is younger than for dogs with either acute or chronic hepatitis. 4 in 21 affected dogs, the median age was 11 months, with 12 dogs (54%) being 7 months or younger. 4, 110 females appear to be at increased risk. 4, 111 lobular dissecting hepatitis may occur in an isolated dog or in groups of dogs from the same litter or kennel. 111 standard poodles may be at increased risk. 110, 112 clinical features are those of advanced hepatic failure and portal hypertension. 111 the most consistent clinical finding is ascites. liver enzymes are typically increased and hypoalbuminemia and increased sba concentrations are common. 4, 111 liver biopsy is required for diagnosis and to differentiate it from other types of chronic hepatitis and cirrhosis. the lesion is characterized histologically by lobular hepatitis: inflammatory cells (lymphocytes, plasma cells, macrophages, and neutrophils) are scattered throughout the hepatic lobule rather than concentrated in periportal regions. bands of collagen and reticulin fibers dissect around single or small groups of hepatocytes and disrupt hepatic lobular architecture. 111 copper stains are negative or moderately positive, consistent with secondary copper accumulation. specific treatment has not been reported, but general measures for management of chronic liver failure are appropriate. 4 in a small group of dogs with lobular dissecting hepatitis, the mean survival time was approximately 3 months, which was significantly shorter than for dogs with acute or chronic hepatitis. 4 infection of the liver is an important cause of hepatic disease in dogs and cats. 113 the liver may be the primary target of infection (e.g., infectious canine hepatitis, bacterial cholangitis, hepatic abscess) or be beneficial. the prognosis is poor and most dogs die within a month of diagnosis. a recent report described seven american cocker spaniels with histologic features resembling lobular dissecting hepatitis. 33 males and females were equally affected. in contrast to previous reports of hepatitis in cocker spaniels, most dogs in this study improved with corticosteroid therapy. 33 english springer spaniel a preliminary report has described chronic hepatitis in 34 english springer spaniels from norway and the united kingdom. 39 female dogs were overrepresented. copper does not appear to play a role. the prognosis appears to be poor, with most dogs dying 4 to 7 months after diagnosis. 39 hepatic cirrhosis (end-stage liver disease), is characterized by fibrosis, regenerative nodules that alter liver architecture and intrahepatic (microscopic) psss (see figure 61 -19) . 2 hepatic fibrosis is not synonymous with cirrhosis. cirrhosis is common in dogs but less so in cats. 2 cirrhosis can result from postnecrotic scarring after acute massive necrosis or from chronic hepatic injury caused by a variety of insults such as infection (e.g., leptospirosis, cav-1), hepatotoxins (e.g., copper, phenobarbital, aflatoxin), inflammation (chronic hepatitis), or hypoxia. the common denominator is hepatocyte death, which leads to repair by fibrosis and nodular regeneration. when cirrhosis is fully developed, the histologic features of the original inciting injury often are obscured by the cirrhotic changes. substantial hepatic fibrosis (without "cirrhosis") can be seen with long-standing extrahepatic biliary obstruction, noninflammatory fibrosis, congenital hepatic fibrosis (a disorder of biliary system development), and congenital portal vein hypoplasia. 2,103-105 a unique form of macronodular cirrhosis, characterized by noninflammatory regenerative hyperplastic nodules and diffuse vacuolar hepatopathy, is seen in dogs with hepatocutaneous syndrome (superficial necrolytic dermatitis). hepatic fibrosis was once considered irreversible, but is now recognized to be a dynamic process, which exists in a balance between synthesis and degradation. a better understanding of the underlying mechanisms may provide potential therapeutic targets. 106 the major fibrogenic cell in the liver is the activated hsc (ito cell, vitamin a-storing cell), which is normally present in the perisinusoidal space. 106, 107 under the influence of fibrogenic stimuli (inflammation and the immune response, oxidative stress, apoptosis, hypoxia, steatosis), the hsc is activated to a myofibroblast, which produces collagen and other extracellular matrix (ecm) constituents. 106 the cytokine, tgf-β, appears to play a central role in fibrogenesis in humans and dogs. 106, 108 perisinusoidal fibrosis decreases the permeability of normal sinusoids, impairing metabolic exchange between hepatocytes and sinusoidal blood further compromising hepatic function. 106 excess fibrous tissue also limits the ability of vessels and sinusoids to distend, resulting in increased resistance to hepatic blood flow and portal hypertension. when fibrotic septae become vascularized, these microscopic communications (between portal vein or arterial artery and hepatic vein) lead to portosystemic shunting of blood. reversal of hepatic fibrosis and improvement in liver function can occur, especially if the underlying cause of injury is treated or removed. 106 examples in human medicine include antiviral drugs for hepatitis b and hepatitis c and prednisone for autoimmune hepatitis. although fibrosis is potentially reversible, cirrhosis for all practical purposes is not, because of the accompanying architectural changes and psss. 109 these ocular complications occur in approximately 20% of naturally infected dogs, and are caused by corneal endothelial damage and antigen-antibody complexes. ich should be suspected in any young, unvaccinated dog with evidence of alf. ich must be differentiated from diseases with similar clinical signs, such as canine distemper, parvoviral enteritis, and hepatotoxicity. abnormalities on the leukogram are common and vary with the clinical stage of infection. during viremia, neutropenia and lymphopenia are often present. neutropenia is also a common a feature of canine parvovirus, a much more prevalent disease of puppies. rebound lymphocytosis and neutrophilia occur in the recovery stages of ich (7 days after infection). biochemical findings are characteristic of acute hepatic necrosis and include increased serum alt and alp activity, and abnormal liver function tests. hyperbilirubinemia is a less-consistent finding. hypoglycemia may complicate the terminal stages of the disease. coagulation parameters are consistent with dic. other potential findings include proteinuria secondary to glomerular damage, abdominal fluid consistent with an exudate, and an increase in protein and mononuclear cells in the cerebrospinal fluid. the clinical diagnosis of ich is usually suspected on the basis of age, vaccination history, clinical signs, and laboratory findings, and is confirmed by liver biopsy or necropsy findings. additional diagnostic tests that are used less frequently include serologic testing, virus isolation, and direct immunofluorescence. therapy for alf caused by ich is primarily supportive care and control of complications that frequently occur such as dic, he, and hypoglycemia. the prognosis in dogs with ich depends on the severity of hepatic necrosis and the incidence of serious complications such as dic. hepatic regeneration and recovery is possible unless widespread coagulation necrosis destroys entire lobules. ich can be effectively prevented by vaccination. canine herpesvirus causes an acute, afebrile, rapidly fatal disease in neonatal puppies (1 to 3 weeks of age). 116 hepatic necrosis is one manifestation of the widespread multiorgan necrosis and hemorrhage that occurs in this systemic viral infection. clinical signs include acute onset of depression, diarrhea, failure to suckle, crying, and abdominal pain in previously healthy puppies. other findings include petechial hemorrhages and vesicles of the mucous membranes. jaundice is rare. seizures and loss of consciousness may be present in the terminal stages, and most pups die within 24 hours of onset of clinical signs. typical gross pathologic findings include focal areas of necrosis and hemorrhage in the liver, kidneys, lungs, and serosal surfaces of the intestines. microscopically, these areas are characterized by foci of necrosis with occasional intranuclear inclusions. neonates are infected by oronasal exposure to the virus in utero or by secretions from an infected bitch or littermates. neonates are particularly susceptible, possibly because of their low body temperature and immature mechanisms for temperature regulation. the diagnosis of canine herpesvirus is primarily based on the history, physical examination, and pathologic findings. laboratory findings are inconsistent but include neutrophilia or neutropenia, and increased serum alt activity. treatment of affected puppies is generally unsuccessful because of the acute fulminant nature of the disease. maintenance of body it may be one of several organ systems involved in a multisystemic disease process such as feline infectious peritonitis (coronavirus), toxoplasmosis, or histoplasmosis (see table 61 -4). infectious agents can be associated with widespread invasion of organs with a large mononuclear phagocytic component, including the liver, spleen, lymph nodes, and bone marrow, although clinically significant liver disease is uncommon. liver biopsy can be diagnostically useful for identification of these organisms in infected animals. etiology infectious canine hepatitis (ich) caused by cav-1 has long been recognized as a cause of acute hepatic necrosis in dogs. 114 this virus is genetically and antigenically distinct from cav-2, a cause of infectious canine respiratory disease. the incidence of clinical disease caused by cav-1 is now very low because of effective vaccination procedures. neutralizing antibodies to cav-1 are also found in mature, unvaccinated dogs, suggesting that natural exposure to the virus is widespread. cav-1 has a special tropism for vascular endothelial cells and hepatocytes. 114 dogs with sufficient immunity (neutralizing antibodies >1 : 500) do not develop clinical signs of disease. susceptible dogs (titer <1 : 4) develop widespread centrilobular to panlobular hepatic necrosis, which is often fatal. distinctive intranuclear inclusions are present in hepatocytes and the endothelium of other tissues. experimentally, dogs with an intermediate titer (between 1 : 16 and 1 : 500) develop chronic hepatitis that can progress to cirrhosis. 115 whether cav-1 is a significant cause of chronic hepatitis under natural conditions is unknown. cav-1 antigen was demonstrated in formalinfixed liver sections from five of 53 dogs with various naturally occurring hepatic inflammatory lesions, suggesting that cav-1 may play a role in spontaneous chronic hepatitis. 46 other attempts to identify cav-1 in dogs with chronic hepatitis have been negative. 44, 47 clinical examination ich is seen most commonly in unvaccinated dogs younger than 1 year of age. clinical signs vary with the stage of disease. dogs that are peracutely ill do not have clinical evidence of hepatic disease but simply become depressed and moribund, and die within a few hours. dogs with a more extended clinical course (5 to 7 days) have signs associated with acute hepatic necrosis that include vomiting, diarrhea, and abdominal pain. a hemorrhagic diathesis may occur during the viremic phase and is manifested by epistaxis, petechial or ecchymotic hemorrhages of the skin, or excessive bleeding from venipunctures. failure of the liver to clear activated clotting factors and impaired hepatic synthesis of clotting factors probably also contributes to development of dic. signs of central nervous system (cns) dysfunction include depression, disorientation, seizures, and coma and have been attributed to he or nonsuppurative encephalitis. common physical examination findings include fever, enlarged tonsils, pharyngitis, laryngitis, cervical lymphadenopathy, and subcutaneous edema of the head, neck, and trunk. hepatomegaly, abdominal pain, and abdominal effusion can occur. jaundice is rare but can develop in dogs that survive the acute fulminant stage of ich. an uncomplicated clinical course lasts approximately 5 to 7 days before recovery begins. unilateral or, less frequently, bilateral corneal edema and anterior uveitis ("hepatitis blue eye") are complications that may become evident during the recovery period. diagnosis of fip should be supported by cytologic or histologic evidence of pyogranulomatous inflammation. the currently recommended "gold standard" for fip diagnosis is immunohistochemistry performed on effusions or lesions containing infected macrophages. 117 the prognosis for recovery is poor. etiology canine leptospirosis is caused by leptospira interrogans sensu lato, with at least 10 serovars appearing to have clinical significance in dogs. 118 serovars canicola and icterohemorrhagica have been included in vaccines for more than 30 years and the incidence of clinical disease from these serovars has decreased accordingly. an epidemiologic shift in serovars causing clinical disease has since occurred, with increasing reports of disease associated with serovars grippotyphosa, pomona, and bratislava. 118 it has been suggested that serovars icterohemorrhagica and pomona are more likely to be associated with hepatic damage. 118 however, other reports have been unable to correlate serogroups with specific clinical features. 119, 120 chronic hepatitis has been associated with serovar grippotyphosa 50 and serogroup australis. 51 reports of clinical leptospirosis in cats are rare, although antibodies to several serovars have been demonstrated. 118 pathophysiology acute renal failure is the most common clinical disease syndrome in dogs with leptospirosis. 121 the liver can also be a target organ and alf may occur concurrently in 10% to 20% of dogs with acute renal failure or independent from renal involvement. 121 with acute hepatic involvement, the liver is enlarged, friable, and yellowbrown. tissues are often markedly jaundiced. microscopic changes in the liver include intrahepatic cholestasis, liver cell dissociation, and nonspecific reactive hepatitis. 3, 122 hepatic necrosis is an uncommon histologic feature. the liver may not show striking changes, presumably because hepatic dysfunction can be caused by a toxin that produces mainly subcellular damage. organisms can be identified in tissues with a warthin-starry stain. common clinical signs include anorexia, depression, and vomiting. hepatocellular involvement is suggested by jaundice. other findings may include arthralgia or myalgia, pu, pd, fever, and dehydration. widespread petechial and ecchymotic hemorrhages of the mucous membranes, sclera, and skin are caused by thrombocytopenia and dic. the terminal stages include signs of cardiovascular collapse, shock, coma, and death. a diagnosis of leptospirosis should be considered in dogs with acute cholestatic liver disease, especially when accompanied by acute renal failure. hematologic findings vary with the stage and severity of disease. leukocytosis and left shift are frequent, but in the early stages of leptospiremia, leukopenia is more likely. thrombocytopenia can also be seen. coagulation parameters are normal unless complicated by dic. routine serum chemistry and urinalysis findings reflect involvement of the liver or kidney. serum liver enzyme activity is usually increased with hepatic involvement, and the magnitude of the increase in serum alp activity is usually greater than that of serum alt activity owing to intrahepatic cholestasis. other findings include hyperbilirubinemia, bilirubinuria, and abnormal liver function tests. an increase in bun or creatinine may result from renal failure or prerenal uremia. the urinalysis is often compatible with acute nephritis with findings of proteinuria and increased temperature (36.7°c to 37.8°c [98°f to 100°f]) may be helpful. intraperitoneal infusion of 1 to 2 ml of hyperimmune serum obtained from bitches with previously infected litters may reduce mortality rates. vaccination for herpesvirus infection is not routinely performed because of the low incidence of disease. canine acidophil cell hepatitis, which encompasses a spectrum of hepatic lesions ranging from acute and chronic hepatitis to cirrhosis and liver failure, has been reported in great britain. 48, 114 it is caused by a transmissible agent, suspected to be a virus that is distinct from cav-1, although a specific virus has never been identified. the disease is experimentally transmissible by serum or liver extracts from affected dogs. in the experimentally induced disease, acute hepatitis can progress to chronic hepatitis in the absence of clinical signs. episodic increases in serum alt activity and fever spikes correspond with histologic evidence of acute hepatitis. the liver is enlarged and friable in the acute stages, and becomes progressively smaller and nodular with chronicity. the most notable histologic feature, regardless of the stage of disease, is the acidophil cell. acidophil cells are dying hepatocytes with an angular shape, reduced volume, hyperchromatic nucleus, and strongly acidophilic cytoplasm caused by small acidophilic coalescing granules. end-stage hepatic disease is accompanied by typical findings of cirrhosis. most dogs with spontaneous disease are presented with signs of chronic hepatic failure. the duration of clinical signs can exceed 1 year. based on experimental studies, it is speculated that the early mild stages may go unrecognized until advanced hepatic disease and failure is present. biochemical findings are consistent with hepatic inflammation and necrosis, evidenced as increased serum alt activity. with advanced disease, severe hepatic dysfunction is noted. the diagnosis requires liver biopsy. recommendations for specific therapy of acidophil cell hepatitis await further information on the causative agent. supportive measures should be instituted as needed. feline infectious peritonitis (fip) is a highly fatal coronaviral infection of both domestic and wild cats. the liver is one of many organs (kidneys, spleen, pancreas, mesenteric lymph nodes, cns, uveal tract, omentum, serosal surfaces) that can be affected by widespread immune complex vasculitis and granulomatous or pyogranulomatous inflammation (see table 61 -5). 117 clinical findings in cats with hepatic involvement are nonspecific and include lethargy, depression, anorexia, dehydration, weight loss, and fever. jaundice is a common finding. extrahepatic findings include nodular renomegaly, abdominal mass (lymph node), and ascites or dyspnea (pleural effusion). ophthalmoscopic examination may detect chorioretinitis or anterior uveitis, which must be differentiated from similar ocular changes seen with the other systemic disorders that involve the liver such as lymphosarcoma, toxoplasmosis, and the systemic mycoses. serum hepatic enzyme (alp and alt) activities are usually normal or only mildly increased. mild to moderate increase in serum bilirubin concentration is common. other findings indicating hepatic dysfunction include bilirubinuria and increased sba concentrations. hyperglobulinemia, neutrophilia, and mild to moderate nonregenerative anemia are other laboratory features of fip. abdominal and pleural effusions, when present, are usually pyogranulomatous exudates with greater than 3 g/dl protein. serologic detection of a high coronaviral antibody titer may support a diagnosis of fip, but is not definitive because of its lack of specificity. the serum bile acid concentration can be markedly increased (>200 µmol/l). 124 extrahepatic bacterial infection-induced hepatic damage should be considered when evidence of cholestatic hepatopathy is found concurrently with extensive bacterial infection in other organ systems (e.g., pyometra, peritonitis) or with extrahepatic disorders likely to be associated with endotoxemia (e.g., parvoviral enteritis). associated clinical findings that would be compatible with endotoxemic crisis include shock, fever or hypothermia, hypoglycemia, neutrophilia or neutropenia with left shift, toxic changes of the neutrophils, and hyperbilirubinemia that is disproportionately increased in comparison to serum alp activity. 124 it is important to recognize that jaundice can occur secondary to extrahepatic infection from a diagnostic standpoint, so that the clinician is not misled into considering that the cause is a primary hepatic or biliary disease. in jaundiced patients with evidence of an inflammatory process, key differential diagnoses include acute pancreatitis, extrahepatic bacterial infections, and primary hepatobiliary disorders such as leptospirosis (dogs only), cholangiohepatitis, cholecystitis, ruptured gallbladder mucocele, and hepatic abscesses. specific therapy for hepatic disease is not usually required, and hepatic damage is reversible with control of sepsis. morbidity is related to the underlying disease process and not overt hepatic failure. etiology hepatotoxicity can be caused by a variety of drugs (prescription or over-the-counter), herbal and dietary supplements, or biologic toxins or chemicals (see box 61-1). 17, 126, 127 the liver is uniquely susceptible to xenobiotic substances because it is directly exposed to them following absorption from the gi tract. the liver is also vulnerable to toxic injury because it plays a central role in the metabolism of many substances. hepatic metabolism renders lipophilic substances more hydrophilic, which promotes excretion via the urine or bile. 17 the process is controlled by phase i and phase ii reactions. phase i reactions are catalyzed by the cytochrome p450 enzyme systems, which activate or detoxify (oxidize, reduce, or hydrolyze) a drug or toxin. phase i reactions may lead to generation of unstable chemically reactive intermediates, which can be toxic. phase ii reactions conjugate drugs or metabolites and produce products that are nontoxic. during biotransformation, the liver can either reduce or enhance the toxicity of the parent compound. for example, after carbon tetrachloride ingestion, the liver converts the nontoxic parent compound into toxic metabolites, which subsequently cause severe hepatocellular damage. genetic polymorphisms of phases i and ii enzymes have the potential to influence drug metabolism in the individual animal. there has been an increased awareness and recognition that drug-induced liver injury can be a significant cause of liver disease in dogs and cats (see box 61-1). this information has been gained from isolated case reports, retrospective clinical studies, and experimental studies. unfortunately, for many of these drug reactions, characterization of the clinical and pathologic features are lacking because only small numbers of affected animals have been described, and liver biopsies are not typically obtained when drug withdrawal results in clinical and biochemical improvement. it is possible that drug-induced liver injury is underrecognized in dogs and cats, as in humans, drug-induced hepatic injury accounts for more than 50% of the cases of alf in the united states, and is the most frequent reason cited for withdrawal of an approved drug from the market. 17 leukocytes, erythrocytes, and granular casts. increased serum creatine kinase activity may indicate leptospiral-induced muscle damage. leptospirosis is most easily diagnosed in the clinical setting by demonstration of a fourfold rise in serum antibody titer (microscopic agglutination test) in paired samples taken at initial presentation and 2 to 4 weeks later. the rise in titer indicates recent or active infection and differentiates a titer from previous exposure or previous vaccination. the optimum treatment regimen for leptospirosis is unknown. traditionally, intravenous ampicillin, 25 mg/kg every 6 hours (with dose reduction in dogs with renal failure), or penicillin g (25,000 to 40,000 units/kg iv q12h), has been used for initial treatment of leptospirosis. 121 doxycycline (5 mg/kg po q12h for 2 to 4 weeks), was recommended as followup therapy to eliminate organisms from renal tubules. however, doxycycline, 5 mg/kg orally or intravenously every 12 hours for 2 weeks, appears to be effective in clearing all phases of leptospiral infection and may be the most effective treatment strategy. 121 management of fluid, electrolyte, and acidbase imbalances is important supportive therapy. the prognosis generally depends on the degree of renal dysfunction and is poor when oliguria develops. c. piliforme (formerly known as bacillus piliformis), a spore-forming gram-negative bacteria, is a rare cause of multifocal hepatic necrosis and necrotizing ileitis in dogs and cats. 123 the infection is mainly opportunistic in stressed or immunocompromised animals with a predisposing disorder (e.g., canine distemper, feline panleukopenia, feline leukemia) or familial hyperlipoproteinemia in kittens. 123 clinical signs include an acute onset of anorexia, lethargy, depression, and abdominal discomfort. jaundice may be observed, especially in cats. these signs rapidly progress to a moribund state; death occurs within 24 to 48 hours. marked increases in alt activity may be detected. histopathology reveals multifocal periportal hepatic necrosis and necrotic ileitis or colitis. bacilli are best seen with special staining techniques (warthin-starry or giemsa), or methylene blue-stained impression smears of fresh tissue. organisms appear as large, slender, intracellular filamentous organisms within hepatocytes surrounding areas of necrosis and in intestinal epithelial cells. routine culture techniques are ineffective for isolation of this organism. the disease is rapidly fatal, and successful therapy has not been reported. 123 extrahepatic bacterial infection associated with sepsis and endotoxemia is an important cause of acute functional cholestatic hepatopathy. however, morbidity is generally related to the underlying infection and not overt hepatic failure. 113, 124 studies in humans and experimentally in dogs, suggest that endotoxemia and the subsequent release of cytokines induces functional changes that interrupt the transport and excretion of conjugated bilirubin. 113, 125 microscopically, hepatic lesions are often mild and nonspecific. intrahepatic cholestasis, characterized by bile canalicular plugs and bile pigment accumulation in hepatocytes, is the most consistent finding. 124 a mild periportal lymphocytic infiltrate can be seen, with scattered foci of macrophages or neutrophils, and occasional individual necrotic hepatocytes. total serum bilirubin concentrations as high as 30 mg/dl can be seen and are disproportionately high compared to the mild to moderate increases in serum alp activity. increased serum alt activity is a less consistent finding. hepatic injury caused by drugs, herbal and dietary supplements, biologic toxins, or chemicals can occur via a number of mechanisms, which influence the histologic pattern of disease. 17, 20 although hepatic necrosis is the most common histologic response, drug-and toxin-induced liver injury can also potentially mimic the full spectrum of acquired hepatic disorders, including acute and chronic hepatitis, granulomatous hepatitis, cholestatic hepatopathy, vacuolar hepatopathy (lipid or glycogen accumulation), hepatic fibrosis and cirrhosis, and venoocclusive disease. hepatic necrosis occurs when covalent binding of a drug or toxin to intracellular proteins disrupts cellular functions, or formation of drug-enzyme adducts stimulates an immunologic response (antibody-or t-cell-mediated cytotoxicity). hepatic inflammation (typically lymphoplasmacytic, but may be granulomatous or eosinophilic), suggests an underlying immunoallergic mechanism. intrahepatic cholestasis occurs when drugs or toxins interfere with hepatic transport proteins at the canalicular membrane, which interrupts bile flow. hepatic lipid accumulation results when direct damage to mitochondria disrupts fatty acid oxidation and energy production, and is the predominant hepatic lesion seen with stanozolol hepatotoxicity in cats, 132 and tetracycline in dogs and cats. 126 hepatic vacuolation caused by glycogen accumulation occurs in dogs treated with corticosteroids, and typically does not cause significant clinical evidence of hepatic dysfunction. damage to the sinusoidal epithelium can result in peliosis hepatic or venoocclusive disease. mechanisms of drug-induced liver injury (and also injury from herbal and dietary supplements) can be characterized as either intrinsic (predictable) or idiosyncratic (unpredictable) reactions. 126 intrinsic hepatotoxic reactions are dose-related and occur shortly after a consistent threshold of toxicity is reached. because intrinsic hepatotoxins predictably damage the liver in an exposed population, they can be experimentally reproduced and studied. hepatic injury is caused by a direct toxic effect of the parent compound (or a reliably generated toxic metabolite) on vital cell targets. acetaminophen is an intrinsic hepatotoxin in dogs and cats and is discussed in more detail later in this section. with intrinsic hepatotoxins, lowering the dose, rather than stopping the drug can be tried. in many cases, most such drugs or chemicals, (e.g., carbon tetrachloride, phosphorus, and chloroform), are no longer used for therapeutic purposes once their intrinsic hepatotoxicity is recognized, but accidental exposures could still occur. in contrast, idiosyncratic hepatotoxic reactions occur at therapeutic doses in only a small number of individuals in the exposed population. these reactions are unpredictable and infrequent; most individuals treated with the drug do not have a reaction, even at high doses. however, toxicity may be more pronounced at higher doses in susceptible individuals. 72, 133 examples of drugs causing idiosyncratic drug-induced liver injury in dogs and cats that have a dose-related effect are phenobarbital, itraconazole, amiodarone, and lomustine (ccnu). 134 idiosyncratic reactions are characterized by a variable latency period (5 to 90 days, but may be longer for some drugs, such as phenobarbital, ccnu, amiodarone) from initial drug ingestion to recognition of hepatic injury. because of the infrequent occurrence (approximately 1 in 100,000), the potential for hepatotoxicity may not be recognized in preclinical screening of a new drug, and cannot usually be reproduced in an experimental setting. individual differences in susceptibility to idiosyncratic drug-induced liver injury may reflect genetic differences in either (a) alternate metabolic pathways by which a drug is converted to different (potentially hepatotoxic) metabolites, (b) the ability of the individual to detoxify the toxic intermediates, (c) an underlying the center for veterinary medicine of the food and drug administration, washington, dc, maintains a registry (http://www.fda.gov/ animalveterinary/default.htm) for reporting adverse drug reactions in animals. this service has been useful to accumulate data and to alert veterinarians to suspected drug effects, including hepatotoxicity. however, because reporting of adverse drug reactions is voluntary and the information obtained may be incomplete, only subjective trends can be identified. furthermore, because mild hepatic injury may not be associated with clinical signs, these cases will not be detected unless biochemical testing is performed while the animal is receiving the drug. evaluating the incidence of druginduced liver injury in dogs and cats is further clouded by the fact that there are no pathognomonic clinical, laboratory, or biopsy findings to distinguish drug-induced liver injury from other causes of liver disease. specific drugs that have been reported to cause druginduced liver injury are listed in box 61-1. herbal and dietary supplements (herbs or other botanicals, nutraceuticals, vitamins, minerals) have the potential to cause hepatotoxicity, similar to drug-induced injury. [127] [128] [129] in humans, herbal and dietary supplements are reported to account for 10% of patients with "drug-induced" liver injury. 127 the incidence of hepatotoxicity may be underrecognized, as only a third of humans taking herbal and dietary supplements reported their use of these products to their health care provider. 127 causality is difficult to prove, because herbal and dietary supplements are often dispensed without medical supervision, fda oversight of product quality is minimal, multiple active ingredients contribute to product variability, and product contamination with hepatotoxic substances (toxic herbs or heavy metals) may occur. [127] [128] [129] drug interactions between herbal and dietary supplements and prescription medications may also occur, because medicinal plants can have effects on hepatic p450 enzyme systems. 129 the rate of dietary supplement use in dogs and cats appears to be lower than that reported for humans. 130 however, because these products are often marketed as "all natural," pet owners may assume they are safe and underreport their use to the veterinarian. reports of herbal and dietary supplement hepatic injury in animals are rare. pennyroyal oil, a volatile oil derived from plants of the labiatae family (pennyroyal, squaw mint, or mosquito plant) was associated with fatal acute hepatic necrosis in a dog after topical application for use as a flea repellent. clinical signs occurred within 1 hour after application and included vomiting, diarrhea, hemorrhage, seizures, and death. other reports include the finding of increased liver enzyme activity in dogs consuming st. john's wort, and increased alt activity and hypoglycemia after accidental ingestion of high doses of α-lipoic acid in two dogs. 129, 131 the paucity of reported hepatic reactions does not necessarily mean herbal and dietary supplements are safe, and the clinician should maintain a high level of suspicion regarding their potential toxicity. box 61-1 lists the herbal and dietary supplements that have been incriminated as causing hepatic injury in humans with potential relevance to dogs and cats. for more detailed information on hepatic injury and herbal and dietary supplements, additional sources should be consulted. [127] [128] [129] hepatic injury may also occur after exposure to a wide variety of industrial chemicals, organic solvents, pesticides, heavy metals, and biologic toxins. most information on chemical hepatotoxins is derived from experimental studies in dogs or extrapolated from information in other species. very few clinical case reports are available in the veterinary literature. isolated reports and case series of clinical liver disease in dogs associated with exposure to biologic toxins, such as aflatoxin, amanita mushrooms, blue-green algae, and cycads (sago palms) have been published. selected hepatotoxins are listed in box 61-1. increased liver enzyme activity is a common finding with hepatotoxicity. increased alt activity (more than three times the upper limit of normal, but can be as high as 100 times the upper limit), suggests hepatocellular injury (often necrosis), and is of more concern than an isolated increase in alp activity (reflecting cholestasis), although mixed patterns commonly occur. progressive increases in alt activity or those accompanied by evidence of hepatic dysfunction (hyperbilirubinemia, increased serum bile acids, coagulopathy, hypoglycemia, hyperammonemia, hypoalbuminemia) are more likely to represent serious hepatic injury. when drug-induced liver injury is suspected, the diagnostic approach is determined by the clinical presentation. if clinical signs are absent or mild, a minimum database consisting of complete history and physical examination, complete blood cell count, serum chemistry, and urinalysis should be performed. if the only abnormality detected is increased liver enzyme activity, and these increases correspond to the recent administration of a drug (especially those listed in box 61-1), the drug should be discontinued and serum biochemistries should be repeated in 10 to 14 days. in many instances, clinical and biochemical abnormalities resolve after the suspected hepatotoxic drug is discontinued and a liver biopsy is not performed. further evaluation of the liver, including sba concentrations, abdominal radiographs, ultrasonography, and liver biopsy, may be warranted if biochemical abnormalities persist, or if initial clinical and biochemical findings suggest hepatic dysfunction. with suspected drug-or toxin-induced liver disease, a liver biopsy can be helpful to (a) characterize the histologic changes (are they consistent with previously described lesions caused by this particular drug or toxin?), (b) determine the severity (focal or diffuse necrosis?) or reversibility (is cirrhosis present?) of the lesions for prognostic purposes, and (c) rule out known causes of liver disease. histologic changes secondary to drug-and toxin-induced hepatic injury are nonspecific and similar to those seen with other nondrug-related causes of acute and chronic liver disease. 126 the most common pathophysiologic response is necrosis without inflammation. 126 hepatic necrosis may be centrilobular (zone 3) or panlobular (figure 61-20) . centrilobular hepatocytes have an abundance of p450 enzymes, and are preferentially affected in drug-induced hepatotoxicity when p450 metabolism of the parent drug results in toxic metabolites. 8 drugs or toxins can also cause a variety of other hepatic lesions, such as cholestasis, lipidosis, or mild inflammation. a chronic response to injury is reflected by findings of biliary immunologic or allergic reaction, or (d) an individual's tolerance or ability to "adapt" to hepatocellular injury (mechanisms unknown) with resolution of injury despite continued medication administration. 20, 135 oral medications with substantial hepatic metabolism are more likely to be associated with adverse hepatic events in humans, presumably because of hepatic generation of reactive toxic metabolites. 136 because of the unpredictability of an idiosyncratic reaction and the low incidence of occurrence, a cause-and-effect relationship is difficult to establish. if an idiosyncratic reaction occurs, the drug must be discontinued or it could result in death of the patient. an idiosyncratic mechanism is suspected for most of the drugs that cause hepatic injury in dogs and cats. susceptibility to hepatotoxicity in humans is influenced by a number of factors such as age, sex, nutritional status, and concurrent drugs. 20 the most important factor may be the effect of genetic polymorphisms on hepatic drug metabolism. 20 in humans, preexisting liver disease does not appear to enhance susceptibility to druginduced liver injury, but impacts the patient's ability to recover. 20 this may be because drug-metabolizing enzyme systems are remarkably preserved in hepatic disease. similar information on risk factors for hepatotoxicity in dogs and cats has not been determined. however, because many toxic metabolites are normally detoxified by glutathione, some metabolites may become more toxic when hepatic glutathione stores are depleted (e.g., animals with preexisting chronic necroinflammatory and cholestatic liver disease). 126 a breed predisposition has been suggested for doberman pinschers (sulfonamides, amiodarone, diethylcarbamazine/oxibendazole) and labrador retrievers (carprofen), which may be a reflection of a genetic predisposition. 21, 71, 73, 134, 137 clinical examination the spectrum of drug-and toxin-induced liver injury and, thus, the associated clinical presentation, can vary from subclinical hepatic injury with only increased serum liver enzyme activity, to severe liver damage manifested as alf, or chronic end-stage liver disease. acute rather than chronic liver injury is more likely for most of the drugs and toxins listed in box 61-1. clinical features often include acute onset of lethargy, anorexia, vomiting, diarrhea, pu, pd, or jaundice in a previously healthy animal, which corresponds to hepatotoxin exposure. alf (signs of acute liver disease plus he and coagulopathy) is most likely with drugs or toxins that cause diffuse hepatic necrosis. drug-and toxin-induced injury is an important diagnostic consideration in dogs and cats presenting for acute hepatitis and hepatic necrosis. drugs or toxins also have the potential to cause chronic hepatic disease, if the initial hepatic injury is mild and goes unrecognized, and exposure to the drug or toxin is continued. for example, phenobarbital, ccnu, or chronic aflatoxicosis can cause chronic liver injury in dogs. 70, 72, 138 diagnosis there are no pathognomonic clinical, laboratory, or biopsy findings that distinguish drug-or toxin-induced liver injury from other causes of liver disease. the diagnosis of drug-induced liver injury often relies on the clinician maintaining a high level of suspicion, and obtaining an accurate and thorough medication history (including prescription and over-the-counter drugs, and herbal and dietary supplements), in every animal with unexplained increases in liver enzyme activity or clinical liver disease. a definitive diagnosis of hepatic injury caused by biologic toxins or chemicals is rarely possible in a clinical setting, unless the owner specifically observes ingestion of a substance that is a known hepatotoxin. prevented. treatment of drug-induced hepatic disease consists of discontinuing the suspect drug. after a drug is discontinued, clinical (and biochemical) improvement usually occurs within a few weeks, even with chronic drug administration. however, exceptions can occur. for example, in dogs with amiodarone toxicity, liver enzyme elevations can transiently progress despite discontinuation of the drug, and biochemical abnormalities may not resolve for 6 to 8 weeks. 137 with the exception of nac for acetaminophen, and silymarin for amanita mushroom toxicity, no specific antidotes are available, and treatment of drug-and toxin-induced liver injury is primarily supportive and symptomatic. however, nonspecific hepatoprotective therapy with antioxidants (vitamin e), glutathione replacement (nac, same), or milk thistle (silymarin) may be helpful and are discussed in more detail in chapter 46. use of nac (or same) may be beneficial, as glutathione depletion may predispose to hepatotoxicity (e.g., methimazole) or impair metabolism of toxic metabolites to a nontoxic form. nac has been suggested to be beneficial for treatment of alf associated with toxicities such as diazepam, methimazole, carprofen, and trimethoprim-sulfa. 9 in addition to treating amanita mushroom hepatotoxicity, silymarin may be beneficial in the treatment of carbon tetrachloride and acetaminophen toxicity. 9,140,141 corticosteroids are not typically indicated for treatment of drug-and toxin-induced hepatotoxicity. it is important to consider a drug-induced cause of liver injury because rapid recognition and prompt discontinuation of an hepatotoxic drug can lead to improvement or complete resolution of hepatic disease, depending on the specific drug and the stage of the lesion. when drug-or toxin-induced hepatic injury causes severe or widespread hepatic necrosis, rapid deterioration and death in 3 to 4 days often occur. with less-severe hepatic injury, complete recovery is possible. acetaminophen acetaminophen is well known as an intrinsic hepatotoxin in dogs and cats. 142, 143 although acetaminophen is occasionally used as an analgesic in dogs (therapeutic doses up to 15 mg/kg tid), most toxicity occurs because of accidental ingestion of improperly stored medication (dogs) or owner administration without veterinary supervision (dogs and cats). 142 toxic metabolites of acetaminophen cause oxidative injury to erythrocytes and hepatocytes, resulting in methemoglobinemia, anemia, and hepatic necrosis. in therapeutic doses, acetaminophen is detoxified by a combination of hepatic glucuronidation and sulfation and renal excretion. 144 after acetaminophen overdosage, these pathways become saturated and a greater proportion of acetaminophen is metabolized through the p450 system, leading to production of the toxic metabolite, n-acetylp-benzoquinoneimine (napqi). glutathione detoxifies napqi and thus protects hepatic cellular constituents from its direct toxic effect. however, once glutathione levels are depleted by large amounts of napqi, centrilobular necrosis occurs. toxicity occurs in a dose-dependent manner. there are substantial species differences in both the metabolism of acetaminophen and the toxic manifestations. 142 cats are uniquely sensitive to acetaminophen because of a deficiency of glucuronyl transferase and limited sulfation capabilities. clinical signs in cats may develop after administration of as little as 162.5 mg ( 1 2 tablet). signs of methemoglobinemia usually dominate the clinical picture, such as cyanosis, dyspnea, facial edema, depression, hypothermia, hyperplasia, fibrosis, and cirrhosis. although individual drugs and drug classes may follow the same pattern, there is often not a consistent reaction for any given drug. for example, hepatic injury in dogs secondary to potentiated sulfonamides may cause hepatic necrosis, primary cholestasis, or marked inflammation. 21 for many of the potentially hepatotoxic drugs listed in box 61-1, histologic features have not been fully characterized. it should be emphasized that for most drug-induced disorders, the diagnosis is presumptive and cannot be proved. it can be especially difficult to pinpoint the causative agent when the patient is receiving a combination of drugs. a clinical diagnosis of drug-induced hepatic injury is easier to establish when the hepatotoxicity of the drug has been previously described and the associated clinical and pathologic features have been characterized (see discussion of specific drugs). the diagnosis may be less convincing when the suspected drug has not been previously incriminated as causing liver damage. however, a drug reaction should still be considered, since an idiosyncratic reaction could occur with any drug. the clinician should also maintain a level of suspicion regarding the potential for hepatotoxicity in newly marketed drugs that have not yet been used widely in the population, where idiosyncratic reactions are often first detected. the suspected hepatotoxic drug should be discontinued while a complete diagnostic evaluation is pursued for other causes of liver disease, for which a specific treatment might be available. a diagnosis of drug-induced injury is supported by the following: (a) evidence of liver injury that occurred within the first 3 months of drug therapy (especially if predrug liver enzyme activity was within normal limits); (b) clinical and biochemical improvement when the drug is discontinued; (c) exclusion of other causes of liver disease; (d) reccurrence of hepatic damage after a challenge dose of the same drug (or inadvertent reexposure). it should be emphasized that rechallenge with a suspected hepatotoxic drug is not recommended as a diagnostic consideration, because it is potentially dangerous, especially with a drug that causes acute hepatic necrosis. rechallenge should only be considered if the association of the drug with hepatic injury is highly questionable and there is no alternative drug available for a significant medical condition. with hypersensitivity or immunologic reactions, the hepatic reaction is more rapid and severe with repeated exposure. 20 hepatic injury because of a biologic toxin or chemical is suspected when exposure to a potential hepatotoxin has been documented. a clinical diagnosis of "toxic" hepatic injury is often made when an episode of acute hepatic injury occurs, hepatic biopsy indicates diffuse hepatic degeneration and necrosis, and no other cause for liver disease can be identified. in selected cases, tissues, blood, or food (aflatoxin) samples can be submitted to a toxicology lab to confirm a suspected toxin. toxin-induced injury should also be considered in the absence of known exposure to toxins, because potential hepatotoxins can be present in contaminated dog food or garbage (aflatoxins), pond water (blue-green algae), and many other unobserved sources. when ingestion of a potential hepatotoxin (e.g., toxic mushrooms, sago palms) has occurred within the preceding 8 hours, general procedures for gi decontamination are recommended, including induction of emesis or gastric lavage (within first 3 hours), followed by administration of activated charcoal (1 to 3 g/kg). 126, 139 induction of vomiting is contraindicated if the patient is comatose or debilitated in such a way that the gag reflex is diminished, which could predispose to aspiration pneumonia. whenever possible, the source of toxin exposure should be identified and further exposure or given as alternate-day therapy), with careful monitoring of liver parameters every 2 weeks. 146 hepatotoxicity appears to be at least partly dose related, as dogs receiving higher daily doses of itraconazole (10 mg/kg) are more likely to be affected. icterus and evidence of hepatic dysfunction suggests a more serious, potentially fatal hepatopathy, requiring discontinuation of medication and symptomatic and supportive care. it is recommended that liver enzymes be monitored on a monthly basis in all animals receiving ketoconazole or itraconazole. azathioprine, a purine analogue commonly used for treatment of immune-mediated disorders in dogs, is commonly listed as a potential hepatotoxin. 126 however, few clinical details regarding the hepatotoxic reaction are available. in a clinical study of 12 dogs with atopic dermatitis treated with azathioprine (2.2 mg/kg daily for 8 weeks) as a single agent, an increase in alt or alp activity was noted in the first 2 weeks in 10 (83%) of the dogs. 147 three dogs had clinical signs suggestive of liver disease, which resolved uneventfully when azathioprine was discontinued. in an experimental study of dogs given azathioprine at a dose of 2 to 4 mg/kg po daily for 40 days, all dogs had increased liver enzyme activity (alt >> alp) within 2 to 7 days of initiating therapy. 148 values peaked within the first 2 weeks and then declined, but not to normal, despite continued medication administration. hyperbilirubinemia was absent. liver biopsies in most dogs revealed centrilobular degeneration and necrosis, with intrahepatic cholestasis but no inflammation. 148 these findings raise the possibility that azathioprine may be an intrinsic (dose related) hepatotoxin in dogs, with possible adaptive tolerance to liver injury. it should be noted that doses used in this study exceeded current clinical recommendations of 1 to 2 mg/kg daily or every other day for maintenance therapy. hepatotoxicity is considered a class characteristic of nsaids, despite the fact that there are many different chemical classes of nsaids, and no consistent mechanism of liver injury. 149 with the exception of aspirin, which is an intrinsic (dose-related) hepatotoxin, the mechanism with other nsaids is believed to be idiosyncratic (either immune or as a consequence of toxic metabolites). 149, 150 toxicity does not appear to be related to prostaglandin inhibition like the renal or gi side effects. 149 preexisting hepatic disease has not been shown to be a risk factor for nsaid-induced liver injury. 150 all nsaids have the potential to cause idiosyncratic hepatotoxicity in dogs, but hepatic reactions appear to be rare. 150 carprofen has specifically been reported as a cause of drug-induced liver injury in dogs. 73 labrador retrievers were overrepresented in the series, but it is not clear whether this is a true breed predisposition. 150 clinical signs (anorexia, lethargy, vomiting, pu/pd) occurred within the first 4 weeks of therapy and icterus was a common finding on physical examination. biochemical evaluation revealed marked increases in liver enzymes (alt activity usually exceeded alp activity) and hyperbilirubinemia. hepatic biopsy findings revealed multifocal to diffuse hepatic necrosis, mild to moderate lymphocytic-plasmacytic inflammation, secondary cholestasis, and variable biliary hyperplasia and bridging fibrosis. 73 concurrent renal toxicity (glucosuria without hyperglycemia, proteinuria, granular casts) also was noted in some dogs. most dogs recovered with discontinuation of carprofen and appropriate supportive care, although some dogs died of alf. general hepatoprotective therapy with same or silybin has been recommended, although the benefits are unproven. nac has been suggested for ancillary treatment when carprofen causes alf. 128 and vomiting. although increases in serum alt activity may be detected, centrilobular hepatic necrosis appears to be uncommon. clinical signs in dogs are more likely when doses exceed 200 mg/kg and can be indicative of methemoglobinemia and/or centrilobular necrosis. 142 laboratory features include methemoglobinemia, anemia, increased serum alt activity, and hyperbilirubinemia. intravenous nac is the treatment of choice for acetaminophen toxicity in dogs and cats. 142 nac increases the synthesis and availability of glutathione, which when conjugated to napqi, decreases toxicity. for maximum effectiveness, nac should be given within 12 hours of acetaminophen exposure; however, there may still be a benefit if given 36 to 80 hours after exposure. nac (10% solution) is diluted 1 : 2 or more with saline and given intravenously through a nonpyrogenic 0.25 µm filter at an initial dose of 140 mg/kg over a 20-to 30-minute period. a maintenance dose of 70 mg/kg is given iv or orally every 6 hours for seven treatments. same also serves as a glutathione source and has been shown to have protective effects against acetaminophen-induced oxidative stress on the erythrocytes in cats and dogs. 143, 145 in an experimental study in cats, silymarin (30 mg/kg po) was as effective as nac for treatment of acetaminophen toxicity when given up to 4 hours after exposure. 140 vitamin c (30 mg/kg iv q6h) may be helpful in the treatment of acetaminophen toxicity because of its antioxidant effects. cimetidine (5 mg/kg iv q8h) is also recommended as adjunctive therapy in the early stages (first 16 hours) because it inhibits hepatic p450 enzymes and decreases napqi formation. the antiarrhythmic drug amiodarone is associated with a reversible hepatotoxicity in dogs. 134, 137 doberman pinschers may be at increased risk. 134 toxicity, which appears to be at least partially dose related (doses of 400 mg/day), was identified in 45% of doberman pinschers treated with amiodarone in one clinical series. 134 clinical signs (anorexia, lethargy, vomiting, diarrhea) and biochemical abnormalities (increased alt and alp activity ± hyperbilirubinemia) developed 6 days to 8 months after initiation of therapy. liver biopsy in one dog revealed multifocal hepatocellular necrosis with mild lipidosis and lymphoplasmacytic inflammation. 137 clinical improvement usually occurs within a few days of stopping the drug, but liver enzyme elevations may not return to normal for 3 months. 134 transient progression of enzyme abnormalities despite discontinuing the drug has also been noted, which may reflect the long half-life of amiodarone causing a delay in systemic elimination. 137 biochemical changes precede clinical signs, so monitoring of liver enzymes at least monthly is recommended. the azole antifungal drugs ketoconazole and itraconazole (and rarely fluconazole) are associated with increased liver enzyme activity and icterus in dogs and cats. 146 hepatotoxicity is more likely with ketoconazole than with itraconazole. cats are more sensitive to the hepatotoxic effects than are dogs, but considerable individual variation occurs. histologic findings are poorly characterized but include bile duct proliferation and infiltration of mononuclear cells. transient mild subclinical elevations of liver enzymes (alt and alp activity) are common, and do not necessarily require a change in therapy. a clinically significant hepatic reaction is suggested by alt activity that exceeds two to three times the upper limit of normal, especially when accompanied by clinical signs of anorexia and vomiting. drug therapy should be stopped for 1 to 2 weeks until appetite and liver enzymes return to normal. a rapid recovery usually occurs, and treatment can be restarted at a lower dose (50% of previous dose decreased appetite, weight loss, pu/pd, vomiting, ascites, and pleural effusion. ascites was due to a combination of hypoalbuminemia and portal hypertension. common biochemical abnormalities included increased liver enzyme activity (alt, ast, alp, ggt) and hypoalbuminemia. other less-consistent findings included hyperbilirubinemia, hypercholesterolemia, and increased serum bile acid concentrations. glucosuria (without hyperglycemia) and renal failure were noted in some dogs, possibly attributable to ccnu renal toxicity. 72 liver biopsy findings were nonspecific (hemosiderinladen kupffer cells, hepatocellular vacuolization, mild to moderate periportal inflammation, and fibrosis) but suggested chronicity. the majority of affected dogs died from progressive chronic liver disease. in a recent study, routine monitoring of alt activity prior to each subsequent dose of ccnu suggested that subclinical elevations of alt activity (greater than five times the upper limit of normal) are common. 155 thirty-two of 109 dogs (29%) had increased alt activity, which developed most commonly after one to three doses of ccnu. 155 increases in alt activity were not associated with cumulative dose. the lower incidence of clinical hepatotoxicity in this study (3 of 109 or 2.8%) versus the kristal study (11 of 179 or 6.1%), was attributed to prompt cessation of ccnu treatment in dogs with significant increases in alt activity. 155 however, it was noted that chronic administration of ccnu could be associated with chronic irreversible hepatopathy, in the absence of a significant alt elevation. the mechanism of hepatotoxicity is suspected to be a result of generation of toxic intermediate metabolites (e.g., isocyanates, diazonium hydroxide), and depletion of glutathione may play a role. 126 preliminary results of a clinical study using denamarin (same and sylibin; nutramax labs, lancaster, sc) for prevention of ccnu hepatotoxicity, suggested that dogs receiving denamarin had less-severe liver enzyme elevations. 156 methimazole, an antithyroid drug, is associated with hepatic injury in cats with hyperthyroidism. clinical findings include anorexia, vomiting, lethargy, jaundice, markedly increased serum liver enzyme activity, and hyperbilirubinemia that usually occurs within the first month of therapy. 126 histologic lesions have not been fully characterized, although biopsy findings in one cat revealed hepatic degeneration and necrosis. clinical signs resolve within a week of discontinuing therapy but biochemical resolution may take up to 45 days. reduced hepatic glutathione concentrations, which have been documented in other species with hyperthyroidism, may predispose to hepatic injury. 126 treatment with same may be beneficial. phenobarbital is associated with chronic hepatic disease and cirrhosis in dogs. 70 most dogs have been treated with phenobarbital for more than a year before the liver disease is apparent. the mechanism of hepatic injury is not known but higher doses, higher blood levels (>40 µg/ml), and long duration appear to be important risk factors. 70 clinical signs in dogs reflect chronic liver disease and include sedation, ataxia, anorexia, weight loss, weakness, ascites, jaundice, coagulopathy, and encephalopathy. phenobarbital-induced hepatic injury should be suspected in any dog with a history of chronic phenobarbital therapy and clinical and biochemical evidence of hepatic dysfunction. routine biochemical screening (every 4 to 6 months) of dogs on long-term phenobarbital therapy is recommended for early detection of hepatic injury. however, mild liver enzyme elevations (especially alp) are commonly seen in dogs treated with phenobarbital, who do not have clinical or histologic evidence of significant liver early recognition of hepatotoxicity (including periodic monitoring of liver enzymes during the first 3 months) and discontinuation of drug therapy provides the best opportunity for full recovery. whether dogs with previous carprofen hepatotoxicity can be safely switched to another nsaid without experiencing a hepatic reaction is unknown. oral diazepam has been incriminated as a cause of acute idiosyncratic fatal hepatic necrosis in cats. 151, 152 intravenous diazepam, and oral oxazepam, clonazepam, and zolazepam also have been implicated. 126, 152 onset of signs occurs within 5 to 13 days of initiating therapy. clinical signs and biochemical evaluation are consistent with acute hepatic necrosis and liver failure. most cats die within 15 days of initial administration of the drug. if treatment of a cat with oral diazepam is unavoidable, liver enzymes should be checked before and within 5 days after starting therapy. if liver enzymes are increased, the drug should be discontinued and symptomatic therapy should be started. ancillary treatment of alf with nac may be beneficial. 9 glucocorticoid therapy in dogs is commonly associated with increased serum alp activity and development of a reversible vacuolar ("steroid") hepatopathy as a result of hepatic glycogen accumulation. these hepatic effects can be seen with virtually any glucocorticoid preparation (including topical ophthalmic and otic preparations) and are influenced by drug preparation (e.g., repositol versus short-acting), dose, duration of therapy, and individual dog susceptibility. in contrast, cats are quite resistant to the hepatic effects of glucocorticoids and only rarely develop these hepatic changes. 153 increased serum alp activity can occur within 3 days after initiating glucocorticoid therapy in dogs and is often striking (up to 64 times normal). glucocorticoids are associated with the induction of a specific corticosteroid-induced isoenzyme of alp, which may account for 60% to 100% of the total alp activity. 154 in contrast, serum alt activity is often normal or only mildly increased. in most dogs, glucocorticoids do not cause significant hepatic dysfunction or clinically relevant hepatic disease and biochemical tests reflecting hepatic function (serum bilirubin, albumin, glucose, blood ammonia concentration, and coagulation tests) are typically normal. serum bile acid concentrations are normal or only mildly increased (<60 mmol/l). hepatic glycogen accumulation causes hepatomegaly (which can be detected on abdominal radiographs), and diffuse or multifocal increases in hepatic echogenicity (detected on ultrasonography). the hepatic effects of glucocorticoids are reversible after drug withdrawal. the length of time required for complete resolution is unpredictable, varying from weeks to months. lomustine ccnu [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] is an oral nitrosourea alkylating agent that is used for chemotherapy of lymphoma, mast cell tumor, histiocytic sarcoma, and brain tumors in dogs. idiosyncratic dose-related hepatotoxicity was described in 11 of 179 (6.1%) dogs given an oral dose of ccnu (50 to 110 mg/m 2 ), with a dosing interval of 3 to 6 weeks. 72 the median time to detection of hepatic disease (from the last dose of ccnu) was 11 weeks and ranged from 2 to 49 weeks. delay in onset was noted, with an inverse relationship between the size of dose, and length of time before abnormal serum alt was detected. 72 a cumulative dose effect was suspected. clinical findings of hepatotoxicity included glutathione precursors (nac, same) and vitamin c in treatment. 21 dogs with hepatopathy are less likely to recover (46%) than are dogs with nonhepatic manifestations of sulfonamide hypersensitivity (89%). 159 tetracycline can predispose to hepatic lipid accumulation because it inhibits protein synthesis and interferes with hepatic secretion of triglyceride-rich lipoproteins. 126 however, it does not appear that these hepatic effects are clinically significant in most dogs and cats, although clinically significant idiosyncratic hepatic injury has been reported. increased liver enzyme activity was a common finding in dogs treated with doxycycline (increased alt activity in 39.4%; increased alp activity in 36.4%), but the clinical significance remains to be determined. 160 aflatoxicosis aflatoxins are metabolites, produced primarily from strains of the saprophytic fungus, aspergillus, which cause toxic hepatitis in dogs and many other species. 138, 161 exposure in dogs may occur through the inadvertent use of aflatoxin-contaminated corn or peanut meal during the commercial production of dog food, or after ingestion of homemade pet foods, moldy garbage, or improperly stored dog food. 161 dogs are relatively susceptible to aflatoxins and the liver is the target organ. clinical cases of aflatoxicosis in cats have not been reported. aflatoxin b1 is most commonly implicated in hepatotoxicity and toxic effects are seen when levels exceed 60 µg/kg of food. 161 aflatoxin b1 is readily absorbed from the gi tract and undergoes hepatic metabolism by cytochrome p450 enzymes, to a toxic intermediate (aflatoxin b1 8,9-epoxide) , which binds to essential molecules within the cell, leading to hepatocyte necrosis and decreased protein synthesis. detoxification of aflatoxin b1 8,9epoxide occurs by conjugation to glutathione. depending on the amount consumed, dogs may present with acute, subacute, or chronic liver disease. high-dose exposure is associated with acute hepatic failure, jaundice, dic, and death. repeated exposure to low doses can lead to chronic liver disease and cirrhosis. in 2005, an outbreak of aflatoxicosis occurred in at least 100 dogs, as a result of eating a commercially available dog food manufactured with aflatoxin-contaminated corn. 138 severity of clinical signs varied significantly among dogs. some dogs died suddenly without preexisting signs of illness. other dogs were presented with signs of anorexia, lethargy, vomiting, jaundice, diarrhea (including melena and hematochezia), abdominal effusion, he, and evidence of a bleeding disorder. common biochemical features included increased liver enzyme activity (especially alt), hyperbilirubinemia, electrolyte disturbances, hypoalbuminemia, hypocholesterolemia, and prolonged clotting times. reduced plasma antithrombin iii and protein c activities and hypocholesterolemia were suggested to be the most sensitive biomarkers of aflatoxin ingestion in dogs with minimal clinical signs, possibly reflecting an early effect of aflatoxin on biosynthesis of certain proteins and cholesterol. 138 in dogs with acute or subacute aflatoxicosis, the liver is enlarged and pale yellow with histologic features of diffuse hepatic vacuolation (lipid accumulation), scattered individual hepatocyte necrosis, biliary hyperplasia, and modest inflammation. collapse of zone 3 hepatocytes around the central vein, associated with perivenular inflammation, may explain clinical features of portal hypertension. with chronic low-level exposure, findings include a small liver with regenerative nodules, acquired pss, and histologic evidence of marked biliary hyperplasia and periportal fibrosis. disease. 157 potential indicators of clinically significant liver injury include increases in alt and alp activity that exceed five times the upper limit of normal; alt activity that exceeds alp activity; any elevation in ast activity; or enzyme elevations accompanied by evidence of hepatic dysfunction (hyperbilirubinemia, hypoalbuminemia, hypocholesterolemia, increased sba). hepatic cirrhosis associated with chronic phenobarbital therapy is characterized grossly by a small, nodular liver and histologically by bridging portal fibrosis, nodular regeneration, biliary hyperplasia, and mild inflammation (see figure 61-19) . 70 these lesions are by no means pathognomonic for phenobarbital-induced hepatic damage; however, in the absence of other known causes of hepatic damage, circumstantial evidence would support drug therapy as a likely cause. chronic phenobarbital therapy also is associated with superficial necrolytic dermatitis (hepatocutaneous syndrome) in dogs. 158 liver biopsy changes were typical of those seen with hepatocutaneous syndrome (marked vacuolar change and parenchymal collapse), which are distinct from the characteristic chronic hepatitis and cirrhosis as described above. 158 phenobarbital should be decreased or discontinued if possible in dogs with biochemical and histologic evidence of hepatic disease. in dogs with phenobarbital-associated toxicosis, clinical, biochemical, and histologic improvement can occur if the drug is discontinued or used at a reduced dosage prior to severe, end-stage liver disease. improvement in clinical signs can be noted within days to weeks of decreasing serum phenobarbital levels. primidone also is associated with chronic liver disease in dogs, likely as a consequence of metabolism of primidone to phenobarbital. phenytoin can cause acute or chronic hepatitis in dogs, as well as jaundice and death. the risk of hepatotoxicity is increased with combination therapy of phenobarbital, primidone, and phenytoin. 126 sulfonamides potentiated sulfonamides (trimethoprim-sulfadiazine, trimethoprimsulfamethoxazole, and ormetoprim-sulfadimethoxine) are associated with the acute idiosyncratic drug-induced liver injury in dogs. 21 trimethoprim-sulfadiazine was implicated in over 20% of hepatic drug reactions in dogs that were reported to the center for veterinary medicine between 1988 and 1990. 71 doberman pinschers are suggested to be at risk for development of polyarthropathy from sulfonamide hypersensitivity, but not necessarily the idiosyncratic hepatic reaction. 159 onset of clinical signs occurs within 5 to 36 days (mean: 12 days) from starting the drug. 159 previous exposure to sulfonamides is not required. doses of potentiated sulfonamides are generally higher in dogs who develop the idiosyncratic hepatic reaction, as compared with other systemic manifestations of sulfonamide hypersensitivity (thrombocytopenia, fever, polyarthropathy, other). 159 biochemical findings include increased liver enzyme activity (alt > alp) and hyperbilirubinemia. liver biopsy usually reveals marked hepatic necrosis; however, cholestasis and marked lymphocytic-plasmacytic inflammation also have been described. 21 the pathogenesis of the idiosyncratic hepatic reaction is unclear. dogs in general may be at increased risk for sulfonamide reactions because they lack genes that express the n-acetylation enzymes, which is a major metabolic pathway of detoxification of sulfonamides in humans. 21 however, this does not explain individual risks among dogs. hepatotoxicity may be a result of p450 oxidation of sulfonamides to reactive metabolites, such as hydroxylamine and a nitroso metabolite, which may be associated with hapten formation, t-cell proliferation, or direct cytotoxicity. 21 impaired detoxification of reactive metabolites via a deficiency in glutathione, cysteine, and ascorbate, may play a role, and theoretically supports the use of hepatocyte uptake of amanitins and has been shown to be protective against experimental a. phalloides liver damage in beagles, when given at a dose of 50 mg/kg iv twice, at 5 and 25 hours after exposure. 141 however, an intravenous form of silymarin is not currently available for clinical use in the united states. experimental studies suggest that penicillin g may also reduce hepatic uptake of amanitins, even several hours after ingestions. 162 iv nac may be beneficial as described for treatment of acetaminophen toxicity. overall mortality rate with amanita mushroom toxicity is high. ingestion of toxin-producing blue-green algae (microcystis aeruginosa) is a rare cause of hepatotoxicity and alf in dogs. 164 algae proliferate in shallow, stagnant water, especially in hot, dry weather. dead or dying algae form a thick blue-green scum on the water's surface, and release the toxic principle, microcystins. toxicity is caused by ingestion of algae-contaminated water. signs occur rapidly (within 1 hour of ingestion) and include vomiting, diarrhea, and lethargy, followed by progressive tachypnea and dyspnea, icterus, and coma. biochemical features reflect hepatocellular injury with increased alt and ast activity (that typically exceed increases in alp activity), and hyperbilirubinemia. however, profound or protracted increases in alt activity may not be detected, because microcystins can interfere with transaminase biosynthesis. 7 hepatic lesions consist of massive hepatic necrosis of the centrilobular to midzonal hepatocytes. treatment is symptomatic and supportive. oxidative injury may play a role, 164 which suggests that glutathione supplementation (nac or same) may be of benefit. the prognosis is guarded. cycads (sago palms) are native to tropical and subtropical regions, and are used as houseplants and in residential landscaping. concentrations of cycasin, the primary toxin in cycads, are highest in the seeds and roots, but present in all parts of the plant. 165 ingestion of as few as one to two seeds can be fatal in dogs. following ingestion, cycasin is metabolized by gi bacteria to its active compound, methylazoxymethanol, which causes gi and hepatic toxicity in dogs. 165 most dogs that ingest cycads develop gi signs, including vomiting, diarrhea, and abdominal pain. neurologic signs (weakness, ataxia, depression, proprioceptive deficits, seizures, coma) are also common, but it is not clear if they are a result of a neurotoxin or he. 165 onset of clinical signs ranges from 15 minutes to 3 days and may last from 24 hours to 9 days. 165 hepatic injury is suggested by findings of progressive depression, icterus, he, and excessive bleeding accompanied by increased liver enzyme activity, hyperbilirubinemia, hypoglycemia, and hypoabuminemia. 166, 167 centrilobular hepatic necrosis is found on liver biopsy. 166 no specific treatment is available. mortality has been reported to vary between 32% and 58%. 165, 167 xylitol xylitol, a 5-carbon sugar alcohol used as a sugar substitute, is associated with hypoglycemia and hepatic necrosis in dogs. 168, 169 xylitol is safe in humans and is commonly used in sugar-free gum and other oral care products, and is available as a granulated powder for baking. xylitol was first introduced into the united states in 2002, and since that time, reports of toxicity to the aspca animal poison control center have increased from two dogs in 2002 to 2512 dogs in 2008. 168 ingestion of more than 0.1 g/kg in dogs is associated with a rapid, severe, increase in blood insulin, which results in signs of hypoglycemia within 30 to 60 minutes of ingestion. when amounts exceed 0.5 g/kg, alf may occur within 9 to aflatoxicosis associated with consumption of a commercially manufactured pet food product should be suspected when there is a geographic or temporal cluster of cases in a household, kennel, or region. the history may reveal recent changes in diet or feeding from a new bag. it should be noted that some dogs may consume contaminated food for weeks to months before signs develop. definitive diagnosis of aflatoxicosis is based on chemical detection of increased levels of aflatoxin (>60 µg/kg) in the food. when aflatoxicosis is suspected, the owner should be advised to retain 1 kg of food in an airtight zippered plastic bag (or four cans of food), for laboratory testing. it is also recommended to save packaging information, including product and date code, to help identify contaminated lots of food. if a sample of food is no longer available, serum or liver samples can be submitted for testing for aflatoxin m1 (aflatoxin metabolite), although usefulness may be limited because of the rapid metabolism and excretion of aflatoxin. detection of aflatoxin m1 in urine is only useful if the dog is still consuming the contaminated diet, as levels fall below detectable levels within 48 hours. there is no specific antidote for aflatoxicosis and treatment consists of symptomatic and supportive management of liver failure. nonspecific hepatoprotective therapy with antioxidants (vitamin e), glutathione replacement (nac, same), milk thistle (silymarin), and l-carnitine have been recommended. 138 the prognosis is guarded if clinical signs of aflatoxicosis are present, with a reported mortality rate of 64% in a series of 72 dogs that consumed aflatoxincontaminated dog food. 138 dogs that survive acute liver injury have the potential to develop chronic liver disease. consequently, monitoring of liver function is recommended in recovering dogs and treatment with thiol donors such as same for 2 months has been empirically recommended. 138 amanita mushrooms amanita phalloides (and other varieties such as amanita verna and amanita bisporigera), are poisonous mushrooms found throughout north america that can cause acute hepatic necrosis in dogs and cats. 162, 163 toxicity is attributed to extremely toxic cyclopeptide toxins called amanitins. ingestion of two a. phalloides mushrooms can be lethal to an adult dog. 163 clinical signs occur within 6 to 24 hours after ingestion and are characterized initially by gi signs such as vomiting, bloody diarrhea, and abdominal pain. the late phase (36 to 84 hours after exposure) is characterized by alf (hemorrhage, marked hypoglycemia, he, and terminal coma) caused by severe massive hepatic necrosis (see figure 61 -20) . toxin-induced renal tubular necrosis may also result in renal failure. 162 biochemical features reflect severe hepatic injury and include increased liver enzyme activity (alt exceeds alp), refractory hypoglycemia, and hyperbilirubinemia. diagnosis is usually made based on positive identification of the suspect mushroom, evidence of its ingestion, and consistent clinical features. mushroom pieces in gastric contents can confirm exposure, but are difficult to identify. accurate mushroom identification requires consultation with an experienced mycologist. the suspect mushrooms should be wrapped in paper towels and stored in a paper (not plastic) bag. 162 definitive confirmation can be established by detecting amanitins in liver or kidney tissue (or serum and urine samples collected during the gi phase) by liquid chromatography-mass spectrometry, through the california animal health and food safety laboratory. 163 gi decontamination procedures are recommended as soon as possible after exposure, as described in the "treatment" section of "drug and toxin-induced liver injury". symptomatic and supportive treatment for alf is indicated, including close monitoring and treatment of marked hypoglycemia. silymarin is believed to reduce lower urinary tract disease ammonium biurate uroliths are formed most of the time in the bladder (and rarely in the renal pelvis) and can cause dysuria, urethral obstruction in males, and seldom uroabdomen as a result of chronic inflammation and subsequent devitalization of the bladder wall. 3 ammonium biurate crystalluria is not pathognomonic for portosystemic shunting, and it may occasionally occur in normal dogs and cats, or be found in certain breeds, such as dalmatians, because of an inborn error of metabolism. prolonged recovery from anesthesia or sedation may occur in seemingly normal dogs and cats that have cpss. the liver plays a crucial role in the detoxification process of toxins and drugs, including anesthetics. patients with cpss or apsc have insufficient functional hepatic mass for detoxification. in addition, dogs with portosystemic shunting have increased endogenous benzodiazepine and gabaergic activities. 4 therefore administration of diazepam or barbiturate to these animals may have prolonged and exaggerated effects (see "pathogenesis" section). recurrent fever together with resultant depression and anorexia can be the only clinical manifestation of cpss in some dogs. 5 portosystemic shunting should be excluded in dogs experiencing recurrent fever. this rare manifestation has only been reported in a few dog. 6 its pathogenesis is not understood. elderly dogs with extrahepatic cpss may present with characteristic signs of hypercortisolism (e.g., pu/pd, polyphagia, thin skin, symmetric alopecia, muscle wasting, pot belly) as primary complaints. these are unusual clinical manifestations of cpss. although small body size is observed in cats with cpss, it infrequently occurs in dogs. many dogs with extrahepatic cpss show no or only nonspecific clinical signs throughout their lives and the shunt is detected as a coincidental finding at the time of necropsy. it is not understood why certain dogs become clinically ill and others do not with the same type of portal vein anomaly. cpss never cause icterus, ascites, or spontaneous hemorrhages. portal vein disorders are much less common in cats than in dogs. most cats are younger than 6 months of age when the first signs appear. 7 cats with cpss are often of smaller stature and have unkempt hair coat. episodic salivation and/or central neurologic signs, for example, compulsive pacing or seizures, as manifestations of he are the most typical presenting complains. 8, 9 portal hypertension clinical signs can develop at any age and arise from the presence of increased hydrostatic portal venous pressure, portosystemic 72 hours. 169 alf is not necessarily preceded by early signs of hypoglycemia. the mechanism of hepatotoxicity is unknown, but has been speculated to be caused by cellular depletion of adenosine triphosphate resulting in hepatocellular necrosis, or production of reactive oxygen species causing oxidative injury. 169 in addition to signs of hypoglycemia, dogs with alf may have vomiting, icterus, and evidence of excess bleeding. biochemical findings include markedly increased alt and ast activity, mild to moderate increased alp activity, hyperbilirubinemia, hypoglycemia, hyperphosphatemia, prolonged pt and aptt, and thrombocytopenia. if ingestion occurred within the last few hours, induction of emesis is recommended (unless showing signs of hypoglycemia). activated charcoal may be of limited value in adsorbing xylitol, but is still recommended if large amounts have been ingested. 168 treatment recommendations include hospitalization for observation and monitoring, dextrose supplementation for control of hypoglycemia, and symptomatic and supportive care for complications of liver failure. hepatoprotective therapy with nac, same, and silymarin may be beneficial. the prognosis is good for recovery in dogs with uncomplicated hypoglycemia and guarded to poor for dogs in liver failure. however, survival after liver failure does not necessarily correlate with amount of xylitol ingested. 169 vascular hepatic diseases include congenital and acquired disorders of the portal vein. congenital anomalies result from (a) hypoplasia or aplasia of the portal vein, (b) macroscopic communications between the portal vein and a systemic vein, or (c) between the portal vein and an artery. acquired diseases result from conditions that increase the hydrostatic pressure in the portal vein (i.e., portal hypertension). macroscopic venous connections between the portal and systemic venous systems result in portosystemic shunting (i.e., blood flows from the portal to the systemic veins) via a congenital portosystemic shunt (cpss) or acquired portosystemic collaterals (apsc). neurologic signs hepatoencephalopathy (he) is a reversible central neurologic manifestation of hepatic insufficiency. 1 cpss causes chronic he. the following grades of chronic he are recognized 2 : grade 1-depression, behavior changes; grade 2-ataxia, compulsive pacing, circling, hypersalivation, head pressing, blindness; grade 3-stupor and seizures; and grade 4-coma. chronic he is characterized by periods of severe (grades 2 to 3) signs (lasting usually several hours to a few days alternating with longer periods (days to weeks) of no or mild (grade 1) symptoms. 2 periods of cortical blindness are accompanied by apparent mydriasis. signs of he may be triggered by ingestion of protein-rich meals. pd means excessive fluid intake (in dogs >100 ml/kg body weight/24 h and in cats >50 ml/kg body weight/24 h). pu may be more difficult to diagnose than pd, as pet owners cannot readily measure urine volume. repeated low specific gravity of morning urine (<1.025 in dogs and <1.030 in cats) is compatible with pu. macroscopic connections exist between major vessel systems or their tributaries. single or multiple portosystemic venous connections allow the portal venous blood to flow directly to the systemic venous system without first flowing through the hepatic sinusoids. this toxin-rich blood will be delivered to all cells of the body through the following route: gut > portal vein > shunt > systemic vein > right heart > lungs > left heart > arteries. a connection through a single, or rarely double, large-bore vein without the presence of portal hypertension is considered to be a cpss. single or multiple connections in the presence of portal hypertension are apsc. whenever a macroscopic venous connection is present between the portal vein (or one of its tributaries) and a systemic vein, the blood will flow from the portal vein to the systemic vein, because the pressure in the portal vein is higher (8 to 10 mm hg) than that in the systemic veins (0-5 mm hg). 8 normal portal venous pressure is approximately 8 to 10 mm hg (10 to 13 cm h 2 o). increased pressure in the portal venous system results in portal hypertension. anatomically, portal hypertension can be classified 17 as (a) prehepatic (i.e., portal vein), (b) intrahepatic or (c) posthepatic (i.e., hepatic veins, thoracic caudal vena cava, or heart). posthepatic (postsinusoidal) portal hypertension. all cardiac diseases that result in right-sided congestive heart failure cause posthepatic portal hypertension. these diseases include the (a) congenital or acquired severe insufficiency of the tricuspid valve (e.g., dysplasia, myxomatous degeneration, annulus dilation caused by dilated or arrhythmogenic cardiomyopathy, pulmonary hypertension of various etiologies), (b) pericardial diseases (pericardial tamponade caused by idiopathic or neoplastic effusion or constrictive pericarditis), (c) various congenital anomalies such as cor triatriatum dexter, atrial septum defect, tricuspid or pulmonic stenosis, (d) intracardiac tumors affecting the right heart, and (e) caval syndrome (caused by dirofilaria immitis heart worms). kinking or compression of the thoracic caudal vena cava (by diaphragmatic hernia or a mass) occurs rarely. compression, stenosis or thrombosis of the hepatic veins (the so-called budd-chiari syndrome) does not occur in dogs and cats. 13,13a in posthepatic portal hypertension the portal and caval pressures increase equally. therefore no apsc develop, and the blood ammonia concentration remains within reference range. the high postsinusoidal hydrostatic pressure causes a large amount of protein-rich hepatic lymph to be filtered through the hepatic capsule into the abdominal cavity causing accumulation of a modified transudate. modified transudate has high protein content because the fenestrated endothelial cells of the hepatic sinusoids keep only 10% to 20% of the plasma proteins in the capillary lumen. marked generalized hepatomegaly and dilated jugular and hepatic veins as well as caudal vena cava are hallmarks of postsinusoidal portal hypertension. intrahepatic portal hypertension. chronic acquired parenchymal liver diseases lead to hepatocyte necrosis and subsequent collagen deposition. the resultant disorganization of the hepatic architecture and contraction of the connective tissue results in obstruction of the intraparenchymal vessels. the underlying disease processes include viral, bacterial, protozoal, immune-mediated, or copperassociated chronic hepatitis; toxic, drug-induced, or idiosyncratic liver damage; lobular dissecting hepatitis; and chronic bile duct shunting via apsc, and the underlying disease that led to portal hypertension. clinical signs of portosystemic shunting are similar, regardless whether it is congenital or acquired in origin (see "clinical manifestations" section). accumulation of a large amount of pure transudate or modified transudate (clear, straw-colored, or slightly turbid blood-tinged fluid) in the abdominal cavity is commonly detected with physical examination and abdominocentesis. the absence of free abdominal fluid does not exclude portal hypertension. depending on the etiology and the anatomic location of the underlying disease that led to the development of portal hypertension various signs may be seen such as jaundice, periodic vomiting, and anorexia. jaundice and hemorrhagic diathesis are absent in congenital vascular disorders. congenital arterioportal fistula causes transudative ascites in puppies between 2 and 6 months of age. 10 primary hypoplasia of the portal vein (phpv), also described as "noncirrhotic portal hypertension," 11 may result in ascites, vague gi signs, and he, or may be entirely subclinical. 12, 13 when the phpv is not severe enough to cause portal hypertension, clinical signs may not be obvious. this mild form of phpv is also known as hepatic microvascular dysplasia 14 and may be detected serendipitously if plasma bile acid concentrations are measured for unrelated reasons. portal hypertension is rare and is not typically associated with ascites in cats. 15 portosystemic shunting because of cpss or apsc tend to cause similar signs, for example, periodic salivation and seizures, as manifestations of chronic he. the most common acquired disease causing intrahepatic portal hypertension is biliary cirrhosis caused by chronic bile duct obstruction. jaundice and acholic feces are typical findings in extrahepatic cholestasis. the liver receives its blood supply from the portal vein (approximately 75%) and the hepatic artery (approximately 25%). with diversion of portal venous blood flow, a compensatory increase of the hepatic arterial flow occurs. arterial vasodilation is mediated by adenosine from energy-depleted hepatocytes. the portal vein transports blood from the spleen and the gi tract to the liver. 16 the smallest portal venous and hepatic arterial branches terminate in the capillary system of the liver, the so-called hepatic sinusoids. a large amount of plasma is filtered through the fenestrated walls of the sinusoids to the space of disse. from here the filtered plasma is taken by lymphatic vessels to the systemic venous system. the remaining sinusoidal blood is then collected by the hepatic veins, which enter the caudal vena cava. normally no the hepatofugal portal flow prevents the splanchnic venous blood from entering the liver and causes the development of alternative pathways and often times accumulation of pure transudate in the abdomen. interestingly, phpv always accompanies congenital arterioportal fistulas both in dogs and cats. 5, 10, 25 when a cpss and arterioportal fistulas are concomitantly present, apsc and ascites do not develop. 25 arterioportal fistula can be extrahepatic in cats. acquired portosystemic collaterals. in healthy mammals, multiple nonfunctional venous connections may exist between the portal and systemic veins. these virtual communications become functional when their lumen becomes sufficiently widened. gradual dilation takes place when sustained increase of the portal pressure takes place without the simultaneous increase of caval pressure. the resultant apsc are multiple, usually thin, tortuous veins with species-specific anatomical locations; however, large-bore veins may also develop. these latter cases should not be misinterpreted as simultaneous cpss and apsc. in the presence of an existing cpss, no apsc would develop even if hepatic cirrhosis develops as there is an already existing portosystemic connection that is able to drain 100% of the portal blood without allowing the development of portal hypertension. 26 splenorenal collaterals are consistently present in almost every dog with apsc. 10, 27 these apsc drain the portal venous blood via the splenic vein through acquired connections to the left gonadal vein. the left gonadal vein enters the left renal vein, which later empties into the caudal vena cava. these splenorenal apsc are thought to prevent the spleen from undergoing congestion, 17 which is why splenomegaly is not a feature of canine pre-and intrahepatic portal hypertensive disorders. 10 shunting of the portal blood is not only detrimental for the brain, but for the liver as well. normal hepatic development and function requires sufficient amount of portal venous perfusion of the hepatic sinusoids. increased arterial perfusion is unable to compensate for portal hypoperfusion. regardless of whether the insufficient portal venous perfusion is caused by cpss or by prehepatic portal hypertension, the result is the same: reduced hepatic mass and function. in both cases, histopathologic evaluation of the liver shows stereotypical reaction: small or invisible portal branches, increased number of arterioles and sometimes bile ductules in the portal tracts, hepatocellular atrophy, and periportal sinusoidal dilation. 13 this secondary portal vein hypoplasia is reversible and is histologically indistinguishable from phpv. because primary and secondary portal vein hypoplasia show identical microscopic features, histopathologic evaluation of liver biopsy specimens is unable to diagnose simultaneous phpv and cpss. glycine and gaba are the most important inhibitory neurotransmitters, whereas glutamate and dopamine are the most abundant excitatory neurotransmitters in the brain. in he, a net increase in inhibitory transmission occurs. 28 this results from upregulation of gaba receptors and downregulation of dopamine receptors. 29 activation of gaba receptors causes opening of chloride channels, which leads to hyperpolarization of the postsynaptic membrane. in the presence of gaba, benzodiazepines increase the frequency, whereas barbiturates increase the duration of the chloride channel opening. 30 the cause of increased gabaergic tone in he is thought to be gut derived, 4 but gaba may also be formed in the neurons from glutamate. 28 endogenous benzodiazepines are proven to be obstruction. the resultant increase in portal pressure may cause development of apsc as well as ascites. modified transudate may accumulate in the abdominal cavity when the disease process predominantly obstructs the postsinusoidal hepatic venules. however, when the disease process affects predominantly the intrahepatic portal vein branches (presinusoidal portal hypertension) pure transudate will accumulate in the abdomen. primary hypoplasia of the portal vein is caused by insufficient development of the intrahepatic portal venous branches, the left portal vein branch, or the whole portal venous system. 12, 18 when the hypoplasia is severe enough to cause intra-or prehepatic portal hypertension, apsc develop. this condition is also known as "noncirrhotic portal hypertension." if hypoplasia is not severe enough to cause portal hypertension, clinical signs will not develop. because no portosystemic shunting is present, the results of rectal ammonia tolerance test (att), portal scintigraphy, and portography are all normal (see "differential diagnosis" section). if either macroscopic or microscopic portosystemic communications is present, hyperammonemia or abnormal att should also be present, but this has never been documented. the only abnormality that could be detected in these dogs is elevation of serum bile acids levels. 14 the most plausible explanation for this is that the hepatic clearance of bile acids by the hypoperfused liver is probably less effective than that of ammonia. the disease can only be diagnosed by histopathologic examination of liver biopsy specimens. this condition also has been described as "hepatic microvascular dysplasia." although the terms noncirrhotic portal hypertension and hepatic microvascular dysplasia have been used to describe this syndrome, both have been replaced by the term phpv. 13 the occurrence of phpv in the cat is very rare and poorly documented. the most common congenital cause of portal hypertension in cats is congenital hepatic fibrosis as a part of polycystic kidney and liver disease complex. 19 this disease may cause clinical signs with or without the presence of macroscopic hepatic or renal cysts. 20 prehepatic portal hypertension. narrowing of the portal vein lumen may be caused by (a) extravascular compression by a tumor, enlarged lymph node, cyst, abscess, or hematoma, (b) idiopathic circumscribed stenosis, 21 or (c) intravascular obstruction by a thrombus or parasites, all of which can lead to portal hypertension. 22 portal vein thrombosis is always secondary either to neoplasia, or to a systemic disorder that causes hypercoagulability such as nephrotic syndrome, immune-mediated hemolytic anemia, hypercortisolism, acute pancreatitis, peritonitis, or sepsis. 23 parasites in the portal vein also may occur, for example, heterobilharzia americana in north america 24 and schistosoma japonicum in east asia. 13 depending on the degree of portal vein occlusion, increased portal pressure with apsc and ascites may develop. as the hydrostatic pressure in the sinusoids does not increase (in contrast to posthepatic portal hypertension), the resultant ascitic fluid will have a low protein content. the narrowed lumen of the portal vein causes reduced portal flow to the liver, resulting in reduction in the size of the liver. portal vein thrombosis itself rarely causes clinical signs and is usually a coincidental finding. when the liver receives little portal venous blood, an insufficient amount of substrate (i.e., ammonia) is available for hepatic urea production. this theoretically results not only in a low plasma urea concentration, but also in a lower renal medullary urea concentration, which impairs renal concentrating ability and causes pu. increased basal plasma concentrations of acth and cortisol as well as increased urinary cortisol-to-creatinine ratios are invariably present in dogs with portosystemic shunting. [43] [44] [45] [46] cortisol interferes with the action of arginine-vasopressin at the renal tubule, causing a nephrogenic-type diabetes insipidus. 47 hypersecretion of acth (and α-melanocyte stimulating hormone [α-msh]) has been shown to arise predominantly from the intermediate lobe of the pituitary. 43, 48 the hormone secretion of this lobe is regulated by tonic dopaminergic inhibition. acth-hypersecretion can be explained by the production of "false" neurotransmitters (e.g., octopamine), whose effect is about one-fiftieth that of dopamine on the dopamine receptors. 35 central diabetes insipidus also contributes to pu in dogs with he. impaired release of arginine-vasopressin from the posterior lobe of the pituitary is caused by a reduced magnitude of response and a highly increased threshold to increased plasma osmolality. 45 release of arginine-vasopressin is inhibited by the gaba inhibitory neurotransmitter system, whose activity is increased in he. 29, 45 pseudohyperaldosteronism cortisol and aldosterone have similar affinities to bind aldosterone receptors. however, cortisol is normally inactivated by 11β-hydroxysteroid dehydrogenase in tissues where aldosterone action is required. 49 high serum bile acids concentrations inhibit this enzyme, and cortisol can bind to aldosterone receptors resulting in increased mineralocorticoid effect. 45 plasma cortisol concentrations are 10-fold those of aldosterone, causing constant and inappropriate pseudohyperaldosteronism. the resultant sodium retention causes secondary water retention and subsequent pu by pressure diuresis. hypokalemia caused by hyperaldosteronism also contributes to pu 50, 51 according to the following mechanism. the presence of aquaporin-2 channels in the renal collecting ducts' cell membranes is necessary for water reabsorption. intracellular signaling pathways through cyclic adenosine monophosphate regulate the insertion of these channels. hypokalemia decreases the sensitivity of cyclic adenosine monophosphate to arginine-vasopressin, which results in decreased insertion of aquaporin-2 channels into the cell membrane. 50 this leads to nephrogenic diabetes insipidus and pu. congenital portal venous anomalies in dogs are typically associated with enlarged kidney volume. increased renal gluconeogenesis as a compensation of insufficient hepatic gluconeogenesis may cause the kidneys to enlarge. 52 in addition, increased systemic circulating growth factor concentrations released from the pancreas may play a role in this increased volume. 53 normally, these growth factors act only in the liver, as they do not reach the systemic circulation in high concentrations. behavior changes and abnormalities in the thirst center due to he may contribute to pd; however this is difficult to prove in individual patients. produced in the intestines of dogs with cpss. 4 their source is not quite clear: they could arise from the diet, intestinal flora, or by endogenous modification of inactive gut precursors. 4 in all forms of portosystemic shunting the concentration of aromatic amino acids are increased in the systemic circulation whereas the concentration of branched-chain amino acids is decreased. high aromatic amino acid concentration results from impaired hepatic clearance and increased production caused by muscular breakdown exacerbated by hyperglucagonemia. 28, 31 the decreased concentration of branched-chain amino acids results from their increased utilization for gluconeogenesis. 28, 32 this amino acid imbalance may result in the development of "false" neurotransmitters (e.g., octopamine) in the brain, which contribute to the development of he. 33, 34 these "false" neurotransmitters have a fraction of dopamine's excitatory effect on dopamine receptors. drugs with dopaminergic effect, such as bromocriptine, may improve signs of he. [35] [36] [37] intestinal toxins such as ammonia and bacteria are normally inactivated by hepatocytes and hepatic macrophages (kupffer cells), respectively, so that toxin-and bacterium-free blood can enter the systemic circulation. in patients with portosystemic shunting the majority of the portal venous blood bypasses the liver, allowing the toxin-and bacterium-rich blood to enter the systemic circulation. ammonia is a neurotoxin and can contribute to the clinical signs of he. 38 other toxins that are believed to play a role in he are endogenous benzodiazepines, gaba, tryptophan, glutamine, serotonin, mercaptans, indoles, and skatoles. 4, 34, 39 hyperammonemia also leads to impaired glial function through increased intracellular glutamine concentration. 38 glutamine is made in the glial cells through incorporation of ammonia into glutamate by glutamine synthetase. 28 chronically increased glutamine concentrations cause swelling of the glial cells resulting in so-called alzheimer ii type degeneration of astroglias. 40, 41 glial dysfunction contributes to the development of he. 40, 42 during hyperammonemia, the reserve capacity of the astrocytic glutamine synthetase is exceeded, and ammonia can enter the neurons. 42 in the neurons ammonia inhibits glutaminase, an enzyme that converts glutamine to glutamate. because glutamate is an excitatory neurotransmitter, its reduced level contributes to the development of he. 28 portosystemic shunting or an atrophic liver alone is insufficient to allow he to develop; he can only develop when they are both simultaneously present. 28 the reserve capacity of the liver ensures that even in advanced chronic parenchymal liver diseases, he does not develop in the absence of portosystemic shunting. alkalosis and hypokalemia can worsen the signs of he. in alkalosis the nh 3 + h + = nh 4 + reaction shifts to the left, causing the more lipophilic ammonia to enter the cells of the cns. during hypokalemia, potassium ions (k + ) move from the cells to the extracellular space in exchange for h + , which later results in extracellular alkalosis and intracellular acidosis. intracellular acidosis causes nh 4 + trapping within the cells. this might explain why the blood concentration of ammonia is not closely related to the severity of the clinical signs of he in individual cases. several mechanisms contribute to the development of pu/pd in portosystemic shunting. high concentrations of sodium and urea in the renal medullary interstitium are essential for the production of concentrated urine. these create a high osmotic gradient between the renal tubular lumen and interstitium, which is necessary for water reabsorption. to result from reduced enzyme activity of the urea cycle. as no clinical signs are present, no treatment is required. the condition resolves spontaneously with age because these pups develop enhanced incorporation of ammonia into glutamine. peritoneal absorption of ammonia-containing urine can cause hyperammonemia, however in such a patient the clinical signs of acute uremia predominate. the presence of urease-producing bacteria (e.g., staphylococci) are necessary to split urinary urea to ammonia. 67 fulminant hepatic failure ingestion of blue algae or certain mushrooms (e.g., a. phalloides) results in peracute insufficiency of hepatic function. these animals develop hepatic coma and die shortly thereafter. hyperammonemia, dic, and icterus are present in most affected patients. arginine is an essential amino acid in the cat. in anorectic cats hyperammonemia may develop along with hepatic lipidosis because of the insufficient amount of hepatic arginine needed for urea synthesis in the urea cycle. 68 thus, in contrast to adult dogs, hyperammonemia in cats can occur without portosystemic shunting. methylmalonic acidemia associated with cobalamin deficiency in dogs, 69a a cat 69 and a suspected "transient hyperammonemic syndrome" in a german shepherd dog 70 have been reported as extraordinarily rare causes of hyperammonemia. if blood sampling and sample processing are performed inappropriately hyperammonia may be erroneously diagnosed. high bile acids concentration may result not only from portosystemic shunting, but also from any primary or secondary hepatic diseases associated with intra-or extrahepatic cholestasis. 71 therefore, the presence of high serum bile acids concentrations is very sensitive, but not a specific indicator of portal venous disorders. 63 the simultaneous presence of hyperammonemia and a large amount of pure or modified transudate in the abdominal cavity indicates the presence of severe pre-or intrahepatic portal hypertension with apsc. biochemical and cytologic analysis of the peritoneal effusion, ultrasonography and central venous pressure measurement may be useful in identifying the source of ascites. 17 suspected intrahepatic causes should be further evaluated by histopathologic examination of liver tissue. in any animal that is presented with central neurologic signs, portosystemic shunting (along with other metabolic encephalopathies, e.g., hypoglycemia or electrolyte imbalance) should be investigated with appropriate blood tests. no single finding is pathognomonic for diagnosing vascular liver disorders. therefore, a combination of history, physical hyperammonemia results in a higher filtered load and urinary concentration of ammonia. high urinary concentration of uric acid results from decreased hepatic conversion of uric acid to allantoin because of reduced hepatic mass and function. the portal vein carries bacteria and endotoxins from the intestines, which are normally phagocytized by the kupffer cells. shunting of portal blood permits these bacteria and/or endotoxins to enter the systemic circulation causing bacteremia and/or endotoxemia, and subsequent fever. 5 systemic antibiotics may result in temporary resolution of clinical signs, however give no definitive cure. if a cpss occurs within hepatic parenchyma it is called intrahepatic. 54, 55 intrahepatic cpss arise either from the left or right portal branches. a left divisional intrahepatic cpss results from the failure of postnatal closure of the embryological ductus venosus. 56, 57 the intrahepatic cpss arising from the right portal branch and all extrahepatic cpss are thought to be developmental anomalies with poorly understood etiology. in dogs the anatomy of the shunting vessel is fairly consistent: intrahepatic cpss may be left, right, or central divisional, whereas extrahepatic shunts drain the portal venous blood via the splenic or the right gastric vein to the caudal vena cava or the azygos vein. 15 the course of the shunting vein in the cat is much more variable. 15, 58 the inherited nature of the disease has been established in several breeds including the irish wolfhound and the yorkshire and cairn terriers. [59] [60] [61] phpv is a congenital anomaly of unknown etiology. although arterioportal fistulas may develop as a result of neoplasm or trauma (e.g., shot wound or liver biopsy), only the congenital form has been reported in dogs and cats. congenital hepatic fibrosis caused by polycystic kidney disease (pkd) is a genetic disease. 19, 62 differential diagnosis high fasting venous blood ammonia concentrations together with high fasting serum bile acid concentrations are very specific and sensitive tests for diagnosing portosystemic shunting. 63 urea cycle enzyme deficiency reduced activity of one or more enzymes of the urea cycle may result in elevated blood ammonia concentration. 10, 64 serum bile acids levels should remain within reference range in patients with urea cycle enzyme deficiencies. normal hepatic scintigraphy and liver histopathology results should confirm the absence of portosystemic shunting. certain metabolites (e.g., citrulline) and enzyme activities (e.g., argininosuccinic acid synthetase) can be measured in urine and liver biopsy specimens, respectively, to establish a definitive diagnosis. the condition is rare in dogs. one suspected feline case has been reported. 65 irish wolfhound puppies healthy irish wolfhounds commonly have moderate hyperammonemia (<120 µmol/l) at the age of 6 to 7 weeks. 66 this is thought chloride (nh 4 cl) solution is administered at a dose of 2 ml/kg fifteen cm into the descending colon using a soft feeding tube. 75 venous blood ammonia is measured before and 20 and 40 minutes thereafter. in the presence of portosystemic shunting the ammonia concentration will increase at least twofold by the 20-and/or 40-minute sampling times. normal att result excludes portosystemic shunting (figure 61-21) . the degree of increment is a semiquantitative measure of the degree of portosystemic shunting. it should be noted that rectal administration of nh 4 cl solution may cause transient irritation of the colonic mucosa during the first 10 minutes. rectal att is a safe procedure, with signs of he rarely occurring. att should not be performed in patients with very high (>150 µmol/l) blood ammonia levels. postprandial measurement of ammonia is thought to decrease the possibility of iatrogenic hyperammonemic he; however the increase of blood ammonia concentration after feeding takes longer and its peak time point is poorly predictable. 76 markedly increased preprandial (i.e., 12-hour fasting) bile acids concentrations are often found with portosystemic shunting as a result of interrupted enterohepatic circulation of the bile acids. 77, 78 high 12-hour fasting plasma bile acid concentration is a sensitive, but nonspecific indicator of portosystemic shunting. specificity can be increased by simultaneous measurement of venous ammonia. as plasma bile acids concentrations are invariably high in icteric animals, the presence of apsc can only be justified or excluded by measuring blood ammonia concentration or performing an att. in case of a normal 12-hour fasting bile acids concentration (<15 µmol/l), measuring an increased postprandial plasma bile acids concentration (>25 µmol/l) 2 hours after a meal increases the sensitivity, but not the specificity of the bile acids test in diagnosing portosystemic shunting. 79 meal-induced gallbladder contraction causes an endogenous bile acid load, which will be absorbed in the ileum and will substantially increase the plasma bile acids concentration when portosystemic shunting is present. an abnormal postprandial increase of bile acids occurs also in cholestatic liver diseases. in patients with portosystemic shunting hypoalbuminemia, hypoproteinemia, hypocholesterolemia, and low plasma urea concentration may be found as a result of their reduced hepatic synthesis. hypoglycemia may be seen as a result of reduced hepatic glyconeogenesis and glycogen storage. low creatinine concentration reflects the increased glomerular filtration rate. 53 none of these biochemical changes is specific for portosystemic shunting; furthermore, many of these findings may be present in healthy pups. the cause of mild increase of plasma alt and alkaline phosphatase activities is not fully understood. microcytosis with or without mild nonregenerative anemia and leukocytosis may accompany portosystemic shunting. relative iron deficiency is thought to cause the microcytosis [80] [81] [82] and bacteremia may induce the leukocytosis. although aptt is often moderately prolonged in dogs with cpss, no spontaneous bleeding tendency or hemorrhage occurs. 83, 84 hemoabdomen is a possible postoperative complication after surgical attenuation of a cpss. acquired parenchymal hepatic diseases examination, laboratory tests, diagnostic imaging results and histopathology of liver biopsy specimens are often required to establish a specific diagnosis. intrahepatic cpss tend to cause clinical signs between 2 months and 1 year of age in large-breed dogs. bernese mountain dogs, irish wolfhounds, hovawarts, and retrievers are predisposed. australian cattle dogs and male dogs more likely have right-sided than leftsided intrahepatic cpss, 72 whereas irish wolfhounds tend to have left-sided intrahepatic cpss. intrahepatic cpss in small-breed dogs are very rare. extrahepatic cpss usually occur in small breeds and can cause clinical signs at any age. 73 the most commonly affected breeds are maltese dogs, miniature schnauzers, dachshund, yorkshire terrier, jack russell terrier, and cairn terrier. no clear sex predisposition is known. the waxing-waning nature of central neurologic signs is suggestive of chronic he. symptoms may improve spontaneously. physical examination often fails to detect abnormalities. occasionally an enlarged left kidney is palpated. the presence of ascites detectable with physical examination should exclude cpss. some authors suggest that copper-colored iris is a typical finding in cats with cpss; however, no reports exist about its positive or negative predictive value. dogs with intra-or prehepatic portal hypertension may have ascites. jaundice is absent in congenital portal hypertensive disorders, but may be present in acquired parenchymal liver diseases. blood ammonia concentration fasting (12-hour) venous hyperammonemia is a very specific and sensitive indicator of portosystemic shunting. as a single test, increased blood ammonia level is usually sufficient to diagnose portosystemic shunting. because ammonia is formed from amino groups of proteins and urea also in the collection tube, the blood sample (in an ethylenediaminetetraacetic acid tube) should be placed directly on ice after sampling and the measurement should be performed within 30 minutes. because contamination of the sample with airborne ammonia may cause false-positive results, samples should be taken using a closed system (needle on a syringe or directly into a vacutainer). hemolysis may artificially increase ammonia concentration because erythrocytes contain two to three times more ammonia than the plasma. measurement of ammonia should be performed in a clean location, where the air is not contaminated with ammonia from congested urine or cigarette smoke. a number of analyzers offer the possibility of ammonia measurement; however some of them are unreliable. 46 measuring arterial ammonia concentration provides no additional value. if venous ammonia concentration is within the reference range or only slightly elevated, rectal att is the cheapest and quickest test to exclude or justify the presence of portosystemic shunting in patients with a suggestive history. most of these "suspicious" animals also have high fasting plasma bile acids levels. 74 a 5% ammonium anomalies, a high-resolution grayscale ultrasound is often sufficient to establish a definitive diagnosis. 10 although certain secondary changes (such as small liver, large kidneys with hyperechoic medulla, and sediment or stones in the urinary bladder) suggest the presence of cpss, diagnosing a cpss requires the visualization of the anomalous vein from its origin to its termination. the urinary bladder should always be evaluated for the presence of urinary calculi. ultrasonography can be used not only during the initial diagnostic workup, but also during the surgical treatment and postoperative followup of cpss. 86 intraoperative grayscale ultrasonography facilitates localizing the course of an intrahepatic cpss, 26 whereas intraoperative color and spectral doppler examination helps to determine the optimal degree of shunt attenuation. 87 scintigraphy free 99m tc-pertechnetate administered into the colon is absorbed into the portal vein and appears in the liver first in animals without portosystemic shunting, or in the heart in patients with portosystemic shunting. 88 isotopically labeled albumin macroaggregates may be directly administered into a splenic vein with ultrasound guidance. 89 the macroaggregates are trapped in the first capillary bed, which is normally the liver. the fraction of portal may lead to spontaneous hemorrhages because of the presence of dic. plain radiographs give very limited additional information (such as small liver, possibly enlarged kidneys, and sometimes visible uroliths) to the history and laboratory results, so they don't have to be part of the routine diagnostic workup of vascular liver diseases. pure ammonium biurate uroliths are radiolucent. abdominal ultrasonography is the first choice of diagnostic imaging modality, once the presence of portosystemic shunting has been established by fasting hyperammonemia or abnormal rectal att (see figure 61 -21). the major advantage is that ultrasonography requires no sedation or anesthesia. its drawback is in the operator dependence. by using a systematic examination protocol one can accurately differentiate cpss from apsc, and intrahepatic from extrahepatic cpss, as well as readily diagnose arterioportal fistulas and prehepatic portal hypertensive disorders. 15, 85, 86 although colorflow doppler highly facilitates evaluation of portal vascular injection of iodinated contrast agent into the portal vein or into one of its tributaries under fluoroscopy used to be the only form of diagnostic imaging available to diagnose anomalous vessels. angiography allows identification of extra-and intrahepatic cpss as well as apsc. 90 the major drawback of selective portography is its invasiveness and the need for general anesthesia. whereas mesenteric portography requires catheterization of a mesenteric vein during laparotomy, 91 splenic portography can be performed by ultrasoundguided percutaneous injection of contrast material into a parenchymal splenic vein. visualizing portal vein segments with hepatofugal flow or shunt segments with hepatopetal flow (figure 61-22) is problematic, as no contrast reaches these parts of the vessels. blood that bypasses the liver will be trapped in the pulmonary capillaries. splenic venous injection of isotopes makes exact calculation of the shunting fraction possible (i.e., activity in lungs/activity in liver + lungs); however, the procedure requires anesthesia. scintigraphy is the gold standard diagnostic imaging method to justify or exclude the presence of portosystemic shunting. however, differentiating congenital from acquired portosystemic shunting or intrahepatic from extrahepatic cpss is not possible. information provided by a scintigram is a "yes" or "no" answer to the question: does the patient have portosystemic shunting? this answer can be reached much more easily and without using radiopharmaceuticals by documenting a single high blood ammonia concentration or by rectal att. images (a, b) . arrows indicate the direction of blood flow. a and b, intraoperative color-flow doppler ultrasound images of the portal vein at the point where the congenital splenocaval shunt originates. note that the diameter of the shunt (sh) is larger than that of the portal vein (pvcaudsh). the portal vein cranial to the origin of the shunt (pvcrsh) becomes narrower than the portal vein caudal to the shunt origin (pvcaudsh) because of hypoperfusion. a and c, because blood always flows toward the lowest resistance, 100% of the portal venous blood flows through the shunt (sh) to the caudal vena cava (cvc). note that the blood from the gastroduodenal vein (gdv) finds lower resistance to flow caudally toward the shunt, than toward the liver. this creates a hepatofugal flow (i.e., flow away from the liver) in the portal vein segment between the entering point of the gdv and the origin of the shunt (pvcrsh). note that the portal vein becomes even narrower cranial to the point where the gdv enters it (pvcrgdv). the diameter of the various portal vein segments varies because of the varying amount of blood that flows through a certain segment. b and d, note that partial occlusion of the shunt increases the resistance in the shunt to such an extent that the blood from the splenic vein (splv) finds lower resistance to flow through the shunting vessel toward the portal vein. this reversed flow in the shunting vessel (*) prevents the portal venous blood from shunting. thus, even though the shunt is only partially closed, it is nonfunctional. also note that the blood in the whole length of the portal vein is forced to flow toward the liver (i.e., hepatopetal flow direction), establishing normal perfusion of the sinusoids. a portion of the splenic venous blood will continue to flow through the attenuated shunt to the cvc, but the splenic blood contains no more toxins than any systemic vein, so no hyperammonemia develops. rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. pine receptor increases the effect of gaba on its gaba areceptor. 4 the cornerstone of therapy in cases of cpss (until definitive surgical therapy) and in all cases of apsc is a high-quality, low-protein diet. 32, 33, 98, 99 commercially available renal prescription diets are ideal and preferable to most hepatic diets, as the protein content is higher in the latter. 28 to decrease colonic transit time and reduce the production of ammonia, oral administration of lactulose is recommended. the dosage of lactulose should be titrated in individual patients to yield soft feces, but not diarrhea. an initial dosage 0.5 ml/kg q12h is recommended. if diarrhea results the dosage should be reduced. lactulose also stimulates the growth of colonic bacteria that can incorporate ammonia into bacterial protein. additional use of antibiotics (e.g., neomycin, metronidazole) has been recommended by some to diminish ammonia-producing colonic flora. antibiotic administration is usually not necessary to control clinical signs of he; moreover, neomycin is thought to antagonize the action of lactulose and may cause the release of endotoxins. 100 furthermore, chronic antibiotic use may contribute to the development of antibiotic resistance. surgical closure of apsc is generally impossible because of their multiple nature, but also contraindicated, as apsc are usually a compensatory mechanism to resolve high portal venous pressures. surgical narrowing (i.e., banding) of the caudal vena cava to reduce shunting by increasing the caval pressure is currently not recommended. in cases of cpsss, attenuation or complete closure of the shunting vein is the choice of treatment. the goals of shunt occlusion are (a) reducing portal flow via the cpss and (b) simultaneously increasing portal venous perfusion of the liver. 86 the shunt should be attenuated as close as possible to the point where it enters the systemic circulation. attenuating intrahepatic cpss is more risky because of the possibility of major bleeding because of excessive dissection of hepatic parenchyma. there are several techniques described, of which none are perfect. the major problem with shunt reduction is that blood from the portal vein will be redirected to newly perfuse the liver and its vasculature. poorly developed portal branches have insufficient capacity to accept even normal amounts of portal venous blood, and in the case of complete shunt occlusion, splanchnic congestion will develop. the goal of surgery is to sufficiently reduce the amount of shunting blood without causing portal hypertension to develop. 86 although complete shunt occlusion would theoretically be ideal, partial shunt attenuation is often sufficient because (a) partial attenuation often results in functional closure 18 (see figure 61 -22), and moreover (b) complete anatomic occlusion may follow in many cases. 101 partial occlusion of extrahepatic cpsss often results in a better outcome because the chance for development of postligation portal hypertension is much smaller compared to complete occlusion. 86 the liver, which may be less than 30% of its normal size, grows very quickly following successful surgery. regeneration may take 2 to 3 weeks to complete in uncomplicated cases. the regenerating liver receives progressively more portal blood flow usually resulting in spontaneous complete closure of the cpss. helical ct, especially the multiscale versions, makes excellent images of the abdominal vasculature (including cpss) relatively quickly after intravenous injection of iodinated contrast agents. 92 three-dimensional reconstruction provides impressive anatomic details of shunting vessels. 93 the drawback is the limited availability of the new-generation scanners and the need for patient anesthesia. although magnetic resonance angiography can provide highquality images of the abdominal vessels, 94 it has not become popular because of its limited availability and high costs. patents require general anesthesia and the examination lasts longer than ct angiography. characteristic mri changes have been described in the brains of dogs and cats with cpss, 95 but these findings are more of research than of clinical interest. histopathologic examination of liver biopsy specimens is essential in identifying the underlying disease process in intrahepatic portal hypertensive disorders. in the routine diagnostic workup of canine cpss liver histopathology gives no additional information. currently, the presence of coexistent phpv cannot be diagnosed preoperatively in dogs with cpss. 18 this is because the histologic findings of liver biopsies are identical in the following conditions: cpss, phpv, cpss with phpv, congenital arterioportal fistula, any prehepatic portal hypertensive disorder (e.g., portal vein thrombosis). 13 however, taking liver biopsies for histopathologic examination is recommended in cats before deciding about surgical closure of a cpss. this is because congenital hepatic fibrosis as part of pkd can be present simultaneously, especially in persian and persian crossbreeds. 20 surgical shunt attenuation is not recommended when congenital hepatic fibrosis and a cpss are simultaneously present in a cat. patients with grades 2 to 4 he may present on an emergency basis. the source of ammonia and other protein breakdown products, which cause the clinical signs of he, is the colon. the purposes of the treatment are (a) to reduce the production and amount of ammonia in the colon by removing its content with warm water enema, and (b) to inhibit the absorption of ammonia by administering lactulose syrup into the emptied colon. lactulose may be given as a retention enema with a soft feeding tube 0.5 to 1.0 ml/kg deep rectally). lactulose is a nonabsorbable disaccharide, which is metabolized by the colonic bacteria into short-chain fatty acids. these fatty acids acidify the intraluminal content causing to form ammonium ion (nh 4 + ) from ammonia (nh 3 ). because of its polarity, nh 4 + will not pass the enterocystic membrane, and is instead trapped in the colonic lumen. 96 it is also essential (c) to correct the acid-base and electrolytic disorders of the patient with iv fluid therapy as alkalosis and hypokalemia can worsen the signs of he. seizures caused by he can be controlled with intravenous propofol. administration of benzodiazepines (e.g., diazepam) and barbiturates should be avoided as they can worsen the clinical signs. 97 binding of a benzodiazepine molecule to its neuronal benzodiazeacute portal hypertension. variable degrees of portal hypertension necessarily develop subsequent to any cpss attenuation. if portal hypertension is severe, circulatory collapse may develop because of sequestration of blood in the splanchnic veins. 116 slight abdominal enlargement as a result of ascites requires no intervention. this usually resolves spontaneously within 1 week of surgery. severe ascites with signs of shock (e.g., depression, tachycardia, hypotension, prolonged capillary refill time, hemorrhagic diarrhea) necessitates emergency surgery and removal of the ligature or constrictor ring from around the shunt. these signs usually develop within 24 hours postoperatively. unfortunately, the prognosis following emergency surgery is poor. portal vein thrombosis. this rare postoperative complication causes sudden onset of shock usually within several days of shunt attenuation. 87, 117 exaggerated shunt occlusion, severe portal hypertension, and stasis of portal venous blood are believed to be the cause of the thrombosis. no survivals have been reported. chronic portal hypertension. when the growth of the liver and development of portal branches is insufficient following partial shunt closure, chronic portal hypertension may induce formation of apsc. this may occur as late as 4 to 8 weeks postoperatively. in such cases, evaluation usually reveals partially patent shunt and increased portosystemic shunting as a result of newly formed apsc. the development of apsc has been documented with all surgical methods. apsc can develop as a result of underdeveloped portal branches or exaggerated shunt attenuation. 18 in most dogs, these apsc remain clinically silent because the hepatic mass has substantially increased following shunt attentuation. 18 all patients should be re-evaluated at 1 month after shunt attenuation by measuring fasting blood ammonia concentration and performing an abdominal ultrasonography. if fasting blood ammonia concentration is within the reference range, rectal att should be performed. if fasting hyperammonemia is present or the att is abnormal, ultrasonography should identify whether shunting occurs via the narrowed cpss, apsc, or both. in the latter cases, lifelong conservative therapy with dietary modifications and lactulose is recommended. a normal att result implies complete shunt occlusion and the pet will generally have a very favorable prognosis. a second surgery to reach further shunt attenuation should only be attempted in patients with persistent clinical signs that have high fasting blood ammonia concentration or abnormal rectal att result 3 months after the first surgery. 18, 86 spontaneous gradual shunt closure would not occur beyond 3 months after surgical ligation. this rare and usually fatal complication of shunt attenuation causes generalized seizures 1 to 3 days postoperatively, almost exclusively in cats and small-breed dogs (often in maltese dogs). 31, 100, 118, 119 its occurrence is unpredictable. the pathogenesis is unknown, but the sudden decrease of endogenous benzodiazepine ligands is thought to play a role (i.e., "benzodiazepine withdrawal syndrome"). 4 because cerebral edema is suspected to be the initial disorder, intravenous mannitol (0.5 to 1.0 g/kg iv, during 20 minutes) may be administered when a patient shows subtle central neurologic signs during the first three postoperative days. once seizures have developed, the prognosis is usually very poor. most patients are euthanized because of uncontrollable seizures or persistent neurologic defects. propofol gradual shunt attenuation is believed to reduce the risk of portal hypertension by allowing the portal venous branches to adapt gradually to the increased flow. 102,103 apsc do develop with gradual shunt attenuation techniques (e.g., cellophane banding and ameroid constrictor ring). cystic calculi should be removed during the same surgical procedure, because resolution of ammonium biurate uroliths with dietary management can only be achieved in 30% of cases. the shunting vessel is identified by midline laparotomy and narrowed or completely occluded by a nonabsorbable ligature. 104 portal hypertension is determined by direct measurement or estimated based on subjective criteria including severe intestinal cyanosis, increased intestinal peristalsis, reduction in arterial blood pressure, and compensatory tachycardia as a result of stasis in the splanchnic circulation. 105 intraoperative doppler ultrasonography will greatly facilitate determination of the optimal degree of shunt attenuation in extrahepatic cpss, helping to prevent severe portal hypertension. 86, 87 cellophane banding instead of a ligature, a 3-mm-thin, three-layer-thick cellophane band is placed around the shunting vessel with or without narrowing of the shunt diameter. 106 gradual shunt occlusion takes place as a result of inflammation induced by the cellophane. 107, 108 ameroid constrictor ring a metal ring filled with a thick layer of casein is placed around the shunting vessel. swelling of the casein occurs as it absorbs fluid. because of the outer metal ring, the casein can only expand centripetally causing gradual occlusion of the shunt over 1 to 3 months. 102, 109, 110 the major drawback of this simple technique is that the rate and magnitude of occlusion is uncontrollable and kinking caused by the weight of the device may cause acute fatal portal hypertension. moreover, because of its relatively large size it can only be easily applied on extrahepatic cpss. 111, 112 laparoscopy laparoscopic shunt narrowing (by clips) is a less-invasive alternative for shunt ligation (see chapter 28) . 113 with this minimally invasive intravascular technique metal spirals with thrombogenic fibers are placed in the shunting vessel. the coils are delivered via a catheter, which is inserted through the jugular vein into the shunt under fluoroscopic guidance in anesthetized animals. 114, 115 the coils cause thrombus formation in the shunt resulting in its partial or complete occlusion. to prevent coil dislodgment from the shunt, an intravascular stent is often placed in the caudal vena cava to cover the point where the shunt enters the caudal vena cava. coiling is an especially attractive method for treating intrahepatic shunts because liver dissection is thereby avoided. in my opinion, the safest and most effective way for attenuation of extrahepatic cpss is doppler ultrasound-guided partial attenuation via surgical ligature. 86, 87 for intrahepatic cpss, coil embolization appears to be an excellent method. ameroid constrictor and cellophane banding would be ideal in extrahepatic cpss, when the portal vein cranial to the shunt origin is severely hypoplastic and the patient is at risk for severe portal hypertension. spironolactone alone does not resolve ascites, furosemide may be added to the therapy. abdominocentesis for removal of abdominal effusion is not recommended because of loss of protein and exacerbation of starling forces permitting further fluid accumulation. dietary and medical management relieves clinical signs only temporarily in most symptomatic young dogs. 99 without surgical shunt attenuation, gradual deterioration of liver function occurs; consequently, conservative therapy alone offers a guarded prognosis in young dogs. in older animals (>6 years) with newly reported signs of cpss, lifelong conservative treatment may be recommended because of the significantly higher complication rates of surgical therapy in these patients. 105 the prognosis depends on (a) whether the cpss is intra-or extrahepatic, (b) the coexistence of phpv, (c) the extent of shunt attenuation, (d) experience of the surgeon, and (e) the age of the dog at the time of diagnosis. intrahepatic cpss generally has poorer prognosis. complete resolution of clinical signs can be expected in approximately 60% to 80% of dogs with extrahepatic cpss in the hands of an experienced surgeon. this same parameter is approximately 50% to 70% for intrahepatic cpss. [102] [103] [104] [105] with extrahepatic cpss, an excellent prognosis can be expected when blood flow is hepatopetal (i.e., toward the liver) in the portal vein segment, which is cranial to the shunt origin. this can be established preoperatively with doppler ultrasound. 86 in cats, regardless of the shunt type and the surgical method, the success rate is approximately 30% to 50% because of the development of postoperative central neurologic signs. 105, 122, 123 the prognosis of portal hypertensive disorders depends on the underlying disease. in the majority of acquired diseases the underlying disorder is chronic and so severe that even stopping the disease process will not cause regression of apsc. primary liver neoplasms are infrequent in the dog and cat, with an estimated prevalence in necropsy studies of 0.6% to 2.6% in the dog and 1.5% to 2.3% in the cat. liver metastases are more frequent than primary hepatic tumors in the dog, and tend to originate from the spleen, pancreas, and gi tract. primary hepatobiliary tumors are more common than metastatic disease in the cat. [1] [2] [3] [4] [5] the etiology of liver cancer in dogs and cats is incompletely understood. potential causes such as aflatoxins, nitrosamines, food additives, parasites, and radioactive compounds have been reported. [6] [7] [8] liver cancer in the dog has many clinical, pathologic, and histologic homologies with liver cancer in humans. [9] [10] [11] [12] in human medicine, chronic diseases of the liver, such as hepatitis b or c infection, as well as cirrhosis, are often associated with (1 to 5 mg/kg, iv) followed by constant rate infusion [cri]) may be used to control seizures. 120 prophylactic treatment with phenobarbital does not reduce the risk of development of postligation neurologic complications, 118 but may be used in long-term seizure management. preventive use of potassium bromide has not been shown to reduce the possibility of postoperative seizures. in every patient with central neurologic signs in the early postoperative period, hypoglycemia and he should be excluded by measuring venous glucose and ammonia concentrations, respectively. blindness and other types of central neurologic signs may develop in cats shortly after surgery. the pathogenesis of these changes is unknown. hemorrhage from liver biopsy sites or shunt dissection in cases of intrahepatic cpss can lead to hypovolemic shock and death in the early postoperative period. dogs with hepatic insufficiency are prone to develop hemorrhagic complications because of decreased concentrations or abnormal synthesis of coagulation factors. 84 postoperative portal hypertension may also contribute to bleeding tendency from hepatic parenchymal dissection. hemoabdomen should be carefully differentiated from severe portal hypertension and septic peritonitis as they all can cause shock and variable degrees of abdominal distention. coagulation parameters (e.g., pt, aptt) should be routinely monitored after surgery and if they are abnormal or if there is evidence of clinical bleeding, fresh-frozen plasma transfusion should be administered (10 to 20 ml/kg, iv). toy-breed dogs are prone to develop hypoglycemia during or shortly after surgery. 100 blood glucose concentrations should be regularly monitored and hypoglycemia should be treated with glucosecontaining infusions. specific treatment should address the underlying parenchymal liver disease based on the histologic results of liver biopsy. he can often be controlled with dietary modification and lactulose. currently, no specific treatment exists for portal venous hypoplasias. renal or hepatic prescription diets and lactulose may alleviate clinical signs of he in case of apsc. diuretic agents may be useful in dogs with ascites. liver lobe resections have been reported in animals with congenital arterioportal fistulas. however, simultaneous presence of phpv in the whole liver prevents postoperative resolution of portal hypertension and the portosystemic shunting via apsc. 10, 25 therefore, the pet owner should be educated that partial hepatectomy may not result in complete recovery of portosystemic shunting, and that lifelong dietary support and/or lactulose will likely be required. severe abdominal effusions should be treated with diuretic agents. 121 the first choice is spironolactone (1 to 2 mg/kg q12h), an aldosterone receptor antagonist as (a) chronic hepatic insufficiency is associated with hyperaldosteronism, (b) concurrent hypercortisolemia is associated with cortisol binding to the mineralocorticoid receptor, and (c) potassium-sparing diuretics prevent development of hypokalemia and alkalosis, both of which would worsen signs of he. if show no clinical signs, and liver cancer is suspected only with increases in serum liver enzyme activities. 3, 5, 7, 23 the most common physical examination findings are cranial abdominal mass (35%), abdominal bloating (30%), and jaundice (18%). jaundice is a less common finding in cases of metastatic cancer. other manifestations include neurologic signs as a result of he; paraneoplastic syndromes such as hypoglycemia and myasthenia gravis; and skin alterations consistent with hepatocutaneous syndrome. 7, 24 no clear breed predisposition is observed with canine liver cancer, although poodles, fox terriers, miniature schnauzers, labrador retrievers, and male dogs are overrepresented in some reports of hepatocellular carcinoma. 1, 12, 19, 25, 26 labrador retrievers and female dogs are overrepresented in reports of bile duct carcinoma. 1, [26] [27] [28] whether male and female cats are equally at risk for bile duct carcinomas is unsettled, 28, 29 but male cats appear to be at greater risk for bile duct adenomas. 30, 31 neuroendocrine tumors are generally observed in younger animals. 1, 32 diagnosis definitive diagnosis of liver cancer can be made only by liver biopsy. asymmetrical enlargements of the liver detected on physical examination, abdominal ultrasonography, or survey radiography should not be assumed to be neoplastic in origin. similarly, laboratory data do not distinguish hepatic neoplasia from other liver pathologies. 33 the clinical approach to an animal with suspected liver cancer should include basic information such as a complete blood cell count, serum biochemistry, coagulation tests, urinalysis, thoracic and abdominal radiographs, abdominal ultrasound ( figures 61-23 and 61-24) , and fine-needle or core biopsy of the liver. a tnm tumor classification system has been used for staging of liver cancer where t represents tumor (t0, no evidence of tumor; hepatocellular tumors; however, there is no established association between hepatic tumors and viral infections in the dog or cat. moreover, canine hepatic cirrhosis does not appear to predispose to hepatocellular carcinoma (hcc). 1 a possible association between hookworm or whipworm infection and liver cancer has been reported, and cats with chronic cholangitis may have an increased predisposition to biliary carcinoma. 7, 13 several liver mitogens and tumor suppressor genes such as epidermal growth factor, tgf-α, vascular endothelial growth factor (vegf), p53, and tgf-β and its receptors (tgf-β-r) have been associated with liver cancers in humans and these may play a similar role in the dog. [14] [15] [16] [17] in human medicine a small percentage of hccs or cholangiocarcinomas originate from hepatic progenitor stem cells. dogs diagnosed with hcc or cholangiocarcinoma do demonstrate activation of hepatic progenitor stem cells in response to liver injury, but hepatic progenitor stem cell expression in liver tumors is relatively low. 18 the hypothesis of cancer development as a multistep process applies to liver tumors in the dog and the cat, as well. 7, 19 precancerous lesions, such as dysplastic nodules, can be identified before the development of overt malignancy in humans. dysplastic nodules are characterized by cell atypia, cellular crowding, trabecular thickness, microacini, and histochemical markers. 20 dysplastic nodules have not been reported in the dog or cat and further studies are needed to understand the chronology of hepatic malignancy in domestic animals. preliminary reports of histochemical markers have been reported in dogs with hyperplastic hepatic lesions and hepatocellular and biliary neoplasms. 11 liver tumors cause damage to the liver by several mechanisms: inflammatory effects, obstruction of the biliary system, obstruction of the vascular compartment or adjacent organs, and spontaneous rupture with hemoabdomen. 21 hepatic tumors are usually resistant to chemotherapy. 7, 19 in one recent study, p-glycoprotein was more highly expressed in hcc than in cirrhosis, which is consistent with the known resistance of hcc to chemotherapy. p-glycoprotein, which is encoded by the multidrug resistance gene (mdr-1), is normally expressed in tissues with excretory function, including the jejunum, kidney, liver, and adrenal gland. 22 primary liver neoplasms are usually classified according to their cellular origin and macroscopic appearance. with respect to cellular origin, these tumors may be hepatobiliary, hematopoietic, sarcomas, or metastases of other tumors (box 61-2). in relation to macroscopic appearance, they can be classified as lobular, multiple nodular, or diffuse (). the combination of histopathologic and morphologic classification has consequences for prognosis and treatment strategy in these animals (tables 61-6 and 61-7) , and the clinician must always address these factors to arrive at correct management decisions. in dogs, malignant tumors are more common than benign lesions. in cats, biliary neoplasms are the most common presentation, particularly intrahepatic benign forms. 6, 7 most animals with liver neoplasia present with nonspecific clinical signs such as anorexia and weight loss. less-frequent clinical signs include vomiting and diarrhea, pd and pu, pale mucosal membranes, and acute weakness because of anemia and hypovolemia coincident with tumor rupture. 7, 19 up to 25% of affected animals diffuse or infiltrating, multiple coalescent nodules in all the lobes, or diffuse disappearance of the liver parenchyma; lobular, nodule or large mass in a single liver lobe; nodular, several nodules throughout the liver parenchyma, or several affected liver lobes. t1, tumor involving one lobe; t2, tumor involving more than one lobe; and t3, tumor invading neighboring structures); n represents regional lymph nodes (rlns) (n0, no evidence of rln involvement; n1, rln involved; n2, distant ln involved); and m represents distant metastasis (m0, no evidence of metastasis; m1, distant metastasis detected). although recommended, this system has not been universally adopted. 34 table 61 -8 illustrates the most typical hematologic and biochemical findings in dogs and cats affected with liver neoplasia. leukocytosis is a result of inflammation and necrosis of large tumors; anemia tends to be moderate and nonregenerative and is thought to be caused by chronic illness, inflammation, or iron deficiency 19 ; thrombocytosis is attributable to a paraneoplastic syndrome characterized by thrombopoietin production, iron deficiency, or anemia. 7 serum liver enzyme elevation is a frequent, but not universal, finding in animals with liver neoplasia. it should be noted, however, that the degree of serum enzyme elevation does not correlate with the degree of liver involvement or severity of disease. in one survey, animals with primary liver tumors tended to have greater elevations in serum alt and alp activities than animals with metastatic disease, while the latter tended to have greater elevations in serum bilirubin and ast. 35 it also has been suggested that an ast-to-alt ratio of less than 1 is more compatible with carcinoma, while a ratio of greater than 1 is more indicative of a sarcoma or carcinoid. 1 other reported biochemical changes include hypoglycemia, hypo-or hyperalbuminemia, and increased serum bile acids. hypoglycemia as a paraneoplastic syndrome associated with hepatocellular carcinoma is attributed to the secretion of insulin-like growth factor ii. 36 unlike dogs, cats usually present with a high incidence of serum creatinine and bun elevations. 28, 29 coagulation factor abnormalities are more commonly associated with hemangiosarcoma, although dic may be evident in end-stage liver cancer or in decompensated patients. coagulation studies should always be performed before undertaking invasive diagnostic procedures. 23 serum α-fetoprotein has been evaluated in the dog, and increases are reported in 75% of animals with hepatocellular carcinoma, and in 55% of those with biliary carcinomas. the use of this biomarker is limited by the fact that it is increased in cases of hepatic lymphoma and other liver pathologies, and only very dramatic elevations in α-fetoprotein may be taken to indicate hepatocellular carcinoma. [37] [38] [39] abdominal radiographs often reveal a mass effect in the cranial abdomen, although this finding will depend upon the size of the neoplasm and the number and size of metastatic tumors. other reported findings include dorsal displacement of the stomach, hepatomegaly, loss of abdominal detail (because of the presence of free abdominal fluid), and, occasionally, biliary tract calcification. thoracic radiography should be considered as part of the staging procedure for animals with metastatic disease. 25, 40 changes in the ultrasound density of the liver may take a variety of forms (box 61-3). most changes are not pathognomonic for a given disease process, and the final diagnosis is established only on the basis of clinical findings, laboratory testing, and results of cytology or histopathology (see . ultrasound is also very useful for evaluating other abdominal structures, and for the staging of cancer. [40] [41] [42] • diffuse or multifocal liver neoplasms tend to present with hepatomegaly, but this depends on the degree of infiltration. liver carcinomas can be diffuse or affect multiple lobes, with variable ultrasound characteristics depending on the presence of necrosis, inflammation, hemorrhage, or cavitation. in these malignant tumors it is common to observe a mixed echogenicity pattern. lymphoma can affect the liver without detectable ultrasound changes, or cause diffuse hypoechogenicity, hyperechogenicity, or mixed echogenicity with or without hypoechoic nodules. consequently, if lymphoma is suspected, even if the liver ultrasound findings appear normal, fine-needle aspiration cytology is recommended. histiocytic neoplasms are more often associated with multiple nodules and hypoechoic masses, although diffuse liver hypoechogenicity has also been described. mast cell infiltration of the liver tends to produce diffuse hyperechogenicity. • nodular patterns • benign nodular hyperplasia is common, particularly in dogs, and accounts for many of the focal liver lesions identified at ultrasound exploration. it has been estimated that 25% to 36% of all nodular masses detected in the liver are nodular hyperplasia. • benign liver adenomas or hepatomas can manifest as a focal mass of variable size and of normally hyperechoic characteristics. • the liver is a frequent location of metastatic spread, fundamentally through the portal system that drains most of the abdominal structures. • primary liver neoplasms such as hepatocellular carcinoma can present as focal or multifocal masses, although less often so than in the case of metastases. focal hypoechoic lesions with a hyperechoic center or core (referred to as target or bull's-eye lesions) are usually associated with metastases, although some benign processes, such as nodular hyperplasia, can generate similar patterns. • biliary obstruction: ultrasound has become an important tool for evaluating biliary obstruction in icteric dogs and cats. primary tumors of the liver, biliary tract, duodenum, or pancreas are capable of causing biliary obstruction. liver cytology has obvious limitations in that it cannot distinguish between liver adenomas and regenerative nodules, and even some hepatocellular carcinoma aspirates may be composed entirely of normal-appearing hepatocytes. in many cases it may prove necessary to resort to ultrasound-guided biopsy, laparoscopy, or exploratory laparotomy. however, cytology may prove useful in determining the presence of lymphoma, mastocytoma, and histiocytic sarcoma, as well as contribute to the initial classification of tumor type. concordance rates between cytology and histopathology findings may be good for some disease processes, but the reported concordance rate varies from 14% to 86%. 44, 45 the treatment to be provided and the prognosis of animals with primary liver cancer depend on the cell of origin, degree of malignancy, and clinical presentation. the clinician should quickly determine if surgery, chemotherapy, radiation therapy, or palliative care is the treatment of choice in individual patients. palliative treatment is the option for animals that are not surgical candidates, for example, tumors with poor response to systemic chemotherapy, and for whom pain management and general liver failure treatment are the best recommendations. the success of newer options such as chemoembolization, metronomic therapy, antiangiogenic drugs, and tyrosine kinase inhibitors in the treatment of these patients has not been clearly established. [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] the macroscopic presentation is clinically very important (see figure 61 -26), as 100% of the diffuse forms have metastasis at the time of diagnosis, versus 37% of the isolated (massive or nodular) clinical presentations. 27 it should be noted, however, that some dogs with massive hcc present without metastasis, and deaths in these cases may be unrelated to hcc. 1, 26, 54 histopathologic subtype and anaplastic characteristics in general influence the prognosis and predictability of metastasis. 1, 26, 55 metastatic spread usually affects the regional lymph nodes, lungs, and peritoneum. 26, 56 high-field mri scanning has an accuracy of 94% in differentiating malignant from benign lesions with a sensitivity and specificity of 100% and 90%, respectively. mri classified malignant hepatic lesions as hcc in all confirmed cases and correctly predicted the histologic grade of five hcc lesions. these results suggested that mri is a useful modality for abdominal imaging in veterinary patients, and that mri accurately differentiates benign from malignant focal hepatic lesions. 43 liver cytology is useful in the initial evaluation of hepatomegaly and usually permits differentiation between primary tumors, metastatic disease, and focal infection (see . however, cytology does not distinguish between benign focal inflammatory disease and progressive chronic liver disease, and it cannot establish the extent and distribution of disease. likewise, a definitive diagnosis of regenerative nodular hyperplasia cannot be established, and the technique is unable to differentiate a benign inflammatory reaction from cell changes associated with other pathologies. contraindications to ultrasound-guided cytology include the following: • coagulation abnormalities-if one or more coagulation test parameters are altered, it is advisable to administer vitamin k 1 via the subcutaneous route 12 hours before cytology. • cavitary masses-the ultrasound detection of a large cavitary lesion in an elderly dog usually contraindicates cytology, a b tumors are more common in cats. 1, 13, 27, 61 three morphologic forms or presentations have been described: lobular, multifocal, and diffuse. in general, only the lobular form should be considered for surgical removal as long as there is no evidence of metastasis. the prognosis for multifocal and diffuse bile duct carcinomas is very poor, surgery is usually not feasible, and most animals die within 6 months of surgery. 29 no effective chemotherapeutic options have been described for these malignancies in dogs or cats. also known as biliary cystadenomas, biliary adenomas, cholangiocellular adenomas, and cholangiomas, these tumors are common findings in aging cats. males appear to be more frequently affected than females (figure 61-28 ). in cats, 50% of these lesions are isolated or lobular and 50% are multifocal. 59 biliary duct adenomas usually do not cause clinical signs unless they grow and compress other structures. 62 despite the benign nature of these tumors, surgical removal is usually recommended because malignant transformation is always possible and because expansion into the porta hepatis may cause life-threatening consequences. 63 liver lobectomy is recommended for cats with single bile duct adenoma or multifocal tumors confined to one or two lobes. in cats, surgical resection of biliary adenomas may provide cure or tumor-free survival of several years. 28, 29, 31, 59, 62, 63 carcinoid tumors neuroendocrine (carcinoid) tumors are infrequent in the dog and cat. in dogs, carcinoids have an aggressive biologic behavior and are usually not amenable to surgical resection as they tend to present as diffuse lesions (see figure 61 -25). 1, 27 carcinoid tumors in dogs have also been described in the gallbladder, and these have been managed successfully with cholecystectomy. 64, 65 carcinoid tumors in cats can be intrahepatic or extrahepatic involving the bile duct and occasionally the gallbladder. 65 the extrahepatic form of carcinoid tumors may cause biliary tract obstruction, icterus, and increases in serum hepatic enzyme activities. biliary tract diversion procedures should be considered for obstructive lesions involving the extrahepatic biliary tract. unlike the circumstance in dogs and humans, female cats are more often affected by these tumors than males (female-to-male ratio of 5 : 1). 66 the prognosis of carcinoid liver tumors in dogs and cats is generally poor, and metastatic disease is present in 90% of the cases at prognostic factors in dogs with massive hcc include need for surgery, liver lobe involvement, serum alt and ast activities, and ratios of alp to ast and alt to ast. 26 liver lobectomy is recommended for cats and dogs with hepatic tumors that have a massive morphologic appearance without metastases. however surgical complications are reported in more than 28% of cases, with a mortality rate of almost 12%. 26 the predilection of massive hcc for left-sided liver lobes has been reported. 27, 54 advanced imaging and intraoperative ultrasonography may provide useful information on the relationship of right-sided and central liver tumors to the caudal vena cava prior to liver lobectomy. 40, 41, 43 even though right-sided liver tumors have a poorer prognosis because of intraoperative death, there is no difference in the survival time after successful surgery. 26 the considerable regenerative capacity of the liver can permit successful resection of up to 80% of hepatic mass if the remaining tissue is functionally normal and critical supportive care is provided. 56 the median survival time for dogs with massive hcc following liver lobectomy is greater than 4 years. without surgery the average life expectancy is 270 days and the prognosis is generally considered poor. 26 tumor recurrence in dogs with massive hcc is rare and reported to be 0% to 13% after lobectomy. 26, 54 the prognosis for dogs with nodular and diffuse hcc is poor. surgical resection is usually not possible because of involvement of multiple liver lobes. no effective systemic chemotherapy or radiation therapy protocols have been described for hcc treatment. hcc is considered chemoresistant in humans although mitoxantrone has been reported to be helpful in some cases. 7, 56, 57 the most likely reason for the poor response to systemic chemotherapy is the expression of p-glycoprotein in hepatocytes. 22 treatment options for nodular and diffuse hcc in humans include liver transplantation and minimally invasive procedures for regional control, such as ablation, chemoembolization, immunotherapy, hormonal therapy, and low-dose metronomic chemotherapy. 56, 58 a recent report recommends therapy with sorafenib, a multikinase inhibitor and antiangiogenic agent. 47, 48 chemoembolization is a procedure commonly used in the treatment of diffuse hepatocellular carcinoma in humans with median survival times of 1 to 2 years compared with 3 to 6 months with systemic chemotherapy. 52, 51 in veterinary medicine, chemoembolization has been reported with moderate success in the palliation of four dogs with hcc. 52, 53 in cats, hepatocellular carcinoma is less frequent, and less data are available. 28, 59 hepatocellular adenomas these tumors are also known as hepatomas and are more common in cats than in dogs. in the dog it is sometimes very difficult to distinguish adenoma from reactive nodular hyperplasia, and biopsy is needed to clarify the diagnosis. the prognosis for adenomas is usually good, but it is advisable to remove focal mass lesions because they can grow and spontaneously rupture with severe bleeding. 19 bile duct carcinoma (adenocarcinoma and cholangiocarcinoma) bile duct carcinoma is the most common liver malignancy in the cat, and the second most common liver malignancy in the dog (see figure 61 -27) . tumor behavior is very aggressive in both species, and metastases are present at the time of diagnosis in 60% to 88% of cases. bile duct carcinomas usually metastasize to the regional lymph nodes, lungs and peritoneum, kidneys, heart, adrenal glands, eye, and bone. 1, 60 bile duct carcinoma can be intrahepatic or extrahepatic, but rarely occurs within the gallbladder. intrahepatic bile duct tumors are more common in dogs, and extrahepatic bile duct must always be considered as a possibility when a hepatic tumor is diagnosed. benign mesenchymal neoplasms, such as fibroma and hemangioma, have been described but are quite rare. 6, 1, 29, 59 lymphoma in dogs the liver can be involved in variable forms of lymphoma, including multicentric, alimentary, and hepatosplenic forms. a study in cats documented that abdominal lymphoma is currently the most common anatomic location and the liver occasionally is the only organ involved. 69, 70 many protocols are recommended for treatment of lymphoma in dogs and cats; most include vincristine, cyclophosphamide, and prednisone, with variable combinations of l-asparaginase, methotrexate, and doxorubicin. careful evaluation of liver function is necessary before starting chemotherapy because many drugs undergo hepatic metabolism and altered hepatic clearance may lead to unpredictable and potentially increased toxicity. 5 surgical resection with liver lobectomy is recommended for cats with primary hepatic myelolipoma and the prognosis is excellent with prolonged survival time and no reports of local recurrence. 7 in dogs with advanced disease, mast cell tumors can metastasize to the liver. primary visceral mast cell tumors are more common in cats than dogs. the spleen is usually the primary site with metastasis to the liver and bone marrow, and the survival time with splenectomy alone can be a year or more. 71 the overall prognosis for disseminated mast cell tumor in the dog is grave. the median survival time reported in one study was 43 days despite therapy with various chemotherapy agents. 72 canine mastocytoma involving the liver can be controlled with cyclophosphamide, vinblastine, and prednisone. 73 recently, tyrosine kinase inhibitors have shown some promise and ccnu has been shown to be active against feline mast cell tumors. 49, 74 hepatic nodular hyperplasia is a common benign lesion observed in the liver of older dogs that can occasionally be observed in some cats. it is characterized by a discrete accumulation of hyperplastic hepatocytes arising as either macroscopic or microscopic hepatic nodules. it reportedly occurs in 70% of dogs older than 6 years and 100% of dogs over 14 years. [75] [76] [77] the wsava standards for clinical and histologic diagnosis of canine and feline liver diseases include hepatic nodular hyperplasia in its classification system of hepatocellular neoplasia so that it may be differentiated from true neoplasia. 78 the etiology of hepatic nodular hyperplasia is unknown. it has been suggested to be a preneoplastic lesion, 76 but this has not yet been reported in the dog. 77 because of hepatocyte microscopic changes, it is suggested that nutritional and metabolic disorders play a role in the pathogenesis of this lesion. 77 hepatic nodular hyperplasia is characterized microscopically by well-differentiated hyperplastic hepatocytes with increased mitotic activity. [75] [76] [77] hyperplastic nodules may be accompanied by concurrent focal intrahepatic cholestasis, mechanical compression on a higado b the time of diagnosis. 7 a better prognosis is observed with extrahepatic carcinoids with a life expectancy of more than a year. 64, 65 liver sarcomas primary liver sarcomas are rare in the dog and cat. hemangiosarcoma is the most frequent primary hepatic sarcoma in cats and leiomyosarcoma the most common in dogs. 7, 19 there also have been reports of hepatic fibrosarcoma, rhabdomyosarcoma, osteosarcoma, liposarcoma, and histiocytic sarcomas in both animal species. 1, 28, 29, 55 these are usually very aggressive tumors, metastasizing in 86% to 100% of cases to the spleen and lungs, or spreading diffusely within the liver. 5 chemotherapy has not been studied in the treatment of primary hepatic sarcomas, although, similar to other solid sarcomas, response rates are likely to be poor. histiocytic sarcomas respond partially to ccnu, with a mean duration of remission of 85 days and a survival of 172 days. 67 continuous low-dose oral chemotherapy may be an effective alternative to conventional high-dose chemotherapy for adjuvant therapy of dogs with hemangiosarcoma. 50 mass resection may offer some palliation in the circumstance of tumor hemorrhage despite irrefutable evidence of metastasis. a cat with a primary extraskeletal hepatic osteosarcoma was treated with surgery and carboplatin and was alive 42 months after diagnosis with no clinical evidence of disease. 68 on the other hand, metastases feline hepatic lipidosis (hl) is a metabolic syndrome found in obese, middle-aged cats that undergo a period of acute anorexia and catabolism. morbidly obese cats are at increased risk and more than 85% of cats with hl suffer from an underlying disorder that contributes to the initial anorectic event. [1] [2] [3] [4] [5] although the underlying pathogenesis of hepatic lipid accumulation in cats has not yet been completely elucidated, several unique biochemical and nutritional features place this obligate carnivore at risk for fat mobilization and fatty infiltration of the liver during periods of anorexia or starvation (box 61-4). 2, [6] [7] [8] [9] [10] [11] there is a general consensus that reduced caloric intake and protein-calorie malnutrition are important predisposing factors. the result is a rapid mobilization of peripheral fat culminating in fatty accumulation in the liver. 1 intracellular processing of fats is an important function of the hepatocyte. during fasting or starvation, fatty acid metabolism becomes deranged in an obligate carnivore as a result of obesity, catabolism, chronic overnutrition, impaired fatty acid oxidation or vldl secretion, and enhanced hepatic fatty acid synthesis ( figure 61 -29). 1,6-11 hl is a disorder of middle-aged to older cats; domestic short-haired cats are more commonly affected. cats with hl often present with a history of acute stress and/or near-complete anorexia of several days duration. [1] [2] [3] icterus is a variable feature of hl. when serum bilirubin concentrations exceed 1.5 mg/dl, clinical icterus can be observed on the pinnae, mucous membranes, sclera, and hard palate in the cat. in general, most cats with hl are obese at the time of presentation, with many cats being 20% to 30% over ideal body weight prior to an episode of hl. other physical features of hl include hepatomegaly, dehydration, vomiting, and weakness. if he develops as a consequence of hl, neurologic abnormalities such as ptyalism, stupor, coma, ataxia, and seizures may be observed. [1] [2] [3] a minimum database, including complete blood cell count, serum chemistry, and urinalysis, almost always reveals severe liver enzyme elevation and other abnormalities such as nonregenerative anemia, stress leukogram, poikilocytosis, and bilirubin crystalluria. 1 the pattern of liver enzyme elevation is typically cholestatic in nature and characterized by marked increases in serum alp activity, followed by smaller increases in serum alt and serum ast activities. serum ggt activity is often normal in affected cats. increased serum bile acids and bilirubin are often observed in cats with hl, and electrolyte abnormalities, such as hypophosphatemia and surrounding hepatic parenchyma, as well as alterations in the microvascular circulation. vacuolar changes are seen frequently, suggesting a reactive or metabolic condition such us hyperadrenocorticism, lipidosis, or hypothyroidism. 77 nodular hyperplasia affects older dogs with a mean age of 11 years without gender or breed predisposition. hepatic nodular hyperplasia does not appear to cause clinical signs or illness. 77 laboratory findings may include mild to marked increases in serum alkaline phosphatase activity and, less commonly, increases in serum alt activity. liver function tests are usually normal with hepatic nodular hyperplasia. 77 hepatic nodular hyperplasia is usually discovered as an incidental finding during a diagnostic workup for other medical problems. nodular hyperplasia is clinically important because it may easily be confused with primary or metastatic hepatic neoplasia during abdominal ultrasound or at surgery. even microscopically, it may be impossible to differentiate hepatic nodular hyperplasia from hepatocellular adenomas, and a large sample (wedge rather than needle biopsy) may be required to confirm hyperplasia from welldifferentiated hccs. [75] [76] [77] routine abdominal radiographs are generally unremarkable and ultrasonographic features are inconsistent because of the varied hepatocellular morphologic characteristics and size of the nodules. 79 multiple nodules varying in size, distributed randomly among the liver lobes, being superficial or deep within the parenchyma are found in most cases. 77 hyperplasic nodules of hepatocytes need to be differentiated from regenerative nodules. hyperplasic nodules develop in livers of normal mass, whereas regenerative nodules arise as a result of compensatory hyperplasia of surviving hepatocytes in a background of hepatic injury, atrophy, and fibrosis. no treatment is usually required. rupture of large nodules may require emergency mass removal and blood transfusion (rare). hepatic nodular hyperplasia has no significance in the morbidity of affected patients. 77 metabolic disorders of the liver are commonly encountered in companion animal practice. this section focuses on the metabolic liver disease induced by concurrent endocrinopathies (hyperthyroidism, hypothyroidism, diabetes mellitus, and hyperadrenocorticism), lipid disturbances (lipoproteinemias, feline hepatic lipidosis, and hyperlipidemias), and metabolic infiltration (amyloidosis). hepatic lipidosis and hyperthyroid hepatopathy are the primary metabolic hepatopathies in cats. in dogs, steroid (or glycogen vacuolar) hepatopathy is the most frequent metabolic liver disorder; diabetic hepatopathy and hyperlipidemic hepatopathies (lipoproteinemias, hypothyroidism) occur less commonly. • essentiality of dietary arginine 6 • low levels of hepatic ornithine 7 • high dietary protein requirements 7 • lack of hepatic enzyme adaptation to low protein 8 • insufficiency of hepatic glutamate reductase 7 • insufficiency of intestinal ornithine transcarbamylase 7 • diversion to orotic acid metabolism 9 • differences in lipoprotein metabolism (hdls) 10, 11 to 90 kcal/kg body weight in most cats. 1, 18 unless he is present, dietary protein should not be restricted (ideal is 35% to 45% protein on a dry matter basis) and even then protein restriction is controversial as protein is needed to support hepatic regeneration. feeding multiple small frequent meals may help to maintain euglycemia and lessen the metabolic impact on the liver. the protein content of the diet should be considered when he is present (see chapter 32). dairy and vegetable-based proteins are higher sources of branchedchain amino acids than meat-derived proteins and may lessen the signs of he. diets high in fiber generally should be avoided because they decrease the nutrient density of the diet. cats with hl occasionally may experience a refeeding syndrome, a condition that results in metabolic and electrolyte disturbances. 12 with the reintroduction of food, insulin secretion promotes intracellular uptake of phosphorus, potassium, and magnesium. hypophosphatemia can result in muscle weakness and hemolytic anemia. gradual reintroduction of food and correction of electrolytes diminishes the risk of refeeding syndrome. glucose intolerance and hyperglycemia are common in cats with hl and can be addressed by decreasing the carbohydrate content of the diet. canned low-carbohydrate, high-protein formulations without added fiber are ideal for the treatment of feline hl as they provide amino acids, limited carbohydrates, and water, and are easily administered through a feeding tube. small amounts of food should be administered via the feeding tube after residual gastric fluid contents have been removed. trickle feeding can be performed by placing liquefied food into an empty fluid bag and allowing gravity to force flow into the feeding tube. alternatively, a large-bore hypokalemia, may be frequently observed. in particular, the presence of hypophosphatemia should alert the clinician to the possibility of refeeding syndrome. 12 presumptive diagnosis of feline hl can be made on the basis of clinical history, physical examination, clinicopathologic features, ultrasound examination, and liver aspirates. 1, [13] [14] [15] ultrasound examination of the liver often reveals hepatic parenchyma that is hyperechoic to that of falciform fat, but a thorough ultrasound evaluation of the gallbladder, pancreas, intestines, kidneys, bladder, and other abdominal structures is essential to rule out other primary disorders, such as acute pancreatic necrosis, which may be the basis of the anorectic event precipitating an episode of hl. definitive diagnosis is best achieved through liver biopsy 16 ; however, anesthesia and biopsy may not be possible in acutely ill patients because of the presence of coagulopathies from vitamin k deficiency. 17 a liver aspirate that reveals more than 80% fatty infiltration of the hepatocytes may be used for presumptive diagnosis of hl. if there is no response to treatment after 3 to 5 days, liver biopsy may be necessary to rule out other underlying hepatobiliary conditions such as cholangitis. a catabolic state develops quickly in the anorexic cat and prompt measures should be taken to place an enteral feeding tube (table 61-9) . nasoesophageal, esophageal, and gastrostomy tubes can be used for this purpose. the caloric needs should be approximately 60 increases in serum ast and alt activities have been reported in approximately 80% of hyperthyroid cats. 22, 23 liver enzyme elevation has been attributed to increased liver metabolic activity compared to blood flow. long-term untreated hyperthyroidism in human beings can ultimately lead to cirrhosis. [24] [25] [26] middle-aged to older cats are typically affected, and there is no breed or sex predilection. because hyperthyroidism is characterized by hypermetabolism, polyphagia, weight loss, pd, and pu are prominent features of the disease. 22, 23 hyperactivity, tachycardia, pupillary dilation, and behavioral changes are also characteristic of the disease and are associated with activation of the sympathetic nervous system. long-standing hyperthyroidism leads to hypertrophic cardiomyopathy, high-output heart failure, and cachexia. long nails, dermatologic conditions, panting, elevated body temperature, and poor grooming or overgrooming are additional clinical signs of feline hyperthyroidism. clinicopathologic features of hyperthyroidism include erythrocytosis and stress leukogram (neutrophilia, lymphocytosis) caused by increased circulating catecholamine concentrations. increased catabolism of muscle tissue in hyperthyroid cats may result in increased bun, but not serum creatinine. most cats will have decreased urine specific gravity, particularly if they are exhibiting pu as a clinical sign. increased metabolic rate results in liver hypermetabolism, therefore serum activities of liver enzymes (alt, ast) are increased in more than 80% of hyperthyroid cats. 22, 23 diagnosis diagnosis of feline hyperthyroidism is achieved by measurement of serum total thyroxine (tt 4 ) concentration. serum thyroxine concentrations are elevated in more than 90% of hyperthyroid cats, making this a very sensitive test of thyroxine-induced hypermetabolism. 22, 23 false-positive test results are rare to nonexistent, suggesting that hyperthyroxinemia is a specific test for feline hyperthyroidism. in a clinically hyperthyroid cat, thyroid hormones still fluctuate on a daily (and hourly) basis with hormone concentration intermittently decreasing into the normal range. 27 to avoid this type of diagnostic error the clinician should repeat blood sampling 1 to 2 weeks after the first test. nonthyroidal disease can have a significant effect on circulating thyroid hormone concentrations. [28] [29] [30] in the case of persisting nonthyroidal illness (e.g., renal disease), the syringe attached to a syringe pump may be useful in delivering the food through the feeding tube. crystalloid fluids supplemented with fortified b vitamins, including thiamine, riboflavin, niacinamide, d-panthenol, pyridoxine, and cyanocobalamin, should be used. 1 nutritional supplements to enhance antioxidant function, such as vitamin e and glutathione precursors (e.g., same) may also be beneficial. amino acid supplements that support hepatic regeneration and metabolism include carnitine and taurine. 1, [18] [19] [20] carnitine functions in the transport of fatty acids into hepatic mitochondria for energy production. taurine is an essential nutrient for cats and is involved in cns, cardiac, and biliary functions. signs of taurine deficiency may be similar to those associated with he. antiemetic therapy is necessary to control vomiting and facilitate feeding of an appropriate type and quantity of diet (see chapters 23 and 35) . injectable antiemetics, such as maropitant (cerenia, pfizer animal health, kalamazoo, mi), a selective nk-1 receptor antagonist, at a dosage of 1 mg/kg sc or iv on a daily basis, is preferred. 21 oral maropitant at the same dosage or oral ondansetron, a 5-ht 3 receptor antagonist at a dosage of 0.1 to 1.0 mg/kg q12-24 h may be used in cats with larger-bore feeding tubes. persistent vomiting should be investigated to identify feeding tube occlusion or other undiagnosed disease. the prognosis depends upon duration of illness, and the time frame of resolution of hepatic enzyme elevation, hyperbilirubinemia, and other biochemical changes. cats that survive an episode of hl have a greater than 50% reduction in liver enzyme and bilirubin concentrations within 10 days of therapy, whereas cats that die usually do so within 7 days of hospitalization. 1 long-term prognosis for recovery is good with the majority of cats having resolution of hl as long as the underlying disease process (e.g., pancreatitis) is identified and treated. hyperthyroidism in cats is caused by adenomatous hyperplasia of the thyroid gland resulting in increased circulating concentrations of thyroxine and triiodothyronine. 22, 23 hyperthyroxinemia increases hepatic metabolism without proportionate increases in hepatic blood flow with the overall consequence of reduced oxygen delivery to hepatocytes. 24 characteristic lesions of superficial necrolytic dermatitis (hard, cracked foot pads and elbows). painful feet caused by footpad lesions are common. clinicopathologic features include mild nonregenerative anemia, microcytosis (with advanced liver dysfunction), increased serum liver enzyme (alp and alt) activities, hypoproteinemia, hypoalbuminemia, and fasting hyperglycemia. serum bile acids are usually increased. serum glucagon is inconsistently elevated, but plasma amino acid concentrations are often less than 50% of normal. 34, 37 diagnosis abdominal ultrasonography may reveal small, normal or increased liver size; however, there usually is a characteristic "swiss cheese" appearance of the hepatic parenchyma as a result of hepatic degeneration, nodularity, and collapse. 36, 38 pancreatic imaging and biopsy are indicated if a glucagonoma is suspected. symptomatic palliative therapies may be beneficial and include high-protein diets with egg white (approximately 2 to 4 egg whites/ day for a 25-kg dog), zinc (2 mg/kg q24h po) niacinamide (250 to 500 mg/dog q24h po), ursodeoxycholic acid (10 to 15 mg/kg/day po), vitamin e (10 iu/kg daily po), same (20 mg/kg/day po 2 hours before feeding), and fatty acid supplementation. some patients will respond to 10% parenteral amino acid solutions (aminosyn, abbott laboratories, chicago) given at a dose of 500 ml over 8 to 12 hours intravenously through a large-bore central venous catheter. if no response is observed following the initial amino acid infusion, therapy should be repeated every 7 to 10 days for a total of four treatments. prognosis is poor for most cases; however, remissions of longer than 2 years have been reported with intensive amino acid and hepatic support therapy. 37 steroid hepatopathy develops following exogenous corticosteroid therapy, or from endogenous hyperadrenocorticism of pituitary or adrenal origin. the dog liver is uniquely susceptible to both glucocorticoid-and sex steroid-induced liver enzyme elevation, glycogen accumulation, and vacuolar degeneration. 39, 40 pathophysiology in healthy dogs, glucocorticoid administration results in significant liver enzyme (alp and alt) elevation in 2 to 3 days. increased alp and ggt activities develop in parallel as the enzymes undergo induction and release from sinusoidal and canalicular membranes. within 7 days of glucocorticoid administration, the glucocorticoidinduced alp isoenzyme increases significantly. glycogen accumulates within the hepatocyte resulting in a vacuolar degeneration typical of the syndrome. 1, 39, 40 steroid hepatopathy occurs primarily in the dog. there is only one reported case of steroid hepatopathy in the cat. 41 a history of corticosteroid administration or signs consistent with endogenous steroid overproduction (cushing syndrome) are usually evident, for example, pd, pu, panting, potbellied appearance, bilaterally symmetric alopecia on the trunk, and polyphagia. in dogs affected with measurement of unbound thyroxine (t 4 ) or free t 4 may be preferable to repeated tt 4 measurements. free t 4 concentrations are a very sensitive test for the diagnosis of hyperthyroidism with 98% of hyperthyroid cats exhibiting elevated serum free t 4 concentrations. the specificity of free t 4 is not as good as its sensitivity; as many as 12% of euthyroid cats with concurrent illness will have high free t 4 concentrations for reasons that remain unclear. 29 as a result, free t 4 should not be used as a screening test, and free t 4 values should be interpreted in light of the tt 4 concentrations. the combination of a high free t 4 with a low tt 4 is indicative of nonthyroidal illness; however a high free t 4 with a high-normal tt 4 is suggestive of hyperthyroidisim. 31 methimazole (tapazole) is the antithyroid drug most often recommended (2.5 to 5 mg q12h). it is available as a transdermal gel or as an oral tablet. methimazole is often used to prepare the patient for surgical thyroidectomy or radioiodine therapy. antithyroid drugs have several side effects. anorexia and vomiting are common side effects of methimazole, whereas rare side effects include self-induced excoriation of the face, thrombocytopenia, bleeding diathesis, agranulocytosis, development of serum antinuclear antibodies, and cholangitis. bleeding, jaundice, and agranulocytosis necessitate immediate withdrawal of the drug. hepatic injury related to antithyroid therapy such as methimazole is well documented in humans and reported in the cat. 22, 32 mild histologic changes are common, but cases of fulminant hepatic failure with central lobular necrosis have been described. 33 prognosis is excellent with definitive therapy of the hyperthyroidism (surgery or radioactive iodine). hepatic reactions to methimazole will necessitate discontinuation of therapy. the etiology is unknown, but hypoaminoacidemia may play a role in the development of diabetic hepatopathy. 34, 35 fatty acid, niacin, and zinc deficiencies also may be involved in the pathogenesis. increased serum glucagon, originally thought to be the cause of diabetic hepatopathy, is found in only one-third of the reported cases. a much stronger association between the skin lesions of superficial necrolytic dermatitis and glucagonoma, hyperglucagonemia, and poorly regulated diabetes mellitus have been observed in both humans and dogs. 34, 35 pathophysiology hepatopathy is thought to occur secondary to the metabolic abnormalities associated with diabetes mellitus, glucagonoma, or nutritional deficiencies. [34] [35] [36] [37] hepatic features include vacuolar hepatocyte degeneration, hepatic parenchymal collapse, and hepatic nodularity. the disorder is seen most frequently in middle-aged male dogs, and has been reported in one cat. [34] [35] [36] [37] acute presentations may include clinical signs such as vomiting, diarrhea, lethargy, weight loss, pd, pu, icterus, and lameness because of dermatopathy of the footpads. in some cases clinical signs are mild or nonexistent. physical examination may reveal poor body condition, lethargy, and lipoproteinemias etiology genetic abnormalities in lipid metabolism lead to diffuse vacuolar hepatopathy and biliary mucoceles. 1 increased circulating cholesterol and triglyceride cause a vacuolar hepatopathy associated with excess lipid accumulation and/or hepatocyte glycogen synthesis and storage. chronic hypercholesterolemia increases biliary cholesterol content and predisposes to cystic hyperplasia, dysmotility of gallbladder smooth muscle, and biliary mucocele. 1 familial hypercholesterolemia and other hyperlipidemias are found in certain breeds of dogs including the miniature schnauzer, shetland sheepdog, briard, west highland white terrier, scottish terrier, cairn terrier, and beagle. mixed-breed dogs may also be affected. clinical signs are usually associated with necrotizing cholecystitis and may include icterus and cranial abdominal pain. more often, dogs are asymptomatic and biliary mucoceles are identified serendipitously during ultrasound evaluation for some other medical problem (such as pancreatitis). clinical pathology findings usually include hypercholesterolemia or hypertriglyceridemia, and elevated liver enzyme activities, particularly alp. necrotizing cholecystitis may be accompanied by leukocytosis, neutrophilia, and hyperbilirubinemia. diagnosis may be made by characteristic ultrasound findings of nongravitational gallbladder sludge, increased gallbladder wall thickening, "kiwi"-shaped mucosal image, and bi-or trilaminar appearance of the gallbladder wall. the hepatic parenchyma may have a pattern of multifocal hyperechogenicity and hypoechoic nodules. 1 the best treatment for biliary mucoceles is surgical removal of the mucocele and/or cholecystectomy and may become an emergency procedure if the clinical signs of necrotizing cholecystitis are severe. medical therapy following surgical removal is usually necessary and includes a fat-restricted diet and lifelong treatment with ursodeoxycholic acid (15 mg/kg po q24h). prognosis is good for patients undergoing successful removal of the mucocele as long as lifelong medical therapy is continued. in dogs and cats, amyloid deposition is usually secondary to sustained systemic inflammatory response, for example, chronic infection, chronic inflammation, immune disorders, and malignancy. 50 amyloidosis is a familial disorder in the chinese shar-pei dog, and in abyssinian, oriental, and siamese cats. [51] [52] [53] [54] hepatic amyloidosis has also been reported secondary to vitamin a toxicity in cats. 55 pathophysiology deposition of amyloid fibrils within and between hepatic sinusoids results in progressive organ dysfunction. light deposits are found in the space of disse and heavier deposits are often found in the atypical hyperadrenocorticism caused by sex steroid overproduction, dermatologic changes (alopecia, poor hair coat) and reproductive manifestations (perianal adenoma in a castrated male or female dog) are often the only signs suggestive of sex steroid imbalance. atypical hyperadrenocorticism with sex steroid excess may present with no clinical signs except increased serum liver enzyme activities. 1 diagnosis of steroid hepatopathy should be based on a history of exogenous steroid administration or endocrine function testing with or without liver biopsy. classically, liver enzyme elevations consist of moderate to marked increases in alp and ggt, and mild to moderate increases in alt and ast. bile acids may also be increased. 40 the low-dose dexamethasone suppression (ldds) test is considered the screening test of choice for endogenous canine hyperadrenocorticism. 42, 43 the ldds test has a high sensitivity at 92% to 95%. only 5% to 8% of dogs with pdh will exhibit suppressed cortisol concentrations at 8 hours. in addition, 30% of dogs with pdh will exhibit suppression at 3 or 4 hours followed by "escape" of suppression at 8 hours. this pattern is considered diagnostic for pdh, making further testing unnecessary. 43 the major disadvantage of the ldds test is the lack of specificity in dogs with nonadrenal illness. 44 the corticotropin (acth) stimulation test is used to diagnose a variety of adrenopathic conditions, including endogenous or iatrogenic hyperadrenocorticism, as well as spontaneous hypoadrenocorticism. 42, 45, 46 as a screening test for the diagnosis of naturally occurring hyperadrenocorticism, the acth response test has a diagnostic sensitivity of approximately 80% to 85% and a higher specificity than the ldds test. 45, 46 in a study by kaplan and peterson, only 15% of dogs with nonadrenal disease exhibited exaggerated response to acth stimulation. 44 i prefer the acth response test over the ldds test as the acth response test is more accurate for the diagnosis of iatrogenic hyperadrenocorticism (if the history is incomplete) and sex steroid imbalance in addition to pdh or adrenal-dependent hyperadrenocorticism. the urine cortisol-to-creatinine ratio (uccr) is highly sensitive in separating normal dogs from those with hyperadrenocorticism; however, the test is not highly specific for hyperadrenocorticism because dogs with moderate to severe nonadrenal illness also exhibit elevated ratios. [47] [48] [49] an elevated uccr should always be confirmed with an ldds test. in the uccr test, urine is collected for 2 days for a baseline uccr. the animal then is given three doses of dexamethasone (0.1 mg/kg, po q6-8 h) and the final uccr is collected 24 hours after the first dose of dexamethasone. failure of the uccr to suppress into the normal range is diagnostic for hyperadrenocorticism. treatment for exogenous hyperadrenocorticism consists of discontinuation of exogenous steroids by slowly weaning the patient to prevent the development of addisonian crisis. treatment for endogenous hyperadrenocorticism can be achieved with chemotherapy (o,p′-ddd, or trilostane) or surgery (hypophysectomy or adrenalectomy). treatment of sex steroid imbalance can be achieved with mitotane or trilostane. prognosis for steroid hepatopathy is good to excellent if diagnosed early and if corticosteroid injury can be abated by discontinuation of steroid therapy or treatment of the underlying disorder. threefold increases in the frequency of hypothyroidism. 61 the pathogenesis of stone formation in hypothyroidism is believed to involve hypercholesterolemia, gallbladder dysmotility, and bilirubin retention. 61 the most common clinical symptoms of hypothyroidism are lethargy, weight gain, depression, hypothermia, and bradycardia. gi signs such as reflux esophagitis, gastric atony, constipation, diarrhea, and hepatopathy with mucocele formation are rare clinical signs of hypothyroidism in dogs. 62 symmetric truncal or tail-head alopecia are a classic findings in hypothyroid animals. 62 hyperkeratosis, hyperpigmentation, secondary pyodermas, and demodicosis are also observed. clinicopathologic findings such as normocytic normochromic anemia, hypertriglyceridemia, and hypercholesterolemia are seen in the majority of hypothyroid animals because of altered lipid metabolism and binding proteins (increased hdls), decreased fecal excretion of cholesterol, and decreased conversion of lipids to bile acids. 63 total serum t 4 concentration and endogenous thyroid-stimulating hormone (tsh) may be used to confirm the diagnosis of hypothyroidism. this combination of tests has been shown to have the highest specificity, sensitivity, and lowest overall cost. if the tt 4 is in the low normal or below normal range and the tsh is high, the animal is suffering from primary hypothyroidism. 64, 65 if the tt 4 and tsh are both low, free t 4 by dialysis should be determined to distinguish euthyroid sick syndrome (normal free t 4 ) from true secondary hypothyroidism (low canine thyroid stimulating hormone [ctsh] resulting from pituitary tsh deficiency). 66 treatment synthetic thyroid hormone supplementation is the treatment of choice for hypothyroidism. levothyroxine sodium therapy is started at a dosage of 0.02 mg/kg given orally twice daily. 66 thyroid function should be monitored every 6 to 8 weeks for the first 6 to 8 months of treatment and then once or twice yearly thereafter. in stable well-controlled animals, the total treatment may be given once daily with excellent clinical results, as long as adequate peak hormone concentrations are achieved. 67 with thyroid hormone replacement therapy in hypothyroid dogs, the prognosis is excellent. inflammatory disease involving the intrahepatic bile ducts is commonly encountered in veterinary practice. cholangitis is recognized more commonly in cats than in dogs, but both species can be affected. the wsava liver standardization group suggests that cholangitis be considered in the following four groups: neutrophilic cholangitis (nc), lymphocytic cholangitis (lc), chronic cholangitis associated with liver fluke infestation, and destructive cholangitis. 1 sinusoidal lumen. amyloid fibrils are readily detected on routine hematoxylin and eosin or diff-quik staining. amyloidosis is confirmed on examination of congo red-stained aspirates or biopsies under polarized light where the extracellular material shows characteristic green birefringence. 50 concurrent amyloid deposition in the kidneys, liver, spleen, and adrenal glands can occur, but clinical manifestations of liver failure are most common. chronic progressive liver failure with clinical signs of anorexia, weight loss, and lethargy, is the typical clinical course in many cases. some animals may instead present with acute collapse following hepatic rupture and intraabdominal hemorrhage. 50 pallor of mucous membranes, hypothermia, and hepatomegaly are the most frequently recognized physical examination findings. typical laboratory findings include regenerative anemia, leukocytosis, thrombocytopenia, marked elevations in serum alt and ast, and marked prolongations in aptt and pt times. radiography is useful in detecting free peritoneal fluid, hepatomegaly, and irregular hepatic borders. ultrasonography reveals a diffuse, heterogeneous echogenicity with highly echogenic ("sparkling") areas and hypoechoic foci. 50 definitive diagnosis requires tissue biopsy and congo red staining. there are no specific treatments for this disorder. colchicine has been recommended because it may block formation of amyloid in the early stages of the disease, but it is of unproven benefit and has been associated with significant side effects. dimethyl sulfoxide has been recommended because it may promote resorption of amyloid. as there are no specific therapies for this disease, treatment is instead largely symptomatic and supportive. with progressive amyloidosis lesions, the prognosis for long-term survival is poor. a familial hyperlipoproteinemia has been reported in cats that is characterized by fasting hyperchylomicronemia, elevated circulating concentrations of vldls, and hypertriglyceridemia. 56, 57 serum cholesterol is only minimally elevated. the underlying biochemical lesion is a reduction in the activity of lipoprotein lipase, and the disorder is transmitted as an autosomal recessive gene. xanthomas accumulate in the soft tissues, including the liver, but clinical signs are more often related to involvement of the peripheral nerves. dietary fat restriction improves clinical signs in some affected animals. 58 etiology decreased circulating thyroid hormone concentration affect hepatic metabolism and cholesterol turnover in the liver. liver function tests are mildly disturbed in almost 50% of patients with hypothyroidism despite normal histologic findings. 59 pathophysiology decreased hepatic metabolism in hypothyroidism is reflected by reduced oxygen consumption. 33, 59, 60 patients with a common bile duct stone and gallbladder stone have, respectively, sevenfold and infection of bile has been commonly identified in cats with ehbdo, 4, 8 clinical examination. previous literature has highlighted differences in clinical presentation between cats with different forms of cholangitis. however, we have recognized few differences between the various forms 4, 9 and suggest that any statistically significant differences cited previously hold little clinical relevance given the large degree of overlap within data ranges. nc can occur in cats of any age, breed, or sex. clinical signs are nonspecific and include anorexia, lethargy, vomiting, and weight loss. the duration of these clinical signs ranges from a few days to a few months and may be shorter in cats with anc than in those with cnc, 3 but this is not a consistent finding. 4, 9 physical examination findings commonly include dehydration and icterus. fever is present in 19% to 37.5% of cases. 4,10 some reports suggest that fever is more commonly associated with anc than cnc, 10 while others recognize no difference. 4, 9 hepatomegaly is seen in fewer than half of the cases. abdominal pain is noted occasionally. 3, 4, 9 diagnosis. definitive diagnosis is made by examination of liver biopsy specimens, with ancillary diagnostics providing supportive information. hematologic findings are variable and may include poikilocytosis, neutrophilia, and left shift, although these abnormalities are present in fewer than one-third of cases. 3, 4, 9, 10 biochemical analysis commonly reveals increased activity of alt, ast, alp, and ggt ranging in severity from mild to severe. however, increased liver enzyme activity may be absent in some cases. serum total bilirubin is increased in most cases. serum cholesterol may become increased in cases with ehbdo. imaging findings are nonspecific for cholangitis, but may provide useful information regarding concurrent disease. abdominal radiographs are rarely helpful. ultrasonographic appearance of the liver in cats with nc can vary greatly, with the most common abnormality being a diffuse change in echogenicity ranging from hypo-to hyperechoic. 11 dilation of intra-and/ or extrahepatic bile ducts, gallbladder distention, increased gallbladder sediment, and thickening of the gallbladder or bile duct walls may be seen. gallbladder distention and bile duct dilation may indicate ehbdo, but these changes may occur in cats with cholangitis lacking obstruction. ultrasonography will also provide information regarding the presence of concurrent disease, such as pancreatitis and inflammatory bowel disease. wedge liver biopsy during laparotomy is the optimal method for obtaining a definitive diagnosis. other biopsy techniques that may be considered include laparoscopic and ultrasound-guided tru-cut needle approaches. tru-cut needle biopsy diagnoses correlate with wedge biopsies in fewer than 50% of cases. 12 diagnostic accuracy of laparoscopic liver biopsies compared with wedge biopsies have not been evaluated. laparotomy and laparoscopy provide the additional benefit of evaluation and sampling of extrahepatic structures. laparotomy should be performed in any cat suspected of having ehbdo. while the optimal sampling strategy is unknown, biopsies should be obtained from multiple liver lobes, as we have recognized wide ranges of severity between different lobes in the same cat. in patients that are not stable enough for liver biopsy, such as those with hypotension, coagulopathy or he, fine-needle aspiration with cytology offers a less-invasive diagnostic approach as it can usually be performed quickly with light sedation. however, liver cytology correlates with biopsy results in only 39% to 60% of cases. 13, 14 cytology is sensitive for identifying the presence of hl, however this is the most common misdiagnosis when using cytology. 14 cytology is cholangitis is a common hepatobiliary disorder of cats, second only to hl. 2 although varying terminology has created some confusion regarding this syndrome, it is clear that feline cholangitis includes a spectrum of disease processes, including forms displaying neutrophilic inflammation and those lacking neutrophilic inflammation. histologically, nc is characterized by the presence of neutrophils in the lumen and/or epithelium of the bile ducts. 1 the disease is recognized to occur in acute and chronic forms. in acute neutrophilic cholangitis (anc) edema and neutrophilic inflammation are seen in the portal areas, with occasional extension of inflammation to the hepatic parenchyma. in chronic neutrophilic cholangitis (cnc) there is a mixed inflammatory infiltrate consisting of neutrophils, lymphocytes, and plasma cells. varying degrees of bile duct hyperplasia and fibrosis will be present depending on the chronicity of disease. etiology. although the true etiology remains unknown, nc is largely suspected to be caused by ascending bacterial infection from the intestine. 1-3 rates of bacterial isolation using traditional methods have varied greatly, from less than 20% to more than 60% in affected cats. 3, 4 recently, fluorescence in-situ hybridization (fish) with a 16s rdna probe that recognizes bacteria in general has been used to identify and localize bacteria in cats with cholangitis. 5 combining traditional culture and fish, bacteria were isolated in three of three (100%) cats with anc and eight of 13 (61%) with cnc. the localization of the bacteria identified using fish supports translocation of enteric bacteria as the cause of infection. although it appears that bacteria play an important role in the etiology of nc in many cases, it is important to note that they are not identified in all affected cats. some authors theorize that nc, and cnc in particular, may have an immune-mediated etiology with persistent inflammation following an initial bacterial infection or other unknown initiating factor. 3, 6 pathophysiology. nc in cats is commonly associated with inflammatory bowel disease and pancreatitis. 3, 4, 7 the pathophysiology underlying the relationship of these diseases is unknown, but rational theories revolve around the unique anatomy of the feline biliary and pancreatic duct systems. in the cat, the common bile duct and pancreatic duct merge prior to entering the duodenum at the major duodenal papilla. 7 cholangitis may develop secondary to reflux of ascending bacteria from the duodenum during vomiting. pancreatitis may result from bacterial reflux into the pancreatic duct, or from pancreatic duct obstruction secondary to cholangitis. 7 in most reported cases, the inflammatory bowel disease associated with cholangitis is moderate or severe, whereas the pancreatitis tends to be mild chronic interstitial disease. 7 nc is also commonly associated with extrahepatic bile duct obstruction (ehbdo). ehbdo has been identified in 40% of cats with anc and 76% of cats with cnc. 4 cholangitis and/or pancreatitis are the most common cause of ehbdo in the cat. 8 in one study, 64% of cats with ehbdo had cholangitis, representing 93% of cats that did not have a neoplastic cause. 8 it is unknown whether cholangitis is the cause or the result of ehbdo. histologic changes consistent with cnc have been seen in the livers of cats with ehbdo secondary to pancreatic carcinoma, cholelithiasis and surgical occlusion of the common bile duct. 8 in contrast, cholangitis has been implicated as the sole cause of ehbdo resulting from proliferation of mucosa within the common bile duct. 8 bacterial choleretic properties that make it a rational choice for treating cholangitis. because of the possibility of immune-mediated mechanisms in the perpetuation of nc, particularly with cnc, corticosteroids may be appropriate in some cases. initial treatment should always involve antibiotics in cats with nc. failure to improve within 2 weeks of antibiotic therapy, or clinical deterioration prior to that time, warrants initiation of corticosteroid therapy. prednisolone at 1 to 2 mg/ kg twice daily is given initially and gradually tapered to the lowest effective dose. antibiotics should be continued concurrently with corticosteroids for a minimum of 4 weeks. the duration of corticosteroid therapy varies between individual patients. many cases can be gradually tapered off of corticosteroids over 4 to 6 months, while others require lifelong therapy. surgical intervention is required in cats with ehbdo; however, the optimal surgical procedure is unknown. biliary diversion (cholecystocholedochostomy, choledochoduodenostomy, or cholecystojejunostomy) and choledochal stenting are the most common procedures. surgery in cats with ehbdo is associated with significant perioperative morbidity. in many cases, profound hypotension develops intraoperatively after 45 to 60 minutes as a result of decreased vascular responsiveness and decreased myocardial contractility and is often refractory to interventions such as fluid or vasopressor therapy. 8, 18, 19 whichever surgical procedure is chosen, it is clear that anesthesia time should be minimized and long-term medical management will be necessary. biliary diversion is associated with short-term mortality rates of 36% to 57% 8, 18 and is associated with long-term complications. 8, 19 in a small case series describing choledochal stenting in cats with pancreatitis and cholangitis, five of seven experienced long-term survival (≥7 months), but reobstruction occurred in two of seven and chronic vomiting and recurrent cholangitis were reported. 19 prognosis. the prognosis for cats with nc is typically good. 3, 4, 10 survival to discharge was reported in 72% of all cats with cholangitis in one study. 4 median survival time of 29.3 months has been reported in cats with nc, with no difference between anc and cnc. 10 prognostic factors have not been identified. given the high rate of perioperative morbidity and mortality, it seems likely that cats with ehbdo have a worse prognosis than those without ehbdo. thirty percent to 40% of cats with ehbdo secondary to inflammatory disease die within a week of surgery. 8, 18 however, in those that survive to discharge, long-term survival has been reported. 18 the wsava liver standardization group describes lc as a common, slowly progressive, chronic disease of cats characterized histologically by infiltration of small lymphocytes (and occasionally plasma cells or eosinophils) restricted to the portal areas associated with varying degrees of fibrosis and bile duct hyperplasia. 1 they remark that inflammation centered on the bile ducts may be present, but is not a hallmark of the disease. it is also stated that welldifferentiated lymphoma may be difficult to differentiate from lc. based on the existing literature regarding lc in cats, the description from the wsava group includes several clinically and histopathologically different subsets that may or may not revolve around a common pathogenesis. recognition of these different subsets within the umbrella of lc may have therapeutic and prognostic ramifications. several investigators describe a group of cats with lc where inflammation is confined to portal regions and there is a lack of targeting of bile ductules or biliary epithelium. 2, 20, 21 this has been insensitive for identifying cholangitis in cats, diagnosing fewer than 30% of cases. 14 cytologic examination of bile may prove more sensitive for the diagnosis of nc in cats. in five of seven cats with cnc evaluated at this institution, bile cytology revealed neutrophilic inflammation, presence of bacteria, or both. techniques have been described for safely obtaining bile via ultrasound-guided percutaneous cholecystocentesis in lightly sedated cats. 15 samples for aerobic and anaerobic bacterial cultures should be obtained in any cat suspected of having cholangitis. gallbladder bile is preferred to liver tissue as the culture source. in a group of 58 cats suspected of having hepatobiliary disease, bile cultures isolated pathogens in 36% compared with only 14% of liver cultures. 16 in the same study, 22 dogs and cats had both liver and bile cultured and none had a positive liver culture in the absence of a positive bile culture. 16 in a group of cats with cholangitis, bile cultures were more likely to isolate pathogens (75% vs. 33%) and less likely to yield contaminants (4% vs. 29%) than liver cultures. 4 in a small study comparing bile versus liver cultures in 22 cats with various hepatobiliary diseases, bile culture was positive in five (four had cnc) while liver culture was positive in only two. in the two cats with positive liver cultures, the same organism was isolated from bile. 17 it is important to recognize that many cats with nc (and other hepatobiliary diseases that mimic it clinically) are not stable enough to tolerate diagnostic testing. in such patients, the risk of aggressive diagnostics may outweigh the benefits of obtaining a definitive diagnosis. in these cases, the diagnosis may be suspected based on clinical response to supportive care, including broad-spectrum antibiotic therapy. treatment. optimal treatment protocols for cats with nc are unknown and the recommendations herein are based solely on anecdotal clinical experience. antibiotics are the mainstay of treatment. drug selection is ideally based on results of bacterial culture and susceptibility testing. in cases where cultures are not performed, or while results are pending, broad-spectrum coverage should be provided. the most commonly isolated pathogens are aerobic and anaerobic bacteria of enteric origin, 16 including e. coli, enterococcus spp., and clostridium spp., among others. 3, 4, 16 effective empiric antibiotic combinations would include a penicillin, a fluoroquinolone, and metronidazole. the optimal duration of antibiotic therapy is unknown, but we recommend a 4-to 6-week course for initial treatment. supportive care and treatment of specific sequelae of liver disease should be included as indicated. nutritional support is required in many cats and is best accomplished by use of enteral feeding tubes. we recommend placement of esophageal feeding tubes in cats with cholangitis if they are anorexic and stable enough for general anesthesia. in unstable patients, nasoesophageal feeding tubes offer a less-invasive method of providing short-term support. several medications and nutritional supplements (including ursodeoxycholic acid [udca], same, milk thistle, vitamin e, vitamin c, carnitine, taurine, and phosphatidylcholine) have been suggested for treating cats with cholangitis. while most of these compounds have theoretical benefits, a clinical benefit has not been proven. to optimize client compliance and avoid adverse drug reactions, i prefer to minimize the number of medications given to feline patients. because most cats with anc respond well to antibiotic therapy, i rarely include other medications in our treatment protocol. however, in cats with anc that do not quickly respond to antibiotics and in many cats with cnc, i like to use udca. among its theoretical benefits, udca has immunomodulatory and distinction between lc and well-differentiated (small cell) lymphoma can be a challenge even for experienced pathologists. preliminary data using immunohistochemistry and pcr for t-cell receptor clonality has not proven useful in differentiating between the two conditions. surprisingly, cats with both lc and lymphoma had monoclonal t-cell receptors, oligoclonal t-cell receptors, and polyclonal t-cell receptors. 20 using light microscopy, the following features were unique to lc and not present in cats with lymphoma: ductopenia, bile duct targeting by lymphocytes, and the presence of lipogranulomas within portal regions (representing a residual marker of cell death). 20 until more studies are done evaluating molecular techniques, these features may prove useful in differentiating the two conditions. interestingly, bile duct hyperplasia and fibrosis were present in cats with lc and those with lymphoma. this may suggest that an inflammatory state precedes the development of lymphoma, 20 which has been reported anecdotally. treatment. the therapeutic approach to cats with lc should be similar to that described for cats with nc in regards to supportive care and symptomatic treatment of the sequelae of liver disease. because bacteria have been isolated from some cats with lc, i recommend treatment with broad-spectrum antibiotics while awaiting results of bacterial cultures. in contrast to nc, long-term treatment of culture negative cats with antibiotics is not warranted. immunomodulation and immune suppression are the major components of treatment based on a presumed immune-mediated etiology. cats that are culture negative, or that have failed to respond to antibiotics within a few days, should be treated with prednisolone at 1 to 2 mg/kg twice daily. responders should be tapered gradually over 4 to 6 months to the lowest effective dose. other drugs that are useful for immunomodulation include metronidazole and udca. cats that fail to respond completely to corticosteroids and/ or other immunomodulators, or who relapse while being treated, may require additional immunosuppressive drugs. although these drugs have not been well evaluated in cats with lc, chlorambucil and methotrexate are suggested by some authors. 3 cats with small cell lymphoma often respond to combination therapy with prednisolone and chlorambucil, but they may require a multidrug weekly sequential chemotherapy protocol. prognosis. cats with lc have a variable prognosis, 3,6 likely a result of being diagnosed at different stages of a chronic disease process. survival of greater than 5 years has been reported, and many cats that die appear to succumb to disease unrelated to the liver. 22 other cases have been reported that fail to respond to treatment and die more acutely, 6 though this is uncommon in my experience. this is a disease that likely requires lifelong management and monitoring with relapse of illness possible as medication doses taper. trematode parasites of the families dicrocoeliidae and opisthorchiidae may inhabit the gallbladder and bile ducts of cats and rarely dogs. 26 there are multiple species with worldwide distribution. the most commonly identified species include the dicrocoelid platynosomum concinnum and the opisthorchid amphimerus pseudofelineus. [27] [28] [29] p. concinnum is mainly found in tropical and subtropical areas, including the southeastern united states. 26 a. pseudofelineus has a wider area of distribution throughout north and south america. 26 the life cycle is similar for both dicrocoelids and opisthorchids. 26 parasite eggs are ingested by a land snail (subulina octona or eulota [bradybaena] similaris), develop into cercariae and enter a second referred to as lymphocytic portal hepatitis. 2, 21 the connection between this histopathologic finding and clinical disease in cats is unknown, as it may represent a common change associated with aging. it was identified in 82% of cats older than 10 years of age and 96% of cats older than 15 years of age from a necropsy population that did not have primary liver disease. 21 it is also possible that this lesion represents a response to inflammation at a distant site, as it is similar to the lesion of nonspecific reactive hepatitis associated with chronic extrahepatic disease. 1 although clinical signs have been described in cats with lymphocytic portal hepatitis, 10 the common occurrence of concurrent disease in these cats makes it difficult to know if the clinical signs are attributable to the lesions in the liver. in another subset of cats, lc is marked by inflammation targeting bile ductules and infiltrating biliary epithelium, leading to progressive ductopenia. 20, 22, 23 these cases seem more likely to have clinical disease attributable to their liver pathology, although side-by-side comparisons of cases with and without bile duct targeting have not been performed. cats with this form of lc in the united states have a similar clinical picture to cats with nc. 9 in the united kingdom, this lesion has been associated with ascites, icterus, and hyperglobulinemia in young cats and termed progressive lymphocytic cholangitis. 22, 23 etiology. although the etiology of lc is unknown, theories suggest that it is an immune-mediated or infectious phenomenon. genetic factors may also play a role, as persian cats are overrepresented in the united kindgom. 22, 23 immunohistochemistry in affected cats has provided evidence for an immune-mediated pathogenesis, although the inciting antigen is unknown. 20, 23 bacteria have been identified in the liver or bile of fewer than 20% of cats with lc. [3] [4] [5] 17, 20 although helicobacter pylori has been isolated from the liver and bile of cats with cholangitis, the evidence for this organism playing an important role in feline cholangitis is not compelling at this time. 24, 25 pathophysiology. as with nc, concurrent inflammatory bowel disease and pancreatitis appear to be common in cats with lc, 3, 4 although some authors report it to be uncommon. 23 the theory that reflux of duodenal bacteria into the biliary and pancreatic ducts incites inflammation may hold true for cats with lc, although a common immune mechanism must be considered. clinical examination. the clinical picture of cats with lc varies widely and has significant overlap with other forms of hepatobiliary disease in cats, including nc. 4, 9 although some studies describe a predominance of older cats, 3 others describe more younger cats. 22, 23 nonspecific clinical signs, including anorexia, lethargy, vomiting, and weight loss, may be chronic and intermittent. 3, 6, 22 physical examination findings may include icterus, hepatomegaly, or ascites, but none are consistent findings. signs of he (dullness, ptyalism, seizure) may develop in severely affected cats. diagnosis. definitive diagnosis is made by liver biopsy. as discussed for nc, ancillary diagnostics will provide information to support hepatobiliary disease, but are not specific for lc. hematology results may be unremarkable, even though marked lymphocytosis has been described in some cases. 3 activity of serum liver enzymes is increased in many, but not all cases and varies in severity. hyperglobulinemia has been described. 3, 6, 22 abdominal radiographic and ultrasonographic findings are nonspecific, but may aid in the recognition of concurrent disease. amoxicillin-clavulanate, milbemycin oxime, and amitraz prior to the onset of clinical signs. 31, 32 the proposed toxic etiology is based on these case histories and histopathologic similarity to humans with idiosyncratic drug toxicity. other toxic insults and viral infection, such as canine distemper, can also result in destructive cholangitis. 1 affected dogs typically present for signs referable to cholestasis, including anorexia, icterus, vomiting, and acholic feces. 31, 32 activities of serum liver enzymes and total bilirubin are moderately to markedly increased. abdominal ultrasound may be unremarkable, showing only mild dilation of intrahepatic bile ducts. 32 definitive diagnosis is made by liver biopsy. optimal treatment options are unknown, but should undoubtedly involve discontinuation of any medications that preceded illness. corticosteroids for immune suppressive and antiinflammatory effects and udca for immunomodulatory and choleretic effects would be rational treatments options, but their efficacy has not been documented. the prognosis appears to be poor, as six of the eight reported cases were euthanized within 6 weeks and the remaining two had only shortterm followup (<6 months). 31, 32 nc, as described in the cat, has been rarely reported in dogs. [33] [34] [35] [36] [37] bacteria have been isolated from the majority of cases reported, including e. coli, klebsiella spp., proteus mirabilis, streptococcus spp., and clostridium spp. [34] [35] [36] [37] although the bacterial species would support ascending infection from the intestine, the literature is too sparse to make conclusions about the pathophysiology of this disease in dogs. bacterial infection could also spread hematogenously or via translocation from the portal circulation. the clinical presentation and diagnostic findings are similar to those reported for cats with nc. affected dogs present with lethargy, anorexia, vomiting, and icterus usually of acute onset. fever is reported in approximately half of the cases. [33] [34] [35] [36] [37] neutrophilia with or without a left shift is common. activity of serum liver enzymes is typically increased and most dogs have mild to moderate elevation of serum total bilirubin. ultrasonography is nonspecific, with the liver varying from normal to hyperechoic with some heterogeneity. 34, 36 thickening and hyperechogenicity of the gallbladder wall is common. 36 definitive diagnosis is made by liver biopsy with changes similar to those reported in cats with nc. most dogs have some degree of mixed inflammatory infiltrate, similar to cnc. [33] [34] [35] [36] [37] this infiltrate extends into the hepatic parenchyma in the majority of cases. aerobic and anaerobic culture of bile or hepatic tissue should be performed, though bile has been the source of bacterial isolation in most cases. treatment involves antibiotic therapy guided by culture and susceptibility results. duration of treatment should be prolonged, as antibiotic courses of 8 to 12 weeks or greater have been required to completely eliminate bile infections. clinical improvement precedes bacterial eradication. 36 the prognosis appears to be good in most cases, although dogs with concurrent disease may have a worse prognosis. liver cysts arising from the intrahepatic bile ducts are rarely encountered in veterinary practice. although cysts may be acquired secondary to trauma, neoplasia, inflammation, or biliary obstruction, 1,38 the vast majority of cases described in the literature are congenital in origin. congenital cystic liver diseases result in dilation of various segments of the intrahepatic bile ducts, and they are associated with varying degrees of hepatic fibrosis and cysts in other organs (most commonly the kidneys). little is known about inheritance patterns of cystic disease in dogs and cats. the various morphologic patterns of cystic disease likely represent abnormalities of bile duct intermediate host. 26, 27, 29 the dicrocoelids tend to use an arthropod, while opisthorchids utilize fish. typically cats acquire infection by ingesting the second intermediate host. in the case of p. concinnum, the sporocysts leaving the snail intermediate host may be eaten by a paratenic terrestrial isopod host (pill, sow, or dung bugs). 26, 29 cats are infected by ingesting this form in a variety of lizard or amphibian intermediate hosts. cercariae migrate from the cat intestine to the gallbladder and bile ducts, where they develop into adults. eight weeks or more after infection eggs are passed in the feces to complete the life cycle. 27 clinical signs are proportional to parasite burden. cats with light infections are often asymptomatic. 27, 28 clinically ill cats may present with nonspecific signs such as anorexia, lethargy, vomiting, or diarrhea. severely affected cats present with signs of ehbdo such as icterus and acholic feces. [27] [28] [29] [30] preliminary diagnostics are nonspecific for fluke infestation. eosinophilia proportional to the parasite burden may be present. 29 serum liver enzyme activity may be mildly to moderately increased, although it is normal in many cases. [28] [29] [30] abdominal ultrasound often reveals evidence of ehbdo. [28] [29] [30] definitive diagnosis by identification of flukes or fluke eggs in the feces is difficult as small numbers of eggs are shed daily, the eggs have varying morphology at different stages of development, and the eggs are quite small. 27 fecal concentration-sedimentation using the formalin-ether technique is the most reliable method of identifying eggs in stool. 27, 29 eggs may also be identified in cytologic preparations of bile. 28, 30 eggs or adults may be seen in liver biopsy specimens, but they are inconsistently identified. 1, 27, 28, 30 histopathologic changes seen in the liver are characterized by dilation of larger intrahepatic bile ducts associated with papillary projections and marked periductal and portal fibrosis. mild to moderate inflammation may be present within the ducts (neutrophils and macrophages) and in the portal areas (neutrophils, lymphocytes, plasma cells). eosinophils may be present in limited numbers. 1 rarely, chronic cholangitis associated with liver flukes can result in the development of cholangiocarcinoma. 1, 29 optimal treatment protocols have not been established, but praziquantel at 10 to 20 mg/kg daily for 3 days appears to be the most effective. [26] [27] [28] [29] [30] doses as high as 40 mg/kg daily have been used successfully, 28 but this dose has also been fatal in cats. 26 sporadic resumption of egg shedding following praziquantel has been reported, suggesting that it does not completely eliminate infection. 26, 27 for this reason, continued treatment at 12-week intervals has been recommended. 29 symptomatic and supportive therapy should be tailored to the individual patient. cats with ehbdo require surgical decompression. glucocorticoids and udca may have some benefit in controlling inflammation and providing choleresis. although infected cats may remain asymptomatic, patients with ehbdo appear to have a grave prognosis. long-term survival has only been reported in rare cases. 28, 30 cholangitis is rarely reported in dogs. reports of cholangitis in dogs include two distinct entities: destructive cholangitis and nc. destructive cholangitis is characterized histopathologically by a loss of bile ducts (ductopenia) within the smaller portal areas, associated with cholestasis, portal inflammation consisting primarily of macrophages, neutrophils, and occasionally eosinophils, and progressive portal fibrosis. 1, 31, 32 this is a rarely reported lesion of unknown etiology. it has been postulated that the lesion represents an idiosyncratic drug toxicity. however, of the eight cases reported in the literature only three had a prior drug history: two having received potentiated sulfonamides and the other having received adult polycystic disease is most commonly recognized as polycystic kidney disease in persian cats. 44, 45 it has also been reported in cats of other breeds and in dogs. 39, 43 this is similar to autosomal dominant polycystic kidney disease in humans. inheritance in persian cats is autosomal dominant. 45 liver cysts are thought to represent a late defect in the development of peripheral intrahepatic bile ducts. the liver may contain multiple cysts ranging from less than 1 mm to greater than 12 cm in diameter. these cysts typically contain clear, colorless fluid. discrete fibrotic areas containing small, irregularly formed bile ducts, referred to as von meyenburg complexes, may be present. 1 in the kidneys, multiple cysts may form in any segment of the kidney but may involve only a small percentage of the nephron population. this is in contrast to the diffuse cysts seen with congenital dilation of the large and segmental bile ducts and juvenile polycystic disease. 1, 40 hepatic cysts are present in 10% to 40% of cats with polycystic kidney disease, while hepatic fibrosis is recognized in up to 48%. 44, 45 the hepatic cysts are usually incidental findings and the animals are not clinically ill unless they develop renal failure secondary to cysts in the kidneys, which happens in adulthood. biliary atresia is an extremely rare congenital disorder, having been reported in only one dog and one cat. 46, 47 in both cases, the common bile duct was not patent because of atresia. in the dog, the occluded segment of bile duct was histologically comprised of fibrous tissue with minimal inflammation. 47 the etiology is unknown, but this lesion likely represents an embryologic nonfusion of the cranial (hepatic) and caudal (cystic) anlages of the bile ducts during development. 1 other possible explanations include ischemic, toxic, traumatic, or infectious insults occurring pre-or postnatally. 47 affected animals have presented at 4 to 6 months of age with clinical signs of depression, anorexia, vomiting, or lameness associated with rickets because of inadequate vitamin d absorption. 46, 47 affected animals show icterus, hepatomegaly and acholic feces. serum biochemistry abnormalities are consistent with ehbdo. definitive diagnosis is made at exploratory laparotomy. surgical biliary diversion is a viable treatment option depending on the location of atresia, but it was unsuccessful in the one case reported. 47 a guarded prognosis should be given as for any animal undergoing a biliary diversion procedure (see discussion under "neutrophilic cholangitis"). cholestasis is impaired bile flow resulting in the accumulation of bile components in the blood. 1 intrahepatic cholestasis occurs secondary to a variety of primary or secondary hepatobiliary diseases. 1, 48, 49 increased activity of serum liver enzymes, particularly alp and ggt, is common with intrahepatic cholestasis but is not specific for the condition. clinically patients may appear jaundiced, but the predominant clinical sign will be related to the underlying disease process. cholestasis is marked by the presence of bile plugs in canaliculi, phagocytosed bile in kupffer cells, and bile granules within hepatocytes. these changes are easily recognized in cytologic and frozen preparations, but are less apparent in paraffin-embedded specimens, particularly in cats. 1 when cholestasis is identified, ehbdo should be ruled out. this should be easily accomplished by abdominal ultrasonography as animals with intrahepatic cholestasis lack the dilation of intra-and extrahepatic bile ducts that is typical of ehbdo. 48 however, exploratory laparotomy should be considered in highly suspicious cases for confirmation. development at different stages of their formation. the wsava liver standardization group suggests that cystic disorders be classified into one of the following groups: congenital dilation of the large and segmental bile ducts; juvenile polycystic disease/congenital hepatic fibrosis; and adult polycystic disease. 1 congenital dilation of the large intrahepatic bile ducts (i.e., the hepatic ducts and segmental ducts) has been described in dogs. [39] [40] [41] the lesion is similar to that of caroli disease in humans and it is thought to represent an early defect in the formation of the intrahepatic bile ducts. 1, 40 the disease is marked by extreme, diffuse, grossly evident dilation of the extrahepatic portion of the large intrahepatic bile ducts containing pale-yellow viscous fluid. the gallbladder and common bile duct are normal, as these have a separate embryologic origin from the intrahepatic bile ducts. the liver is normal to mildly increased in size, with diffuse cysts of varying sizes throughout. histologically there are areas of marked bridging portal fibrosis containing multiple dilated bile ducts. the lobular architecture of the liver is normal. although concurrent ascending cholangitis commonly occurs in humans, this is rarely reported in dogs. [39] [40] [41] in addition to the hepatic lesions, affected dogs have fusiform, radially arranged renal cysts with moderate to marked fibrosis throughout the renal cortex and medulla. [39] [40] [41] affected dogs are presented early in life, ranging from 13 weeks to 3.5 years. [39] [40] [41] clinical signs include vomiting, weight loss/failure to thrive, decreased appetite, lethargy, ascites, and rarely icterus and neurologic signs. the clinical signs are typically chronic in nature. gi signs, neurologic signs, and ascites are likely a result of portal hypertension caused by pressure of the cysts on the portal vein. hepatomegaly may be noted on physical examination. activity of serum liver enzymes is typically normal to mildly increased, although marked increases in activity of alt and alp have been reported. 40 renal azotemia is present in some patients. ultrasonographically, cystic dilations of the intrahepatic ducts (most with associated calcification) are easily recognized, even though the renal cysts are not always apparent. definitive diagnosis is made on the basis of the gross and histologic findings described above. rational treatment options and prognosis are unknown, as only one dog in the literature has been treated. this dog was doing well on a low-protein diet at 5 months of followup. 40 most of the affected dogs have been fairly stable, and supportive care may be warranted despite the appearance of severe cystic disease. juvenile polycystic disease/congenital hepatic fibrosis has been described in litters of cairn terriers, west highland white terriers, and cats. [42] [43] [44] this form is analogous to autosomal recessive polycystic kidney disease in humans, and the inheritance appears to be autosomal recessive in the few families of veterinary patients that have been described. 42, 43 the liver cysts are thought to represent an intermediate defect in the development of the intrahepatic bile ducts. 1, 40 the liver involvement is primarily microscopic including fibrotic portal areas containing abnormally structured, dilated small bile ducts. the result is a grossly enlarged and firm liver. 40 renal cysts are present and are identical to those described for dogs with dilation of the large and segmental bile ducts. 40 affected animals are usually presented at less than 8 weeks of age for abdominal distention because of renomegaly and hepatomegaly. 42, 43 however, one 12-yearold cat with similar lesions has been reported. 44 most affected animals are ill or have died at the time of presentation. 42, 43 liver enzyme activity has been increased in the few animals in which it was evaluated. 43 abdominal ultrasound may identify cysts and definitive diagnosis is made based on histologic findings as described. treatment has not been attempted, and the prognosis appears to be grave. hepatic progenitor cells. 50 they may be identified at intrahepatic or extrahepatic locations. they appear to have a more aggressive course than cholangiocellular carcinomas, with the majority being present in multiple lobes and greater than 90% having metastasized at the time of diagnosis. 52, 54, 55 the major pathogenetic mechanisms of canine and feline extrahepatic biliary tract disease are obstruction, inflammation, and exudation. the major causes of extrahepatic biliary tract obstruction (ehbo) are pancreatitis, gallbladder mucoceles (in dogs), cholelithiasis, parasitic infections (in cats), and tumors. ehbo is more common in dogs than cats. biliary tract inflammation (e.g., cholecystitis, cholangitis) is primarily caused by bacterial infection, but can be nonseptic or parasitic, especially in cats. gallstones may be associated with biliary tract disease (e.g., infection, obstruction), but the majority of them appear to be clinically silent. stones in the gallbladder are termed choleliths while stones in the biliary tract are termed choledocholiths. biliary tract exudation or leakage can be caused by traumatic (primarily of the bile ducts) or spontaneous rupture (primarily of the gallbladder). the latter is caused by necrotizing cholecystitis, which can be caused by sepsis (e.g., infectious cholecystitis), pressure necrosis (e.g., mucocele), or infarction. biliary tract tumors are rare and of uncertain cause. the common bile duct passes through the lesser omentum and the pancreatic parenchyma before entering the mesenteric wall of the duodenum. in the dog, it empties near the opening of the minor pancreatic duct at the major duodenal papilla, whereas in the cat it joins with the major pancreatic duct before emptying into the duodenum. inflammation and edema with pancreatitis may be sufficient to cause compression and obstruction of the bile duct. this is probably the most common cause of canine ehbo. 1 there is, however, no consistent relationship between clinical severity of the pancreatitis and likelihood of ehbo, possibly because pancreatitis can affect different regions of the pancreas. pancreatitis is a rare cause of ehbo in the cat. chapter 60 discusses the breeds at increased risk for pancreatitis and causes. gallbladder mucoceles occur primarily in dogs. in such cases, the gallbladder is filled with inspissated, semisolid mucus that may extend into the bile ducts causing obstruction. mucoceles can exceed the storage capacity of the gallbladder thereby causing pressure necrosis on the wall of the gallbladder. they can also spontaneously rupture (often at the fundus) 2,3 causing bile peritonitis. the cause of gallbladder mucocele is unknown, but might include dysfunction/hyperplasia of mucus-secreting cells in the gallbladder mucosa. mucoceles may become secondarily infected. 2 gallstones are often clinically silent and observed incidentally only at the time of abdominal imaging. they can be associated with cholecytitis, 4 but they rarely cause ehbo because they must be small enough to enter the cystic bile duct but large enough to lodge there. intrahepatic cholestasis is associated with extrahepatic bacterial infection in dogs. 49 this syndrome is well characterized in humans and may occur in other species such as the cat. it represents an important differential diagnosis for hyperbilirubinemia in animals without primary liver disease, as over 40-fold increases in total bilirubin have been reported. the physiology behind this mechanism is incompletely understood, but it is thought to result from reduction of bile salt-dependent and -independent bile flow caused by bacterial toxins and/or inflammatory mediators. 49 tumors of biliary origin in dogs and cats include cholangiocellular adenoma, cholangiocellular carcinoma, and carcinoid. 50 they are uncommon, representing less than 1% of all canine and feline neoplasms. [51] [52] [53] [54] the tumors of epithelial origin, cholangiocellular adenoma and carcinoma, are the most common, comprising 40% of all hepatic neoplasms in dogs [51] [52] [53] and 56% to 80% of all hepatic neoplasms in cats. [54] [55] [56] tumors showing characteristics of both hepatocellular and cholangiocellular carcinoma have been reported rarely. 50, 52 in dogs, 70% to 100% of biliary epithelial tumors are malignant, 51,52 while in cats 35% to 43% are malignant. [54] [55] [56] cholangiocellular adenomas commonly contain cystic components, especially in cats, and have been referred to as biliary or hepatobiliary cystadenomas in this species. 57,58 cholangiocellular tumors arise predominantly from intrahepatic bile ducts in both species. extrahepatic location is more common in cats than in dogs and is always associated with malignancy. [51] [52] [53] [54] 56 in both species, cholangiocellular carcinomas are more likely to present as multiple or diffuse tumors than are adenomas. 51, 52, 54 carcinomas are highly metastatic (70% to 90% rate) with local lymph nodes, peritoneum, and lung being the most common sites of metastasis. 51, 52, 54 the etiology of biliary neoplasia is unknown. affected dogs and cats are typically middle-aged to older, although animals with malignancy may present at a younger age than those with benign disease. 52, 56 clinical signs are usually vague (such as anorexia and lethargy) and somewhat chronic. malignant tumors are more likely to cause clinical signs, as many benign tumors are incidental findings not associated with illness. 51, [56] [57] [58] hepatomegaly or the presence of a cranial abdominal mass may be identified on physical examination. increased activity of serum liver enzymes is more commonly associated with malignancy, often being absent with benign tumors. 51, 56, 58 even though abdominal radiographs will often identify the presence of hepatomegaly or an hepatic mass, ultrasonography is the preferred imaging method for identification of biliary neoplasms. this modality allows for determination of the cystic nature of the tumors 58 and for the evaluation of metastatic potential. fine-needle aspiration and cytology are of limited utility for diagnosis of biliary epithelial tumors. carcinoma was correctly identified via liver mass aspiration in only 20% of cases in one study. 14 however, cytology is recommended as it likely exhibits high specificity, especially for metastatic lesions. surgical excision and biopsy is the optimal diagnostic and therapeutic technique for tumors confined to one or two liver lobes. surgical excision appears to be curative in cats with benign tumors, 56,57 but malignant tumors carry a poor prognosis in both dogs and cats with many cases not surviving to discharge and survival greater than 6 months not reported in any case. 56, 59 adjunctive chemotherapeutic protocols have not been reported. carcinoids, also referred to as neuroendocrine tumors, are far less prevalent in dogs and cats than the tumors of biliary epithelial origin. 52, 54, 55 these tumors are thought to develop from neuroendocrine cells in the epithelium of bile ducts or gallbladder or from anorexia and vomiting as more prominent symptoms. fever is uncommon, icterus inconsistent, and leukocytosis is often insignificant, even with marked bacterial infections of the biliary tract. gallstones are generally asymptomatic. they can be associated with cholecystitis or ehbo. bilious abdomen can be a clinically mild condition, or it can be associated with life-threatening signs. septic bilious abdomen causes extremely severe peritonitis with systemic inflammatory response syndrome (e.g., anorexia, vomiting, abdominal pain, poor perfusion, fever, and death). these patients may be in the initial hyperdynamic state (e.g., red mucus membranes, bounding pulse, fever, or hypothermia) or, if initially undiagnosed, can be in the late hypodynamic state (e.g., pale mucus membranes, weak pulse, and hypothermia). intraperitoneal bile seems to make septic peritonitis more severe. in contrast, some animals with sterile bilious abdomen (e.g., as a consequence of automobile trauma) are essentially normal except for ascites and icterus. plain radiography is occasionally diagnostic of biliary tract disease. some gallstones are radiopaque (figure 61-30 ). 4 finding air in the gallbladder or in the wall of the gallbladder (i.e., emphysematous cholecystitis; figure 61 -31) is diagnostic of infection with a gas-producing bacterium. "porcelain" gallbladder (i.e., a radiopaque gallbladder because of intramural mineralization of the gallbladder) is associated with carcinoma. 15 ultrasound is generally accepted as the most important and sensitive method for diagnosing extrahepatic biliary tract diseases. 7 many patients that are not suspicious for biliary tract disease are fortuitously diagnosed when ultrasound or radiographs are requested for various reasons. 14 in difficult cases (e.g., partial ehbo versus complete ehbo), nuclear scintigraphy techniques can be used 16 ; however, this seems to be rarely required. diagnosis of ehbo generally relies upon the use of ultrasound. dilation of the bile ducts (normal canine bile ducts are ≤3 mm; normal feline bile ducts are ≤2 to 2.5 mm) is primarily caused by ehbo, and is generally seen by 3 days postobstruction. if one is unsure whether the ducts are dilated, repeating the examination in 3 days should be helpful. enlargement of the gallbladder is not diagnostic of ehbo because anorexia and starvation of any cause may do the same thing. however, failing to find a dilated gallbladder parasites occasionally cause obstruction. platynosomum fastosum (i.e., p. concinnum) is a fluke that inhabits the gallbladder and/or bile ducts of cats that are infected by eating lizards or toads. 5 natural infections are found primarily in florida, hawaii, and the caribbean. it may be asymptomatic or may cause obstruction or fibrosis. they are rare causes of cholecystitis. tumors may cause obstruction, and may be one of the more common causes in cats. 6, 7 cholecystitis is most commonly caused by bacterial infection, ostensibly from bacterial migration up the bile duct. various grampositive, gram-negative, aerobic, and anaerobic bacteria (e.g., clostridium, staphylococcus spp., enterococcus spp., streptococcus spp., klebsiella spp., e. coli, helicobacter spp.) have been reported. [8] [9] [10] [11] infections caused by gas-producing bacteria can produce emphysematous cholecystitis. because of a shared biliary and pancreatic ductal system in the cat, hepatobiliary disease is a well-established risk factor for pancreatitis in the cat. aseptic inflammation of the gallbladder (necrotizing cholecystitis) has been reported, and infarction is one such cause. 11, 12 exudation leakage of bile into the abdomen can be a result of mechanical forces (e.g., automobile trauma) that cause a shearing effect resulting in transaction of the common bile duct or one of the other bile ducts. mechanical rupture has also been reported following gunshot trauma. necrosis of the gallbladder raises the risk for rupture of the gallbladder and occasionally the bile ducts. canine and feline gallstones are typically composed of cholesterol, bilirubin, or may be mixed (as opposed to human gallstones which are usually caused by cholesterol). 4 feline gallstones are typically calcium carbonate or mixed stones. 13 biliary tract diseases can cause severe clinical signs (e.g., anorexia, depression, vomiting, icterus, abdominal pain) or they may be relatively asymptomatic. most symptomatic patients have serum biochemical abnormalities (e.g., increased serum alt, alp, and serum bilirubin). hypercholesterolemia is common in patients with ehbo. hyperbilirubinemia by itself does little besides cause icterus; therefore, patients with extremely high serum bilirubin concentrations can be relatively asymptomatic. renal failure has been attributed to excessively high serum bilirubin concentrations, but it is unclear that bilirubin is the cause or that this is common. shetland sheepdogs 14 appear to have an increased risk for biliary tract disease, and cocker spaniels might also (see chapter 62) . besides icterus, clinical signs in patients with ehbo are primarily a result of the cause of the obstruction, not the obstruction itself. canine pancreatitis in particular may cause severe clinical signs (e.g., anorexia, vomiting, abdominal pain-see "complications of liver disease" section). however, not all patients with pancreatitisinduced ehbo have severe pancreatitis. causes of ehbo that are insidious (e.g., mucoceles, tumors, and stones) are often unsuspected until the patient becomes icteric. biliary tract inflammation such as septic cholecystitis is welldocumented in dogs and cats. shetland sheepdogs appear to be at a greater risk of inflammatory biliary tract disease than most other breeds. 14 clinical signs vary, but most animals are clinically ill with cholecystitis is often diagnosed by ultrasound-guided percutaneous aspiration of gallbladder bile for cytology and culture. this is a relatively sensitive and specific procedure for diagnosing biliary tract infection. 2, 18 the finding of a dilated bile duct coincident with a normal-size gallbladder suggests cholecystitis, prior ehbo, or a rare congenital problem such as caroli disease. 19 it is important to note that patients with bacterial cholecystitis may have no ultrasonographic or gross abnormalities. ultrasound may be suggestive, but is relatively insensitive for cholecystitis. 8, 20 consequently, it is probably best to routinely aspirate bile for cytology and culture in patients with hepatobiliary disease. gallstones are relatively easy to diagnose, some can be found by radiographs, but almost all can be found by ultrasonography ( figure 61 -33). with ehbo, the underlying cause is always the primary concern. ehbo in and of itself is not the primary consideration when deciding upon therapy. pancreatitis, for example, is primarily a medical disease (see chapter 60) . surgery is rarely appropriate in the management of pancreatitis even when it is causing ehbo. if deemed necessary, ehbo caused by pancreatitis can be relieved by percutaneous aspiration 21 or placement of a biliary tract stent. 22 if absolutely necessary, a cholecystoduodenostomy may be performed, but this surgery should be avoided if possible. these procedures are seldom necessary because almost all patients with ehbo caused by pancreatitis will experience resolution of the obstruction with medical therapy. gallstones should be removed only if they are causing obstruction or cholecystitis. it is usually better to perform a cholecystectomy 4,13 as opposed to a cholecystotomy; the former has a lower morbidity and mortality rate in people and presumably in dogs and cats as well. biliary tract tumors can seldom be cured surgically. if a patient has ehbo caused by something that cannot be treated medically (e.g., tumor, pancreatic stricture, traumatically torn bile duct), then a biliary bypass procedure can be performed. cholecystoduodenostomy can relieve the obstruction, although the surgery requires special surgical skills. this surgery can predispose the patient to recurrent, ascending cholecystitis or other complications 10,23 and should only be performed in patients that in a patient with clearly dilated bile ducts suggests biliary tract inflammation or prior ehbo. any dog with ehbo should be suspected of having acute pancreatitis until proven otherwise. chapter 60 details the diagnosis of pancreatitis. if acute pancreatitis is eliminated in a patient with ehbo, then one should look for gallstones and tumors, first by imaging and then by exploratory surgery or laparoscopy if imaging fails to provide a diagnosis. mucoceles are readily diagnosed by ultrasound, although there is some debate about what constitutes a mucocele. "classic" mucoceles are described as producing a "kiwi fruit" appearance without gravitydependent bile movement. "sludge" in the gallbladder is a common finding (and it has gravity-dependent movement), but is not clinically significant. 17 the gallbladder of clinically normal dogs may appear abnormal on ultrasound examination (figure 61-32) , while patients with significant biliary tract disease may appear essentially normal. whether or not patients with ultrasonographic abnormalities of the gallbladder will become symptomatic cannot be reliably predicted. the gallbladder is filled with hyperechoic material (arrows) that was not gravity dependent. the dog was asymptomatic for biliary tract disease and was still in good health without therapy for the gallbladder 10 months after this image was taken. . the stones were found fortuitously during an abdominal ultrasound; there was no evidence that they were causing any clinical signs. gb that has a risk of dehiscence. in general, one should either remove the gallbladder, aspirate gallbladder bile, gently express the gallbladder, or leave it undisturbed. if the leakage is associated with septic peritonitis, then that is a true emergency and requires immediate, aggressive medical and surgical therapy. gallstones may be monitored if the patient is asymptomatic. tumors can rarely be resected; they are generally inoperable when found. rare examples exist of patients with biliary tumors being cured surgically. biliary flukes can be treated with praziquantel (20 to 40 mg/kg sq for 3 days). chapter 60 discusses pancreatitis in greater detail. biliary mucoceles that have not ruptured often have a good prognosis; however, one report found a 32% mortality rate in patients without a rupture versus 68% mortality in patients with bile peritonitis. 14 the report authors recommended a more preemptive approach (i.e., versus waiting until the patent is symptomatic). another group found no difference in mortality between rupture and nonruptured mucoceles (i.e., 21%). 3 gallstones generally are innocuous, and even when they are causing signs have a good prognosis as long as rupture has not occurred. bacterial cholecystitis has a good prognosis as long as the gallbladder is intact and not at risk for rupture. septic bilious peritonitis has a very guarded to poor prognosis, depending upon the severity of the peritonitis. gallstones are usually asymptomatic, but can occasionally cause a problem. they are usually easily removed and resolved. biliary tumors are usually a poor prognostic finding 6, 7 because of late diagnosis. flukes can be treated, but without early recognition, extensive tissue injury may or may not resolve after therapy. the liver serves many important functions including metabolism (carbohydrate, protein, lipid, nucleic acid, xenobiotics, porphyrins, vitamins, minerals, glutathione, endogenous hormones), coagulation factor synthesis, biliary secretion, and immune surveillance (see chapter 1). it is not surprising, therefore, that animals with liver disease experience a broad range of complications reflecting perturbations in one or more of these functions. the hepatic portal venous system accounts for up to 75% of the total hepatic circulation and serves to transport nutrients from the gi tract to hepatic sinusoidal capillaries (and hepatocytes) before coalescing once again into central veins, hepatic veins, and caudal vena cava. in health, the multiple branching of the main portal vein, venules, and capillaries reduces the overall resistance to blood flow (circuits in parallel reduce resistance), and therefore the pressure required to perfuse these capillaries is maintained at a low level of less than 5 to 6 mm hg. 1,2 disease processes that obstruct flow through the intrahepatic branches of the portal vein or sinusoids elevate this pressure and result in significant portal hypertension. hepatocyte swelling impedes portal flow in acute pathophysiologic states, and fibrosis further impedes this flow in absolutely require it. generally speaking, ehbo caused by pancreatitis that is not resolving as quickly as desired is not necessarily a good indication for this procedure; patience and medical therapy generally resolve the problem. in fact, pancreatitis can be a complication of surgery in this region. 24 many animals with hepatic disease are concurrently treated with antioxidants and other hepatoprotectants. although most of these drugs will not hurt patients with biliary tract disease (in fact, they may be beneficial if the disease is extending into the hepatic parenchyma, such as cholangitis/cholangiohepatitis), care must be taken before administering udca. udca is a choleretic agent that stimulates bile flow. this could be disadvantageous in a patient with complete ehbo. mucoceles usually need to be removed surgically. 2, 3, 14 medical management may be attempted (e.g., fat-restricted diet plus udca), but there is a substantially increased mortality rate for patients that experience gallbladder rupture (figure 61-34) ; consequently, surgery is probably the safest course. most patients are not at risk of immediate rupture. as long as ultrasonography does not suggest impending rupture, one should make sure the patient is an optimal anesthetic risk. bacterial cholecystitis, uncomplicated by ehbo or stone, should generally first be treated with antibiotics, usually for 4 to 6 weeks. however, some patients with bacterial cholecystitis cannot be cured with antibiotic therapy, ostensibly because the infection has localized in the gallbladder mucosa. these patients consistently relapse after discontinuation of antibiotics, and therefore a cure may be achieved only with antibiotics plus cholecystectomy. when performing this surgery, great care should be taken to avoid causing a stricture or obstruction of the common bile duct. postcholecystectomy bile duct obstruction may prove fatal. it is also very important to be avoid traumatizing the pancreas to avoid severe pancreatitis. 24 biliary tract leakage is treated based upon the underlying cause. if the gallbladder has ruptured, cholecystectomy is usually most appropriate. if the bile duct has ruptured, it is very difficult to successfully anastomose the two ends. in that instance, one generally must perform a cholecystoduodenostomy and ligate both ends of the torn bile duct. it is important to avoid biopsying the gallbladder as clinical signs associated with he in dogs and cats include depression, behavioral changes, circling/head-pressing, ataxia, apparent blindness, abnormal swallowing or salivation, stupor, seizures, and coma (table 61-10) . salivation is much more common in cats than dogs with he. brain dysfunction in he was historically considered to be a neurotransmitter dysfunction but current evidence suggests lowgrade cerebral edema caused by astrocyte swelling is the predominant pathologic change. 8 there is consensus that ammonia is the key toxin in he but blood ammonia concentrations do not always correlate with severity of clinical signs. this is because a large number of other factors interact with the effects of ammonia to precipitate he. astrocytes play a central role because they express glutamine synthetase and detoxify the ammonia that reaches the cns. intraastrocyte accumulation of osmotically active glutamine in he results in astrocyte swelling and thus low-grade cerebral edema. 8 this is largely reversible if the precipitating factors are treated, but edema can become severe and result in irreversible cns changes in severe and acute he. precipitating factors include inflammatory cytokines, benzodiazepine-type sedatives, and disturbances in amino acid metabolism and dopaminergic neurotransmission. [8] [9] [10] the source of ammonia is primarily absorption from the gut, although other sources also exist as a result of interorgan metabolism. gut-derived ammonia was traditionally assumed to be a by-product of intestinal bacterial metabolism in the colon. this remains an important source in some conditions such as melena. however, recent studies in other species suggest that small intestinal enterocyte metabolism of glutamine as their main energy source is the most important source of postprandial ammonia absorption in the portal vein. 9, 10 this is also likely the case in most dogs on normal diets as it is very unusual for undigested protein to reach the colon, although the source of gut-derived ammonia has never been investigated in dogs. in normal dogs, ammonia is transported to the liver via the main portal vein, and further metabolized chronic pathophysiologic states. sustained portal hypertension is associated with many, but not all, of the complications of liver disease. portal hypertension is an important, potentially life-threatening complication of liver disease in the dog. it is rare or poorly documented in the cat. portal hypertension develops most often in dogs with chronic liver disease and cirrhosis. it is occasionally recognized as a congenital lesion in young animals with arteriovenous fistulas, 3 or as a hypoplastic disorder of the intrahepatic portal vein branches resulting in a condition referred to as noncirrhotic portal hypertension. 4, 5 prehepatic portal hypertension can develop secondary to portal vein thrombosis or congenital hypoplasia of the extrahepatic portal vein, but these are less common. sustained portal hypertension may progress to splanchnic congestion, gi ulceration, ascites, and encephalopathy. portal venous hypertension produces vascular stasis and venous congestion and increases the risk of gi ulceration, particularly in conjunction with other risk factors such as anorexia and steroidal and nonsteroidal antiinflammatory drug usage. portal hypertensionrelated ulceration in the dog is typically duodenal although bleeding esophageal varices, similar to those reported in humans, are occasionally observed. 3 glucocorticoids should be used only with great caution in dogs with portal hypertension. reduced systemic blood pressure is another consequence of portal hypertension and splanchnic venous congestion. changes in systemic blood pressure activate the renin-angiotensin-aldosterone system (as described in chapter 8) and renal sodium retention, and thus increase total circulating fluid volume. the increase in circulating fluid volume (the "overfill" hypothesis) is believed to be the triggering event for the development of ascites in animals with portal hypertension. 6 this is why aldosterone antagonists are the initial treatment of choice in ascites caused by portal hypertension (for more details see chapter 8). ascites is a negative prognostic indicator in dogs with chronic hepatitis, 7 although individual animals with chronic hepatitis and ascites can be managed and maintained for many months. with sustained portal hypertension, multiple acquired psss develop and serve as a conduit for portal blood flow directly into the systemic circulation. shunts serve to dissipate some of the increased portal pressure thus reducing the risk of adverse complications such as venous congestion, gi hemorrhage, and ulceration. they do, however, raise the risk of yet another complication of liver disease-hepatoencephalopathy. he is a syndrome of potentially reversible brain dysfunction resulting from impaired liver function. it results from either severe hepatocyte dysfunction or more commonly the presence of portosystemic collateral circulation, either congenital or acquired, where a variable combination of shunting of portal blood and hepatocyte dysfunction contributes to the clinical signs. it can be acute or chronic in presentation. acute he is most often a result of acute fulminating liver failure (see "consequences of hepatocyte and biliary tract injury" section) and carries a poor prognosis. more chronic he is usually a result of congenital or acquired psss. 2 platelet counts, d-dimers, fibrinogen, or protein c) in 24 (57%) of 42 dogs affected with liver disease. 18 coagulation abnormalities are also common in cats with liver disease and one study found abnormalities in 18 (82%) of 22 cats with liver disease. 19 multiple mechanisms of coagulopathy are possible in liver disease patients. in alf cases such as xylitol toxicity in dogs, 20 hl in cats, 21 and cirrhosis in dogs, 18 loss of normal hepatocyte function results in severe coagulation factor deficiency. vitamin k deficiency has also been implicated in coagulopathy particularly in cats in which cholestasis impedes bile salt secretion, emulsification, and micellarization of fat and fat-soluble vitamins and fat-soluble vitamin absorption. 21 concurrent inflammatory bowel disease and pancreatitis exacerbate this condition in many cats with cholangitis. 21 finally, platelet abnormalities (cytopenia and cytopathy) may contribute to coagulopathy in dogs with liver disease. 18, 22 the liver has the unique ability to regulate its growth and mass. hepatocyte loss caused by viral, bacterial, or chemical injury, or partial hepatectomy triggers hepatocyte replication. 23, 24 liver injury not only stimulates hepatocyte turnover but may also stimulate biliary proliferation and activation and proliferation of hscs. these changes usually occur together in an orchestrated wound-healing response. in the case of hepatocyte loss, normally quiescent hepatocytes replicate to restore the liver functional capacity and mass. these are the main cells that regenerate liver mass. however, in severe injury or where hepatocyte turnover is inhibited by senescence, a progenitor cell reserve may also replicate and regenerate liver mass. 24 although hepatocytes are capable of replication, they have very slow turnover in a normal liver and there are negative consequences of long-term increased stimulation and turnover in chronic liver disease. it has been shown that cycling hepatocytes suffer irreversible erosion of telomeres, which leads to senescence. 25 functional capacity is a relative rather than absolute parameter. the set point for growth regulation is the ratio between liver mass and body mass rather than liver mass per se. the optimization of the ratio indicates that the liver reaches a state in which it performs the amount of metabolic work needed to meet the functional requirements of the body. 24 gene expression in the regenerating liver is a multistep process with at least two critical steps: the transition of quiescent hepatocytes into the cell cycle ("priming"), and the progression beyond the restriction point in the g 1 phase of the cell cycle. hepatocytes must first be primed before they can fully respond to growth factors. as many as 70 different genes participate in the early response to hepatectomy, but tnf and il-6 appear to be the major cytokines involved in the priming of hepatocytes. 20 the proliferative effect of tnf on hepatocytes is further influenced by reactive oxygen species, nitric oxide, and glutathione content, and at least four transcription factors (nuclear factor kappa b, stat3, ap-1, and c/ebpβ) play major roles in the initiation of early liver regeneration. 23, 24 progression through the cell cycle beyond the initiation phase requires growth factors, primarily hepatocyte growth factor and tgf-α. the subsequent expression of cell-cycle genes, particularly cyclin d 1 , establishes the stage at which replication becomes growth factorindependent and autonomous. at this point, the hepatocyte is irreversibly committed to replicate and the cell-cycle replication machinery takes over. the regenerative capacity of the residual hepatocytes may restore liver mass and function after as much as 65% to 70% hepatectomy. 23, 24 progenitor cells are only activated in severe liver injury. they appear to reside in a "niche" that is a particular regulatory environment. 24 a recent immunohistochemical to urea by hepatocytes in the krebs-henseleit cycle. with portosystemic shunting or severe hepatocyte dysfunction, ammonia accumulates in the brain (and other tissues) where it is taken up by astrocytes and results in edema as described previously. dogs with he also show disturbances in cns aromatic amino acid metabolism. 11, 12 the aromatic amino acids (tyrosine, tryptophan, and phenylalanine) accumulate in, the cns in portasystemic shunting. in the brain, β-phenylalanine and tyrosine are metabolized to phenylethanolamine and octopamine, both of which can act as false neurotransmitters. however, dietary supplementation with branched-chain amino acids (e.g., leucine, isoleucine, valine) does not convincingly improve he in either dogs or humans. 9, 12 however, some dietary protein sources appear to be better than others in dogs with he. dogs on soya protein diets show a lower plasma ammonia concentration than those fed meat protein. 13 dogs with he have also traditionally been fed a protein-restricted diet. however, protein restriction is no longer advocated in humans with he 9 and it may be the digestibility and type of protein rather than a reduced amount that are most important in dogs. more studies are needed to investigate this. several other metabolic alterations exacerbate clinical signs associated with he, including acid-base disorders, electrolyte abnormalities, particularly hypokalemia, hypoglycemia, hypoxemia, and arginine deficiency (cats). an important trigger in humans and rodents is inflammation: recent studies confirm that inflammatory cytokines are synergistic with ammonia in precipitating he and that controlling inflammation in other organs is an important part of managing the patient with he. 14, 15 there is anecdotal evidence that this is also true in dogs. the liver has significant structural and functional reserve capacity to support ongoing metabolic needs during mild to moderate forms of liver injury. moreover, the liver has the ability to regenerate liver volume and cell mass during the recovery phase of most forms of liver injury. signs of liver failure develop earlier with acute forms of liver injury than with chronic, progressive liver disease. zones 1, 2, and 3 hepatocytes of the hepatic acinus have differing functions. zone 1 (periportal) hepatocytes, for example, have a high capacity to cycle ammonia through the urea cycle thereby reducing the toxicity of ammonia. zone 3 hepatocytes (nearer the hepatic vein) have a lesser capacity for ammonia and instead convert it to glutamine. 16, 17 in health, zonation permits flexibility in hepatic function such that in metabolic acidosis, for example, the liver can rapidly divert ammonia toward glutamine production, which is necessary for h+ ion excretion in the kidney. in severe acute liver injury, this becomes an important "tradeoff" because acute selective destruction of periportal (zone 1) hepatocytes more readily results in signs of encephalopathy because of the reduced ability of zone 3 hepatocytes to detoxify ammonia. in chronic liver disease, if hepatocytes undergo piecemeal necrosis at different rates in different zones, the remaining hepatocytes can assume some of those functions, so that clinical signs of deficiency are not seen until later in chronic disease processes. coagulopathy is a complication of both acute and chronic liver disease in dogs and cats. a recent study reported one or more coagulation abnormalities (prolongation of coagulation times, changes in the cause of chronic hepatitis is usually unknown. 30 there is increasing evidence that fibrosis and even some forms of cirrhosis in humans and rodent models are reversible if the underlying cause is removed. 28, 31 the challenges are removing the cause and also defining the point at which cirrhosis moves from a reversible to irreversible state. increased fibrous septal thickness, smaller nodule size, and reduced cellularity together with increased collagen cross-bridging have all been associated with an irreversible cirrhotic state in rodents and humans. 28 it is unknown whether liver fibrosis or cirrhosis in dogs is reversible clinically. cases of chronic hepatitis in dogs very rarely have sequential liver biopsies over a long period of time to assess progression of disease and noninvasive markers of fibrosis remain to be validated. serum hyaluronic acid is increased in dogs with cirrhosis 32 as is tgf-β 33 but the usefulness of these markers in following progression in clinical cases has not been assessed. identifying a reliable noninvasive marker of fibrosis for sequential studies in humans and dogs remains a challenge. 34 study suggests that canine and human liver progenitor cells are functionally very similar. 26 further characterization of the molecular events regulating hepatocyte replication and liver regeneration should improve outcome in animals affected with severe liver disease. the normal ecm of the liver provides cells with positional information and a mechanical scaffold for adhesion and migration. the ecm consists of collagens, glycoproteins, proteoglycans, glycosaminoglycans and molecules that are bound specifically by the ecm, such as certain growth factors, cytokines, matrix metalloproteinases, and processing enzymes such as tissue transglutaminase and procollagen propeptidases. a normal liver contains a very small amount of fibrous tissue as a percentage of its total mass. 27, 28 acute or chronic liver injury causes a dynamic wound-healing response with both production and removal of fibrosis. 29 hscs are the major source of the collagens that comprise fibrosis and cirrhosis, as well as of the tissue inhibitors of metalloproteinases (timps) that inhibit collagen degradation. it is the balance between collagen production by hscs and its degradation by matrix metalloproteinases that determines the severity and reversibility of the fibrotic response. following acute or chronic liver damage, hscs are stimulated to multiply and to undergo a complete phenotypic transformation from quiescent vitamin a-storing cells to contractile myofibroblasts which synthesize large amounts of ecm. 25, 29 important stimuli for hsc transformation and multiplication in liver injury include oxidative stress; chemokines including platelet-derived growth factor; vegf and tgf-β; adipokines; and parts of the innate immune system including toll-like receptor ligands. 29 the role of adipokines in stimulating fibrosis is increasingly being recognized in humans, where nonalcoholic fatty liver disease can lead to fibrosis and cirrhosis. they are produced by hscs themselves, as well as fat cells, and increased leptin and reduced adiponectin drive fibrosis. 29 their importance in dogs is unknown and it is also unknown whether vacuolar or fatty liver diseases progress to fibrosis in dogs, but these are important questions to answer in the future given the widespread occurrence of obesity in dogs. the contractile function of activated hscs contributes significantly to the development of portal hypertension, 28 and increases in angiogenic chemokines such as vegf and platelet-derived growth factor not only contribute to fibrogenesis by hscs, but also to the development of portal hypertension, so that the two pathologic processes are inextricably linked. 29 activated hscs have greatly increased production of timps, particularly timp1 and timp2, which prevent the action of matrix metalloproteinases in the ecm. the degree of fibrosis and reversibility then depend on the balance between perpetuation of hsc proliferation and secretion and resolution of hsc by either apoptosis or senescence of hscs or, indeed, their reversion to an inactive state. many factors contribute to hsc apoptosis, senescence, or reversion, including reduction in timps and nuclear factor kappa b and increased fas and p53. 29 however, in spite of all this understanding of the molecular mechanisms of fibrosis, a truly effective treatment for hepatic fibrosis in either humans or dogs has yet to be found. 29 it is important to remember that a normal fibrotic response (scar) is important in walling off pathogens and tissue injury and inhibiting this response without removing the inciting cause (e.g., a viral cause) could lead to spread of the pathology. 28 future treatment strategies for fibrosis should therefore incorporate treatment of the underlying cause of disease. this is clearly a problem in dogs where organizational principles of the liver bile formation and enterohepatic circulation functional and morphological relationships between the feline main pancreatic and bile duct sphincters the extrahepatic biliary tract in common domestic and laboratory animals hormonal regulation of bile secretion physiology of cholangiocytes hepatic stellate cell (vitamin a-storing cell) and its relative -past, present, and future hepatic stellate cells wnt antagonism inhibits hepatic stellate cell activation and hepatic fibrosis morphological characterisation of portal myofibroblasts and hepatic stellate cells in the normal dog liver liver fibrosis liver fibrosis the hepatic sinusoidal endothelial cell: morphology, function, and pathobiology vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on hepatic endothelial cells hepatic stem cells and liver development local control of the immune response in the liver immune mechanisms of viral clearance liver regeneration liver regeneration in surgical animal models-a historical perspective and clinical implications il-22 is involved in liver regeneration after hepatectomy fausto n: liver regeneration short-term effect of portal arterialization on hepatic protein synthesis and endotoxemia after extended hepatectomy in dogs liver regeneration pathophysiology: the biological principles of disease skills for communicating with patients teaching and learning communication skills in medicine liver regeneration history, physical examination, and signs of liver disease extrahepatic cholestasis in the dog and the differentiation of extrahepatic and intrahepatic cholestasis historical, physical examination and clinicopathological features of portosystemic vascular anomalies in the dog and cat wsava: standards for clinical and histological diagnosis of canine and feline liver diseases history, clinical signs and physical findings in hepatobiliary disease hepatic ultrasonography and pathological findings in dogs with hepatocutaneous syndrome: new concepts hepatic encephalopathy: management with lactulose and related carbohydrates clinicopathologic evaluation of the liver wsava: standards for clinical and histological diagnosis of canine and feline liver diseases destructive cholangiolitis in seven dogs breed, sex and age distribution in dogs with chronic liver disease-a demographic study hepatic stem cells glycogen storage disease type ia in canines: a model for human metabolic and genetic liver disease energy requirements of adult cats ammonia intoxication in the near adult cat as a result of a dietary deficiency of arginine nutrition of the domestic cat, a domestic carnivore lack of hepatic enzyme adaptation to low and high levels of dietary protein in the adult cat lipid metabolism and hyperlipidemia in the dog a study of the lipid transport system in the cat a new framework for reverse cholesterol transport: non-biliary contributions to reverse cholesterol transport micrornas: fad or future of liver disease? copper-induced hepatitis: the commd1 deficient dog as a translational animal model for human chronic hepatitis copper-associated liver disease esteller a: physiology of bile secretion coagulation disorders in dogs with hepatic disease current cytochrome p450 phenotyping methods applied to metabolic drug-drug interaction prediction in dogs substrate specificity and kinetic properties of seven heterologously expressed dog cytochromes p50 differential expression of cyp3a12 and cyp3a26 mrnas in canine liver and intestine in vitro comparison of cytochrome p450-mediated metabolic activities in human, dog, cat, and horse cytochrome p450s and other enzymes in drug metabolism and toxicity mrna and protein expression of dog liver cytochromes p450 in relation to the metabolism of human cyp2c substrates modulation of acetaminopheninduced hepatotoxicity by the xenobiotic receptor car increased free cortisol in plasma of dogs with portosystemic encephalopathy chronic glucocorticoid excess and impaired osmoregulation of vasopressin release in dogs with hepatic encephalopathy gabaergic inhibition of the pituitary release of adrenocorticotropin and melanotropin is impaired in dogs with hepatic encephalopathy liver sinusoidal endothelial cells: a new type of organ-resident antigen-presenting cell characterisation of 11 beta-hydroxysteroid dehydrogenases in feline kidney and liver central hypertensinogenic effects of glycyrrhizic acid and carbenoxolone feline lower urinary tract diseases metabolic and genetic aspects of urate urolithiasis in dalmatians hypoglycemia associated with nonislet cell tumor in 13 dogs the origins and kinetics of bilirubin in dogs with hepatobiliary and haemolytic diseases bilirubin metabolism in canine hepatobiliary and haemolytic disease plasma coagulation factor abnormalities in dogs with naturally occurring hepatic disease alterations of prothrombin time and activated partial thromboplastin time id dogs with hepatic disease correlation between coagulation profile findings and bleeding complications after ultrasoundguided biopsies: 434 cases (1993-1996) use of a test for proteins induced by vitamin k absence or antagonism in diagnosis or anticoagulant poisoning in dogs hp: circulatory disorders of the liver in dogs and cats diagnostic value of fasting plasma ammonia and bile acid concentrations in identification of portosystemic shunting in dogs evaluation of ammonia measurements in dogs with two analysers for use in veterinary practice ultrasonographic findings in dogs with hyperammonemia: 90 cases comparative studies on plasma and tissue sorbitol, glutamic, lactic and hydroxybutyric dehydrogenase and transaminase activities in the dog pharmacokinetics of liver transaminases in healthy dogs: potential clinical relevance for assessment of liver damage plasma and tissue enzyme activities in the cat hepatic clearance of rat liver aspartate aminotransferase isozymes: evidence for endocytotic uptake via different binding sites on sinusoidal liver cells effects of phenobarbital treatment on serum thyroxine and thyroid-stimulating hormone concentrations in epileptic dogs lobular dissecting hepatitis in juvenile and young adult dogs differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in doberman pinschers chronic active hepatitis in 26 doberman pinschers improvement in liver pathology after 4 months of d-penicillamine in 5 doberman pinschers with subclinical hepatitis subclinical versus clinical hepatitis in the doberman: evaluation of changes in blood parameters copper-associated hepatopathies in dogs hepatitis and copper accumulation in skye terriers the ubiquitous expressed murr1 protein is absent in canine copper toxicosis chronic hepatitis in doberman pinschers. a review copper metabolism and oxidative stress in chronic inflammatory and cholestatic liver disease in dogs copperassociated chronic hepatitis in labrador retrievers molecular regulation of copper excretion in the liver increasing incidence of hereditary intrahepatic portosystemic shunts in irish wolfhounds in the netherlands 1984-1992 inherited congenital extrahepatic portosystemic shunts in cairn terriers canine gastrointestinal disease the liver in systemic disease: an innocent bystander liver: biology and pathobiology a retrospective study of 77 cats with severe hepatic lipidosis, 1975-1990 neuroactive amino acids in hepatic encephalopathy chronic hepatic encephalopathy: studies into the pathogenesis and treatment in the dog the gaba hypothesis of the pathogenesis of hepatic encephalopathy: current status the pituitary-adrenocortical system in canine hepatoencephalopathy chronic glucocorticoid excess and impaired osmoregulation of vasopressin release in dogs with hepatic encephalopathy osmoregulation of systemic vasopressin release during long-term glucocorticoid excess: a study in dogs with hyperadrenocorticism serum acute phase protein concentrations in female dogs with mammary tumors acute phase protein response in dogs with experimentally induced gastric mucosal injury plasma coagulation factor abnormalities in dogs with naturally occurring hepatic disease coagulation abnormalities in 22 cats with naturally occurring liver disease coagulation disorders in dogs with hepatic disease use of a test for proteins induced by vitamin k absence or antagonism in diagnosis of anticoagulant poisoning in dogs: 325 cases (1987-1997) proteins invoked by vitamin k absence and clotting times in clinically ill cats evaluation of plasma protein c activity for detection of hepatobiliary disease and portosystemic shunting in dogs medical management of congenital portosystemic shunts in 27 dogs-a retrospective study nitrogen metabolism and ornithine cycle function postprandial venous ammonia concentrations in the diagnosis of hepatobiliary disease in dogs congenital portosystemic shunts in dogs: 46 cases (1979-1986) congenital portosystemic shunts in maltese and australian cattle dogs sensitivity and specificity of fasting ammonia and serum bile acids in the diagnosis of portosystemic shunts in dogs and cats postprandial venous ammonia concentrations in the diagnosis of hepatobiliary disease in dogs hyperammonemia due to a urea cycle enzyme deficiency in two dogs hyperammonaemic encephalopathy secondary to selective cobalamin deficiency in a juvenile border collie ammonia intoxication in the near-adult cat as a result of a dietary deficiency of arginine evaluation of ammonia measurements in dogs with two analysers for use in veterinary practice clinical investigation of a point-of-care blood ammonia analyzer rectal ammonia tolerance test in the evaluation of portal circulation in dogs with liver disease iron status and erythrocyte volume in dogs with congenital portosystemic vascular anomalies enzyme activities of canine tissue plasma and tissue enzyme activities in the cat enzyme activities in the dog: tissue analyses, plasma values, and intracellular distribution hematologic and biochemical abnormalities associated with induced extrahepatic bile duct obstruction in the cat alkaline phosphate, leucine aminopeptidase, and alanine aminotransferase activities with obstructive and toxic hepatic disease in cats alkaline phosphatase and alkaline phosphatase isoenzymes in the cat tissue sources of serum alkaline phosphatase in 34 hyperthyroid cats: a qualitative and quantitative study kinetics of mrna expression of alkaline phosphatase isoenzymes in hepatic tissues from glucocorticoid-treated dogs elimination of alkaline phosphatases from serum in dog after intravenous injection of canine phosphatases from bone and intestine disappearance rates of intravenously injected canine alkaline phosphatase isoenzymes serum half-life of intravenously injected intestinal and hepatic alkaline phosphatase isoenzymes in the cat solubilization of liver alkaline phosphatase isoenzyme during cholestasis in dogs diagnostic efficacy of serum alkaline phosphatase and gamma-glutamyltransferase in dogs with histologically confirmed hepatobiliary disease: 270 cases (1980-1990) diagnostic value of serum gamma-glutamyl transferase and alkaline phosphatase activities in hepatobiliary disease in the cat benign familial hyperphosphatasemia in siberian huskies hyperphosphatasemia in scottish terriers: 7 cases effect of colostrum ingestion on gamma-glutamyltransferase and alkaline phosphatase activities in neonatal pups the liver as an organ relationship between colloid osmotic pressure and plasma protein concentration in cattle, horses, dogs, and cats glycosylation accelerates albumin degradation in normal and diabetic dogs steroid responsive meningitis-arteritis: a prospective study of potential disease markers, prednisolone treatment, and long-term outcome in 20 dogs the role of acute phase proteins in diagnosis and management of steroid-responsive meningitis arteritis in dogs liver disease in dogs with tracheal collapse comparison of postprandial and ceruletide serum bile acid stimulation in dogs evaluation of urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in dogs urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in cats bile acids diagnostic test believed to contain limitations urine sulfated and nonsulfated bile acids as 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for canine cirrhosis the liver and spleen hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options emphysematous cholecystitis in a siberian husky cholelithiasis in dogs: 29 cases porcelain gallbladder associated with primary biliary adenocarcinoma in a dog plasma lipoprotein changes in experimental cholestasis in the dog gallbladder disease in shetland sheepdogs: 38 cases serum liver enzyme activities in healthy miniature schnauzers with and without hypertriglyceridemia intrahepatic and extrahepatic portal venous anomalies in dogs: 52 cases (1982-1992) hypoglycemia associated with nonislet cell tumor in 13 dogs the origins and kinetics of bilirubin in dogs with hepatobiliary and haemolytic diseases anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs covalently protein-bound bilirubin conjugates in cholestatic disease of dogs serum bile-acids in companion animal medicine direct spectrometric determination of serum bile acids in the dog and cat radioimmunoassay of conjugated bile acids in canine and feline sera absorption of bile acids in dog as determined by portal blood sampling: evidence for colonic absorption of bile acid conjugates dietary influence on bile acid conjugation in the cat periodic gallbladder contraction maintains bile acid circulation during the fasting period: a canine study intestinal transport of bile acids postprandial changes in serum unconjugated bile acid concentrations in healthy beagles evaluation of twelvehour preprandial and two-hour postprandial serum bile acids concentrations for diagnosis of hepatobiliary disease in dogs bile acid concentrations in the diagnosis of hepatobiliary disease in the dog measurement of serum bile-acids concentrations for diagnosis of hepatobiliary disease in cats post-prandial serum bile acid concentrations and ammonia tolerance in maltese dogs with and without hepatic vascular anomalies diagnostic value of fasting plasma ammonia and bile acid concentrations in the identification of portosystemic shunting in dogs serum unconjugated bile acids as a test for intestinal bacterial overgrowth in dogs helical computed tomographic angiography of canine portosystemic shunts comparison of transplenic multidetector ct portography to multidetector ct-angiography in normal dogs use of magnetic resonance angiography for diagnosis of portosystemic shunts in dogs surgical treatment of bile peritonitis in 24 dogs and 2 cats: a retrospective study (1987-1994) fine-needle aspirate cytology suggesting hepatic lipidosis in four cats with infiltrative hepatic disease vacuolar hepatopathy in dogs: 336 cases accuracy of ultrasound-guided fine-needle aspiration of the liver and cytologic findings in dogs and cats: 97 cases morphological classification of biliary disorders of the canine and feline liver diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats sampling and handling of liver tissue influence of biopsy specimen size, tissue fixation, and assay variation on copper, iron, and zinc concentrations in canine livers statistical relevance of ultrasonographic criteria in the assessment of diffuse liver disease in dogs and cats can sonographic findings predict the results of liver aspirates in dogs with suspected liver disease? bleeding after liver biopsy does not correlate with indices of peripheral coagulation lack of increased bleeding after liver biopsy in patients with mild hemostatic abnormalities correlation between coagulation profile findings and bleeding complications after ultrasoundguided biopsies: 434 cases (1993-1996) proteins invoked by vitamin k absence and clotting times in clinically ill cats pivka clotting times in dogs with suspected coagulopathies evaluation of hemorrhage, sample size, and collateral damage for five hepatic biopsy methods in dogs diagnostic correlation of liver aspiration cytology with histopathology in dogs and cats with liver hepatic abscesses in cats: 14 cases percutaneous ultrasoundguided trans-splenic catheterization of the portal vein in the dog transvenous retrograde portography for identification and characterization of portosystemic shunts in dogs use of a percutaneous atrial septal occluder device for complete acute occlusion of an intrahepatic portosystemic shunt in a dog update on hepatobiliary imaging accuracy of ultrasonography in the detection of severe hepatic lipidosis in cats statistical relevance of ultrasonographic criteria in the assessment of diffuse liver disease in dogs and cats cellular features of sonographic target lesions of the liver and spleen in 21 dogs and a cat contrast harmonic ultrasound of spontaneous liver nodules in 32 dogs correlations between ultrasonographic findings and specific hepatic diseases in cats: 72 cases (1985-1997) changes in gallbladder volume in healthy dogs after food was withheld for 12 hours followed by ingestion of a meal or a meal containing erythromycin prevalence of gallbladder sludge in dogs as assessed by ultrasonography ultrasonographic features of extrahepatic biliary obstruction in 30 cats gallbladder mucocele in dogs: 30 cases ultrasonographic diagnosis of portosystemic shunting in dogs and cats ultrasonography of portosystemic shunts in dogs and cats ultrasonographic identification and characterization of congenital portosystemic shunts comparison of 99mtco4(-) trans-splenic portal scintigraphy with per-rectal portal scintigraphy for diagnosis of portosystemic shunts in dogs use of hepatobiliary scintigraphy in the diagnosis of extrahepatic biliary obstruction in dogs and cats: 25 cases (1982-1989) comparative evaluation of the liver in dogs with a splenic mass by using ultrasonography and contrast-enhanced computed tomography magnetic resonance imaging of focal splenic and hepatic lesions in the dog inflammatory canine hepatic disease in: ettinger sj,feldman ec chronic hepatitis, cirrhosis, breed-specific hepatopathies, copper storage hepatopathy, suppurative hepatitis, granulomatous hepatitis, and idiopathic hepatic fibrosis drug-induced hepatotoxicity the pathology of drug-induced liver injury granulomatous hepatitis in dogs: nine cases (1987-1990) drug-related hepatotoxicity idiosyncratic toxicity associated with potentiated sulfonamides in the dog chronic progressive hepatitis in bedlington terriers associated with elevated liver copper concentrations clinical, morphologic, and chemical studies on copper toxicosis of bedlington terriers hereditary copper toxicosis in west highland white terriers the relationship between hepatic copper content and morphologic changes in the liver of west highland white terriers chronic active hepatitis in 26 doberman pinschers association between liver copper concentration and subclinical hepatitis in doberman pinschers chronic hepatitis in doberman pinschers. a review breed, sex and age distribution in dogs with chronic liver disease: a demographic study chronic hepatitis in cocker spaniels-another syndrome? acvim forum proc hepatic accumulation of alpha-1-antitrypsin in chronic liver disease in the dog diagnosis and prognosis of chronic hepatitis and cirrhosis in dogs hepatopathy in seven american cocker spaniels hepatitis and copper accumulation in skye terriers copper-associated liver disease in dalmatians: a review of 10 dogs copper-associated chronic hepatitis in labrador retrievers chronic hepatitis in labrador retrievers: clinical presentation and prognostic factors copper-associated hepatitis in labrador retrievers accuracy of ultrasound-guided fine-needle aspiration of the liver and cytologic findings in dogs and cats: 97 cases comparison of liver cytology and biopsy diagnoses in dogs and cats: 56 cases cytologic evaluation of inflammation in canine liver aspirates influence of biopsy specimen size, tissue fixation, and assay variation on copper, iron, and zinc concentrations in canine livers percutaneous transabdominal hepatic needle biopsies in dogs percutaneous biopsy of abdominal organs under ultrasound guidance diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats morphological classification of parenchymal disorders of the canine and feline liver. 2. hepatocellular death, hepatitis and cirrhosis clinical, pathological and serological features of spontaneous canine leptospirosis. an evaluation of the igm-and igg-specific elisa primary hepatitis in dogs: a retrospective review acute liver failure idiopathic hepatitis and cirrhosis in dogs interpretation of liver enzymes summary of the world small animal veterinary association standardization committee guide to classification of liver disease in dogs and cats therapeutic use of cytoprotective agents in canine and feline hepatobiliary disease hepatic abscesses in dogs: 14 cases (1982-1994) hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options hepatic abscesses in cats: 14 cases percutaneous drainage and alcoholization of hepatic abscesses in five dogs and a cat morphological calssification of parenchymal disorders of the canine and feline liver. 3. hepatic abcesses and granulomas, hepatic metabolic storage disorders and miscellaneous conditions transient acquired fanconi syndrome associated with copper storage hepatopathy in 3 dogs identification of a new copper metabolism gene by positional cloning in a purebred dog population the ubiquitously expressed murr1 protein is absent in canine copper toxicosis copper metabolism and oxidative stress in chronic inflammatory and cholestatic liver diseases in dogs chronic active hepatitis with cirrhosis in the doberman pinscher hepatic copper concentrations in labrador retrievers with and without chronic hepatitis (1980-2008): an emerging syndrome or oversupplementation? canine liver iron, copper, and zinc concentrations and association with histologic lesions hepatotoxicity of phenobarbital in dogs: 18 cases hepatotoxicity associated with pharmacologic agents in dogs and cats hepatotoxicity associated with ccnu (lomustine) chemotherapy in dogs hepatocellular toxicosis associated with administration of carprofen in 21 dogs transient erythropoietic protoporphyria associated with chronic hepatitis and cirrhosis in a cohort of german shepherd dogs upregulation of major histocompatibility complex class ii antigens in hepatocytes in doberman hepatitis chronic hepatitis in dogs: a review of current understanding of the aetiology, progression, and treatment circulating autoantibodies in dogs with chronic liver disease anti-liver membrane protein antibodies in dogs with chronic hepatitis characterization of the inflammatory infiltrate in canine chronic hepatitis phenotypic analysis of hepatic t lymphocytes in a dog with chronic hepatitis histomorphological and immunohistochemical studies of chronic active hepatitis in doberman pinschers phenotypic analysis of hepatic lymphocytes from healthy dogs serum protein electrophoresis as a prognostic marker of chronic liver disease in dogs influence of biopsy specimen size, tissue fixation, and assay variation on copper, iron, and zinc concentrations in canine livers chronic hepatitis: therapeutic considerations chronic hepatitis in the english springer spaniel chronic hepatitis: a retrospective study in 34 dogs effects of corticosteroid treatment on survival time in dogs with chronic hepatitis: 151 cases (1977-1985) ascites is a negative prognostic indicator in chronic hepatitis in dogs autoimmune hepatitis pcr screening for candidate etiological agents of canine hepatitis chronic hepatitis in the dog: the role of immune factors immunohistochemical detection of canine adenovirus in paraffin sections of liver use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis a new transmissible agent causing acute hepatitis, chronic hepatitis and cirrhosis in dogs persistent hepatitis and chronic fibrosis induced by canine acidophil cell hepatitis virus chronic active hepatitis in dogs associated with leptospires chronic hepatitis associated with leptospiral infection in vaccinated beagles chronic hepatitis associated with canine leishmaniosis (leishmania infantum): a clinicopathological study of 26 cases detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease copper-associated liver diseases oxidative stress and liver disease a perspective on copper and liver disease in the dog copper-associated hepatopathies in dogs hepatopathy associated with excessive hepatic copper in a siamese cat copperassociated chronic hepatitis and cirrhosis in a european shorthair cat hepatic copper concentrations in purebred and mixed-breed dogs inherited copper toxicity of the liver in bedlington terriers concurrent hepatic copper 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cholestasis toxic, metabolic, infectious, and neoplastic liver diseases herbal and dietary supplement hepatotoxicity metabolic, antioxidant, nutraceutical, probiotic, and herbal therapies relating to the management of hepatobiliary disorders flatland b: botanicals, vitamins, and minerals and the liver: therapeutic applications and potential toxicities disease prevalence among dogs and cats in the united states and australia and proportions of dogs and cats that receive therapeutic diets or dietary supplements therapy and outcome of suspected alpha lipoic acid toxicity in two dogs hepatotoxicity of stanozolol in cats relationship between daily dose of oral medications and idiosyncratic drug-induced liver injury: search for signals diagnosis and management of copper associated liver disease in dogs improvement in liver pathology after 4 months of d-penicillamine in 5 doberman pinschers with subclinical hepatitis iatrogenic copper deficiency associated with long-term copper chelation for treatment of copper storage disease in a bedlington terrier use of zinc acetate to treat copper toxicosis in dogs dietary management of hepatic copper accumulation in labrador retrievers liver glutathione concentrations in dogs and cats with naturally occurring liver disease population dynamics of inherited copper toxicosis in dutch bedlington terriers (1977-1997) linkage of a microsatellite marker to the canine copper toxicosis locus in bedlington terriers copper toxicosis in the bedlington terrier: a diagnostic dilemma some new aspects of the role of copper in doberman hepatitis morbidity and mortality in 928 doberman pinschers born in the netherlands between 1993 and 1999 hepatic (64)cu excretion in dobermanns with subclinical hepatitis differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in doberman pinschers subclinical versus clinical hepatitis in the dobermann: evaluation of changes in blood parameters lesions of subclinical doberman hepatitis liver disease in west highland white terriers copper associated acute hepatic failure in a dog congenital hepatic fibrosis in 5 dogs idiopathic hepatic fibrosis in 15 dogs acquired portosystemic shunting in 2 cats secondary to congenital hepatic fibrosis hepatic fibrogenesis tenascin-c in chronic canine hepatitis: immunohistochemical localization and correlation with necro-inflammatory activity, fibrotic stage, and expression of alpha-smooth muscle actin, cytokeratin 7, and cd3+ cells transforming growth factor beta-1 signalling in canine hepatic diseases: new models for human fibrotic liver pathologies cirrhosis reversal: a duel between dogma and myth effects of long-term phenobarbital treatment on the liver in dogs superficial necrolytic dermatitis in 11 dogs with a history of phenobarbital administration clinical findings in 40 dogs with hypersensitivity associated with administration of potentiated sulfonamides investigation of doxycyclinerelated side effects in dogs aflatoxicosis in dogs and dealing with suspected contaminated commercial foods toxic mushrooms diagnosis of amanita toxicosis in a dog with acute hepatic necrosis clinical and pathologic findings of blue-green algae (microcystis aeruginosa) intoxication in a dog cycad palm toxicosis in dogs: 60 cases cycad intoxication in the dog cycad intoxication in dogs: survival and prognostic indicators xylitol toxicity in dogs. comp contin educ e1-e4 acute hepatic failure and coagulopathy associated with xylitol ingestion in eight dogs hepatic encephalopathy. current concepts of the pathogenesis portosystemic hepatic encephalopathy related with congenital and acquired hepatopathies in the dog urinary calculi associated with portosystemic shunts in six dogs endogenous benzodiazepine activity in the peripheral and portal blood of dogs with congenital portosystemic shunts recurrent fever as the only or predominant clinical sign in four dogs and one cat with congenital portosystemic 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s-adenosyl-lmethionine (same) for the treatment of acetaminophen toxicity in a dog the role of paraaminophenol in acetaminophen-induced methemoglobinemia in dogs and cats s-adenosylmethionine (same) in a feline acetaminophen model of oxidative injury antimicrobial drug formulary treatment of canine atopic dermatitis with azathioprine: a pilot study factors determining short-and long-term survival after orthotopic liver homotransplantation in the dog hepatotoxicity of non-narcotic analgesics an update on nonsteroidal anti-inflammatory drugs (nsaids) in small animals fulminant hepatic failure associated with oral administration of diazepam in 11 cats acute hepatic necrosis and liver failure associated with benzodiazepine therapy in six cats, 1986-1995 iatrogenic cushing's syndrome and steroid hepatopathy in a cat alkaline phosphatase: beyond the liver prevalence of elevated alanine transaminase activity in dogs treated with ccnu (lomustine) prospective randomized clinical trial assessing the efficacy of denamarin for prevention of lomustine (ccnu)-induced hepatopathy in tumor bearing dogs false neurotransmitter in hepatic coma successful use of bromocriptine in the treatment of chronic hepatic encephalopathy dopaminergic agonists for hepatic encephalopathy direct and indirect enhancement of gabaergic neurotransmission by ammonia: implications for the pathogenesis of hyperammonemic syndromes hepatic encephalopathy: a disorder in glial-neuronal communications l-glutamate and gammaaminobutyric acid uptake in synaptosomes from the cerebral cortex of dogs with congenital chronic hepatic encephalopathy mechanisms of ammonia-induced astrocyte swelling the pituitary-adrenocortical system in canine hepato-encephalopathy increased free cortisol in plasma of dogs with portosystemic encephalopathy (pse) chronic glucocorticoid excess and impaired osmoregulation of vasopressin release in dogs with hepatic encephalopathy fast resolution of hypercortisolism in dogs with portosysemic encephalopathy after surgical shunt closure water transport in the kidney and nephrogenic diabetes insipidus gabaergic inhibition of the pituitary release of adrenocorticotropin and α-melanotropin is impaired in dogs with hepatic encephalopathy mineralocorticoid action: target specificity is enzyme, not receptor, mediated hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex role of aquaporin-2 water channels in urinary concentration and dilution defects renomegaly in dogs and cats. part i. differential diagnosis glomerular filtration rate and renal volume in dogs with congenital portosystemic vascular anomalies before and after surgical ligation morphology of congenital portocaval shunts in dogs and cats congenital interruption of the portal vein and caudal vena cava in dogs: six case reports and a review of the literature functional closure of the ductus venosus during early postnatal life in the dog the anatomy and embryology of portosystemic shunts in dogs and cats portal hypertension associated with primary hypoplasia of the hepatic portal vein in dogs budd-chiari syndrome) as a cause of ascites in a cat use of endovascular stents in three dogs with budd-chiari syndrome characterization of hepatoportal microvascular dysplasia in a kindred of cairn terriers ultrasonographic identification and characterization of congenital portosystemic shunts and portal hypertensive disorders in dogs and cats gross anatomy of the canine portal vein portal hypertension part i. pathophysiology and clinical consequences ultrasonographic evaluation of partially attenuated congenital extrahepatic portosystemic shunts in 14 dogs polycystic kidney and liver disease in cats acquired portosystemic shunting in 2 cats secondary to congenital hepatic fibrosis portal hypertension in a dog due to circumscribed fibrosis of the wall of the extrahepatic portal vein ultrasonographic diagnosis of unusual portal vascular abnormalities in two cats thrombosis of the portal vein in eleven dogs heterobilharzia americana infection in a dog multiple congenital portal vein anomalies in a dog intraoperative ultrasonography of the portal vein during attenuation of intrahepatic portocaval shunts in dogs portosystemic communications in the dog nutritional aspects of the management of chronic hepatic encephalopathy alterations of gaba-a and dopamine d-2 brain receptors in dogs with portal-systemic encephalopathy clinical presentation and management of moxidectin toxicity in two dogs generalized motor seizures after portosystemic shunt ligation in dogs: five cases nutritional support for dogs and cats with hepatobiliary disease effects of a branched chain amino acid-enriched diet on chronic hepatic encephalopathy in dogs cerebrospinal fluid glutamine, tryptophan, and tryptophan metabolite concentrations in dogs with portosystemic shunts evaluation of twelve-hour preprandial and two-hour postprandial serum bile acids disease in dogs hematologic changes associated with serum and hepatic iron alterations in dogs with congenital portosystemic vascular anomalies characterization of iron status in young dogs with portosystemic shunt iron status and erythrocyte volume in dogs with congenital portosystemic vascular anomalies hemostatic profiles in 39 dogs with congenital portosystemic shunts coagulation profiles in dogs with congenital portosystemic shunts before and after surgical attenuation standard planes for ultrasonographic examination of the portal system in dogs szatmári v: ultrasonography of portosystemic shunting in dogs doppler studies before, during and after surgery ultrasonographic assessment of hemodynamic changes in the portal vein during surgical attenuation of congenital extrahepatic portosystemic shunts in dogs use of transcolonic 99mtechnetium-pertechnetate as a screening test for portosystemic shunts in dogs quantification of portosystemic shunting in dogs by ultrasound-guided injection of 99m tc-macroaggregates into a splenic vein portal vein anomalies in the dog: their angiographic diagnosis use of intraoperative mesenteric portovenography in congenital portosystemic shunt surgery helical computed tomographic angiography of canine portosystemic shunts three-dimensional multiscale helical computed tomography techniques for canine extra-hepatic portosystemic shunt assessment use of magnetic resonance angiography for diagnosis of portosystemic shunts in dogs brain magnetic resonance imaging characteristics in dogs and cats with congenital portosystemic shunts medical management of animals with portosystemic shunts improvement of chronic hepatic encephalopathy in dogs by the benzodiazepine-receptor partial inverse agonist sarmazenil, but not by the antagonist flumazenil apparent dietary protein requirement of dogs with portosystemic shunt medical management of congenital portosystemic shunts in 27 dogs -a retrospective study ultrasonographic diagnosis of congenital 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repair key: cord-014527-nvzfpntu authors: nan title: research communications of the 25th ecvim‐ca congress date: 2015-11-09 journal: j vet intern med doi: 10.1111/jvim.13647 sha: doc_id: 14527 cord_uid: nvzfpntu nan cobalamin concentrations were previously investigated in cats, but little information is available concerning the follow up of hypocobalaminemic cats. we aimed to assess the frequency of hypocobalaminemia within a large cohort of cats with gastrointestinal signs and describe the epidemiological, clinical, biological and follow-up characteristics of hypocobalaminemic cats. 1495 cats with gastrointestinal signs and for which a cobalamin assay (simultrac-snb radioassay kit vitaminb12 ò , mpbiomedical) was performed between 2007 and 2014 at the ldhvet laboratory were retrospectively included in the study. 259 (17.3%) cats presented for gastrointestinal signs had an hypocobalaminemia: the majority were domestic short hair, 47% were males (84% castrated) and 53% females (94% castrated), aged from 3 months to 18 years. the main clinical signs included chronic diarrhea (93%), weight loss (71%), polyuropolydypsia (48%), vomiting (43%), polyphagia (38%), fatigability (36%) and dysorexia (19%) with a median duration of 4 months before diagnosis. cobalamin values ranged from 44 to 400 ng/l (median: 296 ng/l). 60% of the hypocobalaminemic cats had also a hyperfolatemia (folate > 12 ng/l) at diagnosis. ft4 was measured in the 64 older cats (> 12 years) and revealed an hyperthyroidism (ft4 > 40 pmol/l) in 20% of the cases. 67/259 hypocobalaminemic cats had a known clinical and biological follow-up (median time follow-up = 44 days): cobalamin significantly improved 1 month after treatment (50 lg/kg im cyanocobalamin in a single dose) for 87% of the cats, even if 16% remained hypocobalaminemic. 62% of the followed cats were clinically improved, of which 85% with an associated higher cobalamin value. clinical and biological improvement after cobalamin supplementation was significantly associated with an increase in folate concentration (p-value = 0.02). however, 33% of the cats with an improved cobalamin value did not show any clinical improvement. hypocobalaminemia is frequently observed in cats as a consequence of gastrointestinal signs. cobalamin concentrations could be used as an indicator of the severity of various gut diseases more than a primary cause, because one third of the cats did not show any clinical improvement despite an improved cobalamin value. a hyperfolatemia appearing after treatment of hypocobalaminemia seems to be a good indicator of a clinical improvement associated with a return to a normal intestinal integrity. disclosures: no disclosures to report. cobalamin malabsorption is common in old cats with weight loss, macronutrient malabsorption and enteric protein loss due to idiopathic chronic enteropathy, ice (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, 55-61, 2010, williams and czarnecki-maulden, proc 23rd ecvim-ca congress, 2013) . high dose oral cobalamin supplementation reverses subnormal serum concentration within 1 week but serum cobalamin can become undetectable within as little as 1 month following cessation of supplementation (williams and czarnecki-maulden, proc 23rd ecvim-ca congress, 2014). the objectives of this study were to determine if serum cobalamin concentrations in cats with ice and the response to oral supplementation and withdrawal are associated with differences in the intestinal microbiome. the study evaluated 46 cats older than 12 years of age that were being fed nutritionally complete and balanced diets that included a fortification of vitamin b12. thirty-one of these cats had ice demonstrated by increased fecal fat (>20%), subnormal fat digestibility (<90%), subnormal serum cobalamin or increased serum methylmalonic acid, but without exocrine pancreatic insufficiency as assessed by assay of serum trypsin-like immunoreactivity. serum cobalamin was determined by competitive binding assay and the fecal microbiome by roche 454 sequencing and analysis by qiime (quantitative insights into microbial ecology), pca (principal component analysis) and opls (orthogonal projections to latent structures). 13 of these ice cats were supplemented with oral cobalamin for 2 months. serum cobalamin concentrations were determined monthly during supplementation and for 3 months after cessation of supplementation. in the 46 cats there was a significant (p ≤ 0.05) association between serum cobalamin and the fecal microbiome, with 12 species being positively correlated with serum cobalamin concentration and 7 species being negatively correlated. serum cobalamin was subnormal (<290 ng/l) in 4 of the 13 ice cats at the start of the supplementation study and subsequently became normal or supranormal. within 1 to 3 months after cessation of supplementation serum cobalamin was subnormal in the 4 original cats and 1 additional cat. it is concluded that serum cobalamin concentration and the responses to oral supplementation and subsequent cessation of supplementation are significantly associated with the composition of the intestinal microflora as reflected in the fecal microbiome. disclosures: the study described in the abstract was performed at nestle-purina facilities and funded entirely by nestle-purina. david williams is a consultant and adviser for nestle-purina, idexx laboratories, and the gastrointestinal laboratory at texas a&m university. he receives royalties from idexx laboratories and has given continuing education lectures sponsored by nestle-purina. inflammatory bowel disease (ibd) is a common cause of chronic gastrointestinal signs in cats. typically, lymphoplasmacytic inflammation is found in biopsies, but a subset of cats with ibd has neutrophilic inflammation. the clinical significance of neutrophilic infiltration is unclear. the aim of this retrospective study was to use fluorescence in situ hybridisation (fish) to look for the presence of any microorganisms within the intestinal epithelium of cats diagnosed with ibd and then to identity those micro-organisms. our hypothesis was that neutrophilic enteritis in cats would be associated with intestinal mucosal invasion by microorganisms, and specifically by campylobacter spp. the study included 27 cats presented to the small animal hospital, langford veterinary services for investigation of gastrointestinal disease which had duodenal biopsies collected endoscopically. thirteen cats were diagnosed with neutrophilic inflammation (study group) and 14 cats with lymphoplasmacytic inflammation (control group). fluorescence in situ hybridisation (fish) targeting either all eubacteria or campylobacter jejuni, coli and upsaliensis was used to identify and count intra-mural bacteria in the intestinal biopsy samples. neutrophils were detected simultaneously using a fish probe to neutrophil elastase. the pixel distance between different bacterial species and neutrophils was measured. all animals in both groups showed the presence of intra-epithelial bacteria and the number of bacteria present did not differ between the control and study groups. similarly, campylobacter jejuni and upsaliensis were present in some animals in each group but numbers did not differ between the 2 groups. in contrast, campylobacter coli was present in significantly more study cats than control cats (p = 0.04; chi-squared test) and the study group showed significantly higher numbers of c. coli in the tissue than the control group (p = 0.02; mann-whitney u test). co-localisation of neutrophils and c. coli was demonstrated with c. coli closer than any of the other bacteria to the neutrophils. this association was statistically significant (p < 0.001; mann-whitney u test). the role of the intestinal virome in health and disease is gaining increased attention in human medicine. the use of next generation sequencing (ngs) technologies has allowed identification of diversity and distribution of the virome. these approaches can equally be applied to dogs.this study aimed to identify and characterise the virome present in faeces of dogs with chronic enteropathy (ce) compared to the virome of healthy dogs (hd). faecal samples were evaluated from 8 hd and 8 dogs with ce (4 food, 3 antibiotic and 1 steroid responsive) using a ngs approach. a viral enrichment protocol, using a series of centrifugation, endonuclease treatments and bacterial filtration were performed. the enriched viral dna and rna were extracted and amplified using sequence-independent single-primer amplification (sispa) protocol, and subsequently sequenced by ngs using the illumina miseq platform at the agrf. two bioinformatic pipelines were used to analyse the viral population. after selecting high quality reads and removing dog and bacterial sequences, sequence information was compared against 2 reference databases. we identified a total of 15,358 viral contigs, with 14,241 dna viral sequences and 1,041 rna sequences across all 16 dog samples. the majority of viral hits from both groups of faecal samples were bacteriophage (73.8% hd and 99.7% ce), from several families mainly from the caudovirales order. after all analyses, only 6 viral eukaryotic families were identified across all samples. two groups of sequences similar to known virus families, reoviridae and papillomaviridae, were identified in both groups (hd 1/8 and 1/8 and ce 2/8 and 2/8, respectively). sequences similar to picornaviridae were identified only in one dog with ce and sequences similar to adenoviridae, parvoviridae and coronaviridae were identified only in healthy dogs (1/8 each).further genomic characterisation and phylogenetic analysis was undertaken on 2 viruses. the 11 genome segments of a rotavirus (reoviridae) isolate were determined. similarly, the sequence of the entire coding region of a kobuvirus (picornaviridae) isolate was determined. preliminary analyses indicated that all rotavirus gene segments exhibited between 55% -98% nt homology to previously reported canine rotaviruses. the kobuvirus sequence exhibited moderate nt homology (55%) to previously described genomes and clustered with other canine kobuvirus sequences available in genbank. in conclusion, viral sequences from a range of different virus families, including both rna and dna families, and known pathogens were identified and characterised, and the largest proportion of viral contigs identified belonged to bacteriophages. disclosures: no disclosures to report. endoscopy is widely used to perform targeted and minimally invasive biopsies for histopathology of the gastrointestinal tract of dogs and cats. only a few studies have focused on the diagnostic contribution of cytological samples of the alimentary tract. the aims of this study were to compare 'imprint' and 'squash' techniques to obtain valuable cytological samples from endoscopic biopsies, and to evaluate the potential interest of cytology compared to histology in reaching the definitive diagnosis. eighteen dogs and 5 cats presenting gastrointestinal symptoms that underwent an endoscopy of their alimentary tract were prospectively included. five biopsies of each area of interest were collected for regular histopathological analysis. an additional biopsy from each area was used to obtain cytological specimens by imprint and squash techniques. cytology samples were all reviewed blindly by the same pathologist. cytology samples of insufficient quality were considered as 'non diagnostic' and were excluded from further analysis. diagnostic conclusions of both cytological and histological analyses were classified into defined categories (inflammation or neoplasia with subcategories, fibrosis, epithelial hyperplasia, and normal) to allow comparison between the techniques. agreement between cytology and histology was determined by cohen's kappa coefficient. from the 23 cases, biopsy specimens were collected from 48 different localizations for histology and 95 cytology slides were obtained. final diagnosis was neoplasia in 5 cases and inflammatory disease in 18. considering imprint technique, 15/47 were considered 'non diagnostic'. for squash technique, only 2/48 of cytological samples were considered as 'non diagnostic'. squash cytology and histology gave the same results in 65.2% of the cases (n = 46) and agreement between the 2 techniques was considered 'moderate' (k = 0.48 (95% confidence interval [ci] 0.32; 0.65)). agreement was 'fair' between imprint cytology and histology (n = 32) (k = 0.39 [95% ci 0.2; 0.58]). gastric spiral organisms (gso) were observed in 8 cases. in 3 cases they were identified only on cytology. amongst these 8 cases, mast cells were identified on cytology in 5 cases, and not on histology in any cases. mast cells were not found in any other cases. this prospective pilot study demonstrated that cytological examination of gastrointestinal biopsy squash samples obtained during endoscopy of the alimentary tract may give relevant information, which can help the clinician to initiate treatment while histopathological analysis is pending. furthermore, it can give additional information (presence of potential pathogens, mast cells) that may not be identified on histopathology. disclosures: no disclosures to report. intramucosal escherichia coli are implicated in the pathogenesis of granulomatous colitis of boxer dogs. clinical remission hinges upon its eradication, most commonly achieved with fluoroquinolones. antimicrobial resistance is not uncommon among e. coli isolated from dogs with gc and impairs successful treatment. published data is lacking on efficacious therapies for gc dogs with fluoroquinolone-resistant e. coli. the aim of this study was to characterize the antimicrobial resistance patterns and molecular characteristics of e. coli isolated from dogs with gc. additionally, to evaluate the clinical outcome of dogs treated with antimicrobials guided by culture and susceptibility results. the study population was 25 (21 boxers and 4 french bulldogs) client-owned dogs with gc. gc biopsies with fish-confirmed intramucosal e. coli were submitted for bacterial culture. antimicrobial susceptibility was determined by broth microdilution. most strains were further characterized by phylogroup and overall genotype using triplex and random amplified polymorphic dna polymerase chain reaction, respectively. treatment and clinical outcomes data were obtained. culture yielded 42 e. coli strains (1-6 per dog, med 2) from 24/ 25 dogs. resistance to fluoroquinolones was identified in 15/24 dogs; this was correlated with resistance to other macrophage-penetrating antimicrobials (p < 0.005). phylogroup a was over-represented among enrofloxacin-resistant strains. in dogs with e. coli isolated at multiple time points, phylogroup changed over time. clinical remission was achieved in 8/9 dogs with fluoroquinolone-sensitive e. coli. dogs with fluoroquinolone-resistance had a more variable response; treatment with meropenem (median 10 mg/kg sq q12 hours for 7 weeks) resolved clinical signs in 5/11. we conclude that antimicrobial resistance is a growing concern. gc-associated e. coli appear genetically diverse. clinical remission can be achieved in the face of fluoroquinolone-resistance though in vitro antimicrobial susceptibility does not consistently predict a positive response. disclosures: no disclosures to report. canine s100a12 has potential as a biomarker of inflammation in dogs. fecal s100a12 concentrations were increased in dogs with chronic gastroenteropathy (ce), and correlated with the severity of clinical and endoscopic disease. a negative outcome was associated with higher fecal s100a12 concentrations in ce dogs, but the response to different forms of treatment and fecal s100a12 has not been reported, and this information will be important to further evaluate the utility of fecal s100a12 as a biomarker for gastrointestinal disease. aim of this study was to evaluate the association between responses to various treatments (i.e., elimination diet, antimicrobial drugs, or corticosteroids/other immunosuppressants) and fecal s100a12 in dogs with ce. fecal samples were collected from dogs diagnosed with ce, and fecal s100a12 was measured in all specimens using an established in-house elisa. based on the response to treatment, dogs were classified as having antibiotic-responsive diarrhea (ard), food-responsive diarrhea (frd), or steroid-responsive/therapy-resistant idiopathic inflammatory bowel disease (ibd). statistical analysis was performed using non-parametric 2-or multiple-group comparisons, the likelihood ratio to evaluate the association between groups of dogs and response to treatment, and a receiver operating characteristic curve to calculate sensitivity and specificity at the optimum cut-off concentration. a total of 64 dogs with ce (median age: 6.3 years; 33 males/ 31 females) were included in the study, the final diagnosis of which were ard (n = 9), frd (n = 30), or ibd (n = 25). response to treatment was complete remission (n = 35), partial response (n = 25), or no response (n = 4). fecal s100a12 concentrations ranged from 1 to 34,500 ng/g, and higher s100a12 levels were seen in dogs with ibd than in dogs with frd (p = 0.010) or ard (p = 0.025). dogs that did not respond to treatment had significantly higher s100a12 levels than dogs with partial (p = 0.005) or complete (p = 0.003) remission, but response to treatment was associated with disease classification (p = 0.020). despite a small number of patients, fecal s100a12 levels of >2,700 ng/g at the time of diagnosis distinguished dogs that failed responding to treatment from those with at least partial remission with a sensitivity of 100% and specificity of 87%. we conclude that, in line with our previous finding that fecal s100a12 may be a useful biomarker of disease severity in dogs with ibd, fecal s100a12 may also have utility in predicting the lack of response to treatment in dogs with ce. the utility of serial fecal s100a12 concentrations to monitor treatment response in dogs with ce warrants further research. disclosures: dr. heilmann and dr. steiner have filed a patent application that includes the s100a12 elisa used for this study. constipation is a common presenting complaint in dogs and cats. differential diagnosis for this clinical sign is well-known but strictures resulting from gastro-intestinal inflammation are not commonly included and have been rarely reported in the literature. acute diarrhea and bone ingestion can lead to anal or rectal stricture which is responsible for the constipation. the aim of this retrospective study was to describe the prevalence of inflammatory rectal and anal stricture in small animals and to describe a simple and effective treatment. medical records of dogs and cats presented for constipation, dyschezia or tenesmus and diagnosed with an inflammatory stricture were obtained from the database of the gastro-intestinal diseases consultation of 2 referral centers in gastroenterology between 2007 and 2014; and were reviewed. signalment, presenting complaint, clinical findings, treatment protocol and outcome were recorded. five dogs and 5 cats were included in the study. of the 5 cats, 4 were purebred kitten between 3.5 and 7 months, and among them 3 were persians. the fifth cat was a 16-year-old female domestic shorthair. three out of 5 cats had history of acute diarrhea and 2 cats had constipation since adoption with unknown history. digital rectal examination under anesthesia revealed stricture in all cats which was treated by bougienage every 5 days and high-fiber diet. of the 5 dogs, age ranged from 5.5 to 14 years; 3 dogs had history of acute diarrhea and 2 had ingested bones in prior days. colonoscopy and biopsies were performed in all dogs and showed a lymphoplasmocytic infiltration in all of them. dogs were treated with digital bougienage every other day until disappearance of the stricture, metronidazole, lubricant laxatives, corticosteroids and highfiber diet. the prevalence of inflammatory stricture in dogs was 8.9% based on dogs presented with the same complaints between 2007 and 2014. for all dogs and cats, clinical signs related to the stricture resolved for the duration of their follow-up. benign strictures secondary to gastro-intestinal inflammation should be systematically included in the differential diagnosis of constipation. strictures are easily palpated on digital rectal examination, which should always be performed during clinical examination. histology should be a routine part of the diagnosis workup to exclude neoplasia. endoscopy-assisted balloon dilatation with concurrent intralesional injection of triamcinolone has been used in dogs and reported in the human literature. the treatment described here is simpler and effective. in dogs, it can be done at home by the owner. disclosures: no disclosures to report. acute pancreatitis is a diagnostic challenge because of anatomic inaccessibility of the pancreas, vague clinical signs and physical examination findings, and inconsistent laboratory results. common, yet non-specific, clinical signs include abdominal pain, anorexia, vomiting, and diarrhea. ultrasonography is the imaging modality of choice to evaluate the pancreas. the purpose of this study was to compare clinical signs with ultrasonographic findings in dogs with acute pancreatitis to account for differences in clinical presentation depending on the region of the pancreas affected as determined by ultrasonography. the hypothesis was that there would be differences in clinical presentation depending on the pancreatic region involved. records of 293 client-owned dogs diagnosed with acute pancreatitis based on history, clinical signs, laboratory testing, and abdominal ultrasonography were retrospectively evaluated. based on ultrasonography, dogs were divided into 3 groups: group 1-41 dogs with changes within the left limb of the pancreas exclusively; group 2-105 dogs with changes within the right limb of the pancreas exclusively; and group 3-147 dogs with diffuse pancreatic involvement. presence of abdominal pain, anorexia, vomiting, and diarrhea was correlated between groups using chi-square and fischer's exact test. no significant differences regarding age, breed and sex were noted between groups. in group 1 pain was noted in 11%, anorexia in 32%, vomiting in 66%, and diarrhea in 41% of dogs. in group 2 pain was present in 10%, anorexia in 42%, vomiting in 42%, and diarrhea in 19% of dogs. in group 3 pain was noted in 20%, anorexia in 31%, vomiting in 52%, and diarrhea in 24% of dogs. pain was noted with a significantly higher frequency in diffuse pancreatic disease as compared to disease restricted to the left or right limb of the pancreas. anorexia was significantly more common with right limb involvement. both vomiting and diarrhea were significantly more common with disease restricted to the left limb as compared to diffuse parenchymal or right limb involvement. despite overlap between groups, these findings indicate that pain response is expected to occur with a higher frequency in diffuse pancreatitis but overall is not a very common clinical sign. anorexia is more prevalent in dogs with pancreatitis of the right limb whereas vomiting and diarrhea both are more evident in dogs left limb pancreatitis. differences between the groups can possibly be ascribed to gastric involvement when the left side of the pancreas is affected. disclosures: no disclosures to report. increased physical exercise has been reported to improve the clinical symptoms of chronic enteropathies, such as inflammatory bowel disease, in human patients. the aim of this investigation was to evaluate the impact of an intervention to increase physical exercise in dogs with chronic enteropathies. twenty-two dogs (11 each in the exercise and control groups) with chronic enteropathies and no response to an elimination diet were included. routine diagnostic work-up (haematology, plasma biochemistry profile, urinalysis, faecal parasitology, abdominal radiographs, and ultrasound) was conducted in all dogs to eliminate underlying causes. all dogs were given oral prednisolone (1 mg/kg/day) for 14 days, followed by a tapering dosage over 10 weeks. after 4 weeks of prednisolone treatment, a certified canine rehabilitation therapist instructed the owners of dogs in the exercise group on how to increase their dogs' physical exercise. the exercise protocol combined aerobic and resistance exercises in low-to moderate-intensity interval training. owners of dogs in the control group were asked to maintain the dogs' routine lifestyles. modified canine inflammatory bowel disease activity scores (cib-dais), based on the parameters of activity level, appetite, vomiting, stool consistency, stool frequency, bloating, and weight loss, were calculated pre-treatment and 4 and 10 weeks post-treatment for all dogs. cibdai scores were compared among timepoints (pre-treatment and 2 post-treatment assessments) and between groups (exercise and control) using multivariate repeated-measures models for multiple comparisons. all dogs showed improvement after 4 weeks of prednisolone treatment. modified cibdais decreased in the exercise (from 18.3 ae 1.7 to 10.3 ae 2.2) and control (from 18.3 ae 1.4 to 11.2 ae 1.1) groups. after 6 weeks of the increased physical exercise intervention, the modified cibdai in the exercise group decreased significantly (3.8 ae 2.3) relative to the first post-treatment assessment (p = 0.022), whereas this index remained similar (11.2 ae 1.1) in the control group. modified cibdais differed significantly between groups after 10 weeks of treatment (p = 0.006). all 7 parameters of the modified cibdai were significantly affected by the intervention of increased physical exercise; the largest difference was found for body weight (p < 0.001, adjusted r 2 = 0.747) and faecal frequency (p < 0.001, adjusted r 2 = 0.693) and activity level (p < 0.001, adjusted r 2 = 0.692). an increased physical activity intervention had positive effects on clinical symptoms in dogs with chronic enteropathies. disclosures: no disclosures to report. corticosteroid therapy is commonly required in veterinary patients for treatment of inflammatory, immune-mediated, neurological and neoplastic diseases. some of these patients also require assisted enteral nutrition via percutaneous endoscopic gastrostomy (peg) tubes. this retrospective case-control study evaluated the complications associated with peg tube use in veterinary patients receiving corticosteroids in a referral teaching hospital. medical records of dogs and cats in which a peg tube was placed in the qmha between january 2006 and march 2015 were reviewed. patients were included if the peg tube was in use for at least 24 hours and if complete medical records, including clinical notes from referring veterinarians, kennel sheets, communication records with patients' owners and notes from tube removal, were available. to be included in the steroid group, patients must have received corticosteroid therapy (> 1 mg/kg/day) for at least 50% of the length of time the peg tube was in use. control patients were not treated with corticosteroids. forty-two cases were included (38 dogs and 4 cats). fourteen patients (12 dogs and 2 cats) were included in the steroid group and 28 patients (26 dogs and 2 cats) were included in the control group. complications were scored in terms of severity as minor (1), moderate (2) and major (3) and compared between groups using the mann-whitney u-test. values of p < 0.05 were considered significant. complications included: serous discharge (n = 17), sanguineous discharge (7), purulent discharge (7), stoma site inflammation (8), peg tube dislodgement (6) , pain around the stoma (4), peg tube blockage (2) and peg tube chewed by the patient on its tip (3) or at the stoma (1). median (interquartile range) of maximum complication scores for control and steroid groups were respectively 1 (2) and 2 (2). the maximum complication scores were not significantly different between groups (u = 129.5, p = 0.06), though patients receiving corticosteroids showed a trend towards higher maximum complications scores than those in the control group. in conclusion, owners of dogs and cats receiving corticosteroids in which a peg tube is planned should be appraised of the possibility of complications beyond those normally associated with tube placement alone. disclosures: no disclosures to report. canine obesity is usually treated with dietary energy restriction, but data are limited regarding nutritional adequacy. the aim of the current study was to compare intake of essential nutrients with national research council recommendations in obese dogs during weight management with a purpose-formulated diet. twenty-seven dogs were included in this non-randomized retrospective observational cohort study. all were determined to be systemically well, and without significant abnormalities based upon physical examination and clinicopathological assessments. the dogs underwent a controlled weight loss protocol of at least 26 weeks, to achieve ideal condition and using a high protein high fiber weight loss diet. median, maximum, and minimum daily intakes of all essential nutrients were compared against nrc 2006 recommended allowances (ra) for adult dogs. median weight loss was 28% (16-40%), median daily energy intake was 61 kcal/kg 0.75 (44-74 kcal/kg 0.75 ), and no signs of nutrient deficiency were observed in any dog. based upon the average nutrient content of the diet, daily intake of the majority of essential nutrients was greater than their nrc 2006 ra (per kg body weight 0.75 ), except for selenium, choline, choline (2/27 dogs) and methionine+cysteine (2/27 dogs), all essential nutrients remained above nrc minimum requirements (mr) throughout the trial. daily intakes of most essential nutrients meet both their nrc 2006 ra and mr in obese dogs throughout a period of weight loss. in light of absence of signs of nutrient deficiency, the significance of the borderline intakes for some nutrients (especially selenium and choline) is not known, and further studies are recommended. disclosures: the following conflicts of interest apply: ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational mate-rial, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin; the diet used in this study is manufactured by royal canin; ss and vb are employed by royal canin. esvcn-o-3 endocrine profile of 402 obese dogs. d. j. rochel, c. amato, p. nguyen, l. jaillardon, b. siliart. oniris, nantes atlantic college of veterinary medicine, food science, engineering, nantes cedex 03, france obesity is a frequent condition of the dog, associated with many endocrine and metabolic disturbances leading to major organ dysfunctions. we therefore aimed to assess biochemical and hormonal profiles of a large cohort of obese dogs. 402 obese dogs were retrospectively included in the study, based on an overweight over 30% the ideal body weight (ibw). endocrine profiles consisted in assessing prolactin, leptin, insulin like growth factor type 1 (igf1), cortisol after an acth stimulation test, insulin, free thyroxine (ft4) and ctsh serum concentrations. obese dogs (64% females of which 20% spayed and 36% males of which 16% castrated) were from 33 different breeds and ranged from 2 to 15 years [median 6 years, 69.9% between 3 and 8 years]. 92% of the dogs suffered from generalized (versus abdominal) obesity and long-term obesity (>1 year) was described in 78% of the cases. the main observed clinical signs were abdominal distension (76%), fatigability (45%), polyphagia (29%), decline of interest for usual activities, (27%) and polyuropolydipsia (21%). biochemical profile was unremarkable except that 93% of the dogs had hypercholesterolemia (cholesterol > 8 mmol/l). a high prolactin value (>10 ng/ml) was observed in 32% of the dogs, a high leptin value (> 10 lg/l) in 45%, a high igf1 value [igf1> 200 lg/l (ibw < 15 kg), > 290 (15 < ibw < 40 kg) and > 500 (ibw>40 kg)] in 63% and a high insulin value in 27% (>40 lui/ ml), without significant correlation with glucose concentration in 59% of the cases. 29% of the dogs had a high cortisol value (>450 nmole/l after acth stimulation) and 57% had alow ft4 (ft4 < 15 pmol/l) with 41% having a high tsh value (> 0, 45 ng/ml). canine obesity is associated with many endocrine disorders including hyperprolactinemia, hyperleptinemia, high igf1 value, hypercortisolemia and/or a hypothyroxinemia associated with a high ctsh value. the endocrine profile could be very interesting for the diagnosis and prognosis of canine obesity and could allow the veterinarian to choose a better treatment, particularly when the diet is unsuccessful. further investigations could be done to assess the prognostic value of the endocrine profile at the diagnosis of canine obesity to control the treatment efficiency. disclosures: no disclosures to report. a better understanding of how dogs undergo healthy ageing would benefit owners and veterinarians alike. in july 2004 a longitudinal study began to evaluate health and longevity in 39 labrador retrievers (12 males and 27 females, all neutered, mean age 6.7 years), continuously fed a fixed plane of nutrition with identical housing, standardised husbandry and veterinary care. body condition score was maintained between 2 and 4 on a 5-point scale. standard veterinary protocols were used for any medical conditions; cancer and severe or life threatening conditions were managed individually based on quality of life assessments. the 'average' lifespan of labrador retrievers was estimated to be 12 years. dogs were classified according to lifespan as 'typical' if they died between 9 and ≤12.9 years of age, 'long' ≥13 to 15.5 years and 'exceptional' ≥15.6 years (corresponding to 30% longer than the average lifespan). data were analysed using linear mixed models with random effects for slopes and intercepts and a fixed effect for lifespan grouping variable. on 31st july 2014, 11 dogs (28%) were classified as exceptional with 5 still alive, typical (n = 13) and long (n = 15). gender and age at neutering were not associated with survival time or risk of death (p≥0.1). body weight change showed a quadratic trend: up to age 9, body weights increased for all 3 lifespan groups but the changes were not significantly different. there was a significant change in body weight from 9 to 13 years as exceptional dogs increased body weight while the long-lifespan dogs lost weight (+0.53 versus à0.91 kg/dog/year, p = 0.007). after age 13 the exceptional and long groups both had similar losses. dual-energy x-ray absorptiometry scans revealed that wholebody fat (g) increased in all lifespan groups to age 13 but the change was significantly slower for the long lifespan dogs when compared with typical dogs which accumulated fat at >3 times the rate. all groups lost a similar amount of whole-body lean tissue (g) through age 13 (p > 0.05). up to age 13 the mean % gain in whole body fat, and % loss of whole-body lean tissue, was slower and the mean change in fat to lean ratio was lower in the exceptional and long-lived dogs compared to the typical dogs (p £0.02). typically aged labradors showed a greater gain of fat tissue, and greater loss of lean tissue, up to 13 years of age than exceptional dogs. disclosures: the eukanuba diet used in this study is manufactured by spectrum brands whilst dmm is employed by spectrum brands. vja is an independent epidemiologist who helped analyze the data and was financially supported by spectrum brands for this work. pjw has participated in veterinary seminars organised by spectrum brands and has received an honorarium for this work. introduction: in healthy animals, the phosphate (p) in combination with calcium homeostasis is regulated in comparatively narrow limits: excessively ingested p is excreted via urine. common knowledge is, however, that p is a progressive factor in chronic renal failure (crf) wherefore typically a p restricted diet is prescribed for affected patients. in 1995, pastoor demonstrated that a p excess (significantly at~890 mg p/mj me; ca/p 0.4/1 for 28 days) impairs renal function even in healthy cats, diagnosed mainly by reduced endogenous creatinine clearance. dietary p originates from meat and other protein sources, bones and cartilages, mineral supplements and technical additives (water binding, palatability a/o texture enhancer etc.). the daily amount ingested with complete diets from the european market often exceeds the recommended daily allowance (rda; nrc 2006 , fediaf 2014 considerably (up to 10times, anonymus 2014). our own studies were done with the aim (1) to survey the reproducibility of the results of pastoor (1995) and (2) to test effects of different ca/p ratios and p sources on parameters of renal function in healthy adult cats. animals, materials and methods: up to 13 adult, healthy cats were appointed to 2 groups in every trial period. firstly, a balanced diet was fed for 28 days including a 10 days balance trial. one group was then switched to a high p diet whereas the other remained on the balanced diet, again completed by a balance trial. after 14 days of wash-out (balanced diet) both groups were switched in a cross-over design repeating the 28 days trial period. this design was carried out repeatedly with high p diets differently composed concerning ca/p ratios and p sources. endogenous creatinine clearance, glucosuria, microalbuminuria, water and mineral balance were determined at each period. the study was approved by the proper authority for animal welfare. results and discussion: the studies confirmed the results of pastoor (1993): a p content of approximately 870 mg/mj me in a diet consumed by healthy cats at maintenance may lead to a decrease of the creatinine clearance. markers of acute tubular damage, i.e. glucose and microproteins in the urine, showed positive results in several trials. the p concentration of a diet alone is no sufficient marker of its tolerance since ca/p ratio and p origin influence the effects. therefore, high p diets cannot be considered safe and should be avoided also in healthy cats. literature the study aim was to investigate serum global proteomes in dogs with overt dilated cardiomyopathy (dcm) and to correlate protein expression in serum with that in ascitic fluid. eight dogs diagnosed with dcm based on echocardiographic evidence including increased left ventricular dimension at diastole and systole, increased e point to septal separation, and decreased fractional shortening were included in the study. serum and ascitic fluid samples were analyzed for proteomes using a label-free lc-ms/ms method. eight dogs from different breed, sex and age served as controls. proteome analyses revealed significantly different expressions of eight proteins in all samples. expressions in serum of apolipoprotein a1, ig heavy chain v, superoxide dismutase and plasminogen were higher (p < 0.001), while expressions of clusterin, hemoglobin subunit ß, apolipoprotein c ii, b 2 glycoprotein i (ß2gpi) were lower (p < 0.001) in dogs with dcm than in control dogs. in addition, apolipoprotein a1, clusterin, hemoglobin subunit ß, ig heavy chain v, plasminogen and ß2gpi were down-regulated whereas apolipoprotein c ii and superoxide dismutase were upregulated in ascitic fluid compared with serum in dogs with dcm. data obtained in the present study suggest that serum and/or ascitic fluid proteomes may help explain some of the pathophysiological mechanisms involved in the progression of dcm. tick paralysis is an important disease of dogs and cats in australia, induced by toxins of the paralysis tick ixodes holocyclus, very commonly occurring from spring to autumn on the eastern seaboard. respiratory failure is one of the major clinical derangements occurring in severe cases of tick paralysis, although its pathogenesis is poorly characterised. there is some suggestion that the respiratory failure is secondary to toxin-induced myocardial dysfunction with the subsequent development of cardiogenic pulmonary oedema. the purpose of this study was to determine cardiac involvement in dogs infested with ixodes holocyclus, through measurement of cardiac biomarkers. a cross-sectional study of 111 client-owned dogs was undertaken. dogs enrolled in the study belonged to one of 3 groups: dogs with tick paralysis and no-mild respiratory compromise (group a), dogs with tick paralysis and moderate-severe respiratory compromise (group b) and a control group of dogs with neither tick paralysis nor respiratory compromise. respiratory compromise was scored using a commonly employed grading system. each animal had the following parameters determined: serum cardiac troponin i (ctni) concentration, plasma n-terminal pro-btype natriuretic peptide (nt-probnp) concentration and serum creatinine concentration. for most dogs, but not all, spo2 was also determined. mean nt-probnp concentrations were significantly lower in dogs with tick paralysis than those in the control group, with no statistical difference detected between dogs with and without respiratory compromise. there was no significant difference in mean ctni concentrations between groups, however there were some high outliers of ctni concentration. creatinine concentrations differed significantly between each group, with the control group having the highest mean creatinine and those in group b having the lowest mean creatinine. there was no significant difference in spo2 between groups. this study showed no compelling evidence of cardiac insult as measured through cardiac biomarkers in our cohort of dogs with tick paralysis; however there was evidence supporting reduced preload in these dogs. in addition, the results of this study suggested that a small subset of patients with systemic hypoxaemia might have some loss of cardiomyocyte integrity. disclosures: employee/salary: gp nicolson, r malik and nj beijerink are employees of sydney university; alh mcgrath is a student at sydney university; ra webster is an employee of the animal emergency service; carrara; s kaye is an employee of queensland veterinary specialists, stafford heights; j li is an employee of northside emergency veterinary service, forestville. grants/research: this study was funded by bequest grants provided by the faculty of veterinary science at the university of sydney. idexx laboratories provided some funding for the laboratory tests. no other disclosures. speaking & consultancies: none related to this presentation. investments/commercial interests: none related to this presentation. gifts, hospitality, travel support: none related to this presentation. other: none related to this presentation. the use of ntprobnp, troponin i (high-sensitivity, ctni) and pdk4 pre-screening for occult dilated cardiomyopathy(odcm) in the doberman pinscher(dp) has been previously reported. the aim of this prospective collaborative study was to identify robust pre-screening recommendations for dp utilizing the current generation of commercially available diagnostic tests. a cohort of asymptomatic dp were evaluated at the american doberman national specialty show in 2012, 2013, and 2014 (n = 449, median age 5 years, range 1-12). evaluations consisted of auscultation, echocardiography (echo), 3-minute ecg (ecg), ntprobnp (cardiopet plus r ), ctni, and pdk4. dp were classified as affected (odcm) if their lvids was > the protect entry criteria with or without vpcs (n = 22). dp were classified as normal (nl) if their lvidd and lvids < protect entry criteria and they had no vpcs (ntprobnp:n = 373, ctni:n = 368, pdk4;n = 253). roc analysis comparing odcm and nl was done for ntprobnp, ctni, and pdk4. overall accuracy (percent correctly classified) was considered for individual tests as well as a variety of combinations. the goal of combining tests was to eliminate false negatives while minimizing false positives. the auc for ntprobnp, ctni and pdk4 was 0.91, 0.90 and 0.65 respectively with the percentage correctly classified equal to 81.8, 80.7 and 56.1 (including 4 false negatives for pdk4) when a cut-off of 548 pmol/l, 0.139 ng/ml or a positive pdk4 (hetero-or homozygous) were used respectively. when the cutoffs for ntprobnp and ctni are used in combination the auc was 0.95 and 91.3% were correctly classified (0 false negatives, 30 false positives). disclosures: research and programatic support from idexx the lab that runs ntprobnp. the study was sponsored by boehringer ingelheim, idexx and the doberman pinscher society of america. with renal disease. r. langhorn 1 , a.s. kloster 1 , l.r. jessen 1 , a. jensen 2 , j. koch 1 . 1 university of copenhagen, frederiksberg c, denmark, 2 copenhagen small animal hospital, valby, denmark cardiac troponins are sensitive and specific markers of myocardial injury. however, their reliability in renal disease has been questioned due to possible renal involvement in troponin elimination. the purpose of this study was to examine whether cardiac troponin i (ctni) is elevated in cats with renal disease and no concurrent cardiac disease, and whether ctni is measurable in urine of cats with normal and compromised renal function. cats presenting with renal disease or primary structural cardiac disease were enrolled in a renal and a cardiac group, respectively. a healthy control group was similarly included. clinical and echocardiographical examination was performed and blood and urine samples obtained for each cat. the mann-whitney u test was applied to evaluate differences between groups. seven cats with renal disease, 13 cats with cardiac disease, and 8 healthy cats were included. serum ctni concentrations were (median [range]) 0.16 [0.026-0.78] ng/ml for the renal group, 0.058 [0.003-3.27] ng/ml for the cardiac group, and 0.016 [0.050-0.14] ng/ml for the control group. the renal group had significantly higher serum ctni concentrations than the control group (p = 0.0059), but was not significantly different from the cardiac group (p = 0.18). urine ctni was measurable in 71.4% (5/7) of cats in the renal group (0.008 [0.005-0.026] ng/ml), 0% in the cardiac group, and 12.5% (1/8) of controls (0.005 ng/ml). it was concluded that elevated serum ctni in cats with renal disease may occur without concurrent cardiac disease. moreover, compromised renal function was associated with presence of ctni in urine. disclosures: no disclosures to report. sudden death (sd) commonly occurs in dog breeds with a high predisposition to vpds and vt, occuring in about 30% of asymptomatic doberman pinschers (dp) and 50% of dp with chf, and reported in 31% of boxers with arvc. in human patients with atrial fibrillation (af) on anticoagulant therapy for stroke prevention (n = 18113), cardiac death (sd and progressive heart failure) has been reported to account for 37.4% of all deaths. the objective of this study was to evaluate the incidence of sd in irish wolfhounds (iw) with dcm and/or af. iw from western europe (n = 1552) were examined by physical examination, standard echocardiography and electrocardiography between 5/1990-10/2014 (av). dogs were longitudinally followed, and owners instructed to report date and circumstances of death. dcm and/or af were diagnosed in 29%. long-term follow-up until death was possible in 134 (80 m, 54f) dogs with dcm and 47 (22 m, 25f) dogs with lone af. based on the initial diagnosis, 4 disease groups were established. results: sd occurred in 21 to 24% of all groups with dcm or af: (1) out of 76 dogs with dcm +af, sd occurred in 25% after median 502 (31-2170) days, median age 6.5ae1.9 years. (2) out of 29 dogs with dcm +sinus rhythm, 20.7% died from sd after median 893 (310-1209) days, median age 7.0ae2.5 years. (3) out of 29 iw with dcm, af +chf, 24.1% died from sd after median 232 (2-1587) days, median age 6.1ae2.3 years. (4) out of 47 iw with lone af, sd occurred in 23.4% after median 956 (482-1707) days, median age 6.1ae2.4 years, of these, 4 dogs had developed dcm prior to death. sudden cardiac death (sd) from cardiac arrest is the most common cause of death in people worldwide, accounting for > 50% of all deaths from cardiovascular disease. ventricular tachycardia (vt)/ fibrillation (vf) is the most common cause of sd, other causes include pulseless electrical activity. the fatal arrhythmia has not recorded in iw. in this study, vpds were recorded at one or more occasions in 4/47 iw with af, and in 6/134 with dcm, while in iws without heart disease vpds were seen in 3.7% of 454 males and in 3.7% of 459 females. in conclusion, sd occurs in 23.3% of iw with lone af before or after development of dcm and chf, and in 23.9% of dogs with dcm. disclosures: no disclosures to report. tachycardia may induce dilated cardiomyopathy (dcm). irish wolfhounds (iw) are commonly affected with dcm and atrial fibrillation (af). the objective of this study was to compare heart rates (hr) of iw with lone af with hr of an age and gender matched control iw cohort that had neither af nor dcm until death and to iw with dcm with either congestive heart failure (chf), af or sinus rhythm (sr). all disease groups had hr recorded before and after 3-6 months of medical therapy. out of 1552 iw with cardiovascular examinations including standard echocardiography and electrocardiography long-term follow-up until death was possible in 134 (80 m, 54f) dogs with dcm and 47 (22 m, 25f) dogs with lone af. based on the initial diagnosis, 4 disease groups were established. dogs received single or combination treatment of metildigoxine, aceis, pimobendan, diltiazem, furosemide, spironolactone, atenolol and sotalol. mean hr during 3 minutes ecg monitor recordings with print-outs were evaluated. the differences in hr in the 4 disease groups before and after treatment versus controls were examined by analysis of variance with post hoc multiple comparisons (dunnett t3). mean hr in 47 (22 m, 25f) control dogs was 120.8ae21.9 bpm. mean hr in 47 iw with lone af was 143.8ae34.7 bpm before, and 128.7ae23.7 bpm with therapy. mean hr of 76 dogs with dcm +af was 147.7ae37.2 bpm before, and 130.1ae31.8 bpm with therapy. mean hr of 29 dogs with dcm +sinus rhythm (sr), was 119.2ae26 bpm before, and 127.9 ae sd 27 bpm with therapy. mean hr of 29 iw with dcm, af +chf, was 181.3ae37.4 bpm before, and median 145.7ae18.6 bpm with therapy. in conclusion, compared to control dogs, untreated iw with chf, with dcm+af, and iw with lone af had statistically significant (p = 0.001) increased hr, but not dogs with dcm and sr, while under medical therapy elevation of hr was only significant (p = 0.001) in iw with chf and dcm. disclosures: no disclosures to report. echocardiography, as a noninvasive method, is being increasingly used as a complementary means of diagnosis in small animal clinical practice. the need for standardization of techniques by ultrasound operators in the measurement of the different echocardiographic parameters is essential for a proper examination. the aim of this work was to check a potential correlation between the values obtained in right parasternal long-axis and short-axis views in 2-dimensional mode and m-mode. twenty persian cats were submitted to a complete physical examination, clinicopathologic tests (hematocrit, total solids and t4 hormone), systolic blood pressure measurement using doppler and echocardiography. seventeen cats fulfilled the criteria inclusion and were included in the study. two-dimensional mode and m-mode echocardiograms were recorded, in systole and diastole, from both short-axis and long-axis views for evaluation of left ventricular internal diameter (lvd), interventricular septum thickness (ivs), left ventricular free wall thickness (lvpw), aorta diameter, left atrium diameter (la), pulmonary artery diameter, shortening fraction (fs) and ejection fraction (fe). statistical analysis included paired t-test (wilcoxon test) and a linear regression analysis with graphical analysis to assess agreement between the 2 methods of data acquisition. there was a highly significant correlation (p < 0.001) between the values obtained in short-axis and long-axis views for the parameter la diameter (longitudinal: 0.96ae0.07 cm; transversal: 0.94ae0.1 cm), a very significant correlation (p < 0.01) for the parameter lvds (longitudinal: 0.56ae0.2 cm; transversal: 0.69ae0.2 cm), and significant correlation (p < 0.05) for the parameters ivss (longitudinal: 0.7ae0.14 cm; transversal: 0.68ae0.18 cm), lvpws (longitudinal: 0.74ae0.18 cm; transversal: 0.66ae0.14 cm) and fs (longitudinal: 55.7ae14.1%; transversal: 48.3ae15.4%), with no significant correlation (p > 0.05) between the 2 methods for the remaining parameters. in conclusion, the data obtained from right parasternal short-axis and long-axis recordings cannot be used interchangeably in the evaluation of diastolic parameters in normal adult cats. disclosures: no disclosures to report. real time three-dimensional transesophageal echocardiography (rt3dtee) is an established imaging modality for interventional cardiac procedures in humans. it has been shown to yield comprehensive views of the cardiac valves and congenital heart defects. it potentially provides a more accurate echocardiographic means of evaluating cardiac chamber volumes and a more precise pre and postoperative tool. rt3dtee was used in combination with conventional 2-dimensional transesophageal (2-d tee) and transthoracic echocardiography (tte) standard imaging protocols. the pulmonic valve anatomy and function was evaluated in 14 client-owned dogs with severe valvular pulmonic stenosis prior to and post-balloon valvuloplasty. the 3-d images were obtained with the phillips ie33 and cx50 cardiac ultrasound systems using a 3-d transesophageal 7-2 mhz xmatrix probe. standard cardiac 5-1 mhz, 8-3 mhz and 12-4 mhz sector array probes were used to acquire 2d tte images. diagnostic images were obtained in all examined patients. rt3dtee did not change the balloon size decision when compared with 2-d tee, but provided additional views, detailed anatomy of the pulmonic valve cusps and commissures, as well as thickness and mobility of the pulmonic valve cusps, when compared to those obtained with 2-d tee or tte. successful balloon valvuloplasty was achieved in 13 of the 14 patients. repeatable artifacts occurred with respiratory excursions and insufficient probe contact. no complications related to rt3dtee were observed. rt3dtee provided enhanced views of the pulmonic valve while aiding in the procedure guidance and evaluation of the results post-balloon valvuloplasty. a better understanding of the anatomy of the pulmonic valve may improve procedure success. immediate visualization of the results post-balloon valvuloplasty may reduce patient risk and fluoroscopy time. a larger sample and further research will be needed to establish guidelines and predict success based on particular valve anatomy. we can conclude that rt3dtee provided additional anatomical and intraprocedural information and was well tolerated in this group of dogs. disclosures: no disclosures to report. pimobendan is an inodilator utilised extensively in the treatment of canine congestive heart failure. several retrospective studies evaluating clinical records have suggested that it is well tolerated in cats; however its efficacy in this species remains ill-defined. moreover, a recent pharmacokinetic study found peak plasma concentrations of the drug to be around ten times greater than those reported in the dog, thus highlighting inter-species differences in the pharmacokinetics and, potentially, pharmacodynamics of this drug. this study was conducted to evaluate the cardiovascular effects following oral doses of pimobendan in healthy cats. a placebo-controlled, randomised, operator-blinded crossover study was conducted in 8 healthy cats (weight range 3.69-4.83 kg) to evaluate the effect of 2 doses of pimobendan (high dose [hd]: 1.25 mg vetmedin chewable tablet po; low dose [ld]: 0.625 mg vetmedin chewable tablet po) and placebo ([pl]: water po) on cardiovascular parameters over time. standard echocardiography (2d, m-mode, and spectral doppler) and oscillometric blood pressure measurements (vethdo) were performed repeatedly for 12 hours following dosing. each measured parameter was evaluated for between-and within-treatment effects over time using linear mixed modeling with reml estimation to account for intercat variability. heart rate was used as a proxy for the level of anxiety experienced by the cats, and adjustment for this was performed through inclusion of heart rate as a fixed effect in the final model. the effect of treatment with pimobendan was most evident in the left ventricular internal diameter in systole (lvids). maximal effects occurred 2 hours following treatment with hd and ld. the predicted mean reduction from baseline following heart rate adjustment at this time for lvids was 1.96 mm (24% reduction) and 1.68 mm (20% reduction) for hd and ld, respectively. although there were no significant differences between hd and ld in the magnitude of effect at any given time point, lvids remained significantly reduced from baseline and the pl group for longer in the hd (40 minutes to 10 hours following dosing) than in the ld group (2 to 4 hours following dosing). significant treatment effects on aortic velocity and fractional shortening were also present, but to a lesser degree. these results demonstrate that treatment with pimobendan results in measurable changes to systolic indices in cats. a dose-dependent increase in duration of effect was also observed. further studies are required to characterise the optimal dose of pimobendan in cats and to evaluate its efficacy in clinical patients. disclosures: this study was funded by grants provided by the faculty of veterinary science at the university of sydney. m. yata received financial support from luoda pharma, the australian postgraduate awards scholarship, and the eric horatio maclean scholarship whilst undertaking this project. none of the authors involved in this study have current affiliations with the drug company that manufactured the product used in this study (boehringer ingelheim pimobendan has positive inotropic, positive lusitropic and vasodilator effects and is licensed for use in dogs with cardiac disease in many countries. numerous studies have shown benefit with the use of pimobendan in canine dilated cardiomyopathy and chronic degenerative mitral valve disease, and whilst not licensed for use in cats, recent studies have reported benefits with the use of oral pimobendan in a variety of cardiac diseases including dilated and hypertrophic cardiomyopathies. an intravenous formulation has been available in the uk since january 2013. the use of intravenous pimobendan in cats in the clinical setting has not previously been described. the aim of this study was to describe the use of intravenous pimobendan in cats with naturally occurring heart failure and report tolerability and side effects/adverse reactions. the hospital data base was searched for the use of intravenous pimobendan in feline patients. signalment, presenting signs, investigations, diagnosis, dose and time of pimobendan administration, concurrent medications, short-term outcome and adverse reactions were recorded. a boarded-certified cardiologist retrospectively reviewed all the cases in order to confirm the diagnosis. all owners had signed consent forms to permit use of off licensed drugs. eight cats were included in the study. median age was 11.9 years (range, 3.2-17.3) . six (75%) were male and 5 (63%) were domestic short-haired. weight ranged from 3.0 to 5.5 kg. all presented with dyspnoea. three out of 8 cats (38%) had a heart murmur and 5 out of 8 (63%) had a gallop rhythm. different heart conditions were diagnosed including 7/8 cats with cardiomyopathy and 1/8 with suspected endocarditis. median dose of intravenous pimobendan was 0.15 mg/kg (range, 0.136-0.150). concurrent drugs administered included frusemide, dalteparin, terbutaline, dexamethasone, amoxicillin-clavulanate, maropitant, midazolam, butorphanol, methadone, glyceryl trinitrate, clopidogrel, aspirin and potassium gluconate. no immediate adverse reactions/side effects were observed in any of the cats. five of the 8 cats were discharged from the hospital between 24 and 72 hours post pimobendan administration. one cat was euthanatized, one died during thoracocentesis and one had a thromboembolic episode between 4 and 8 hours post pimobendan administration. intravenous pimobendan was well tolerated by this clinical population of cats with heart failure. no immediate adverse reactions/ side effects were observed. the intravenous route may be considered as an alternative method of administration of pimobendan in cats with heart failure. disclosures: no disclosures to report. spironolactone (sp) is an aldosterone receptor antagonist, registered in europe for the treatment of congestive heart failure (chf) caused by valvular regurgitation in dogs, in combination with standard therapy. in cats, cardiomyopathy (cm) is the predominant cause of heart failure. to evaluate the safety and efficacy of sp in cats with cm, a double blind, randomized placebocontrolled study has been conducted with cats receiving either sp (1.7 to 3.3 mg/kg po once daily) or placebo for up to 15 months in addition to benazepril and furosemide (dose at clinician's discretion). 20 cats (17 dsh, 1 ragdoll, 1 siamese and 1 burmese) with cm of various types (15 hypertrophic, 2 dilated, 2 unclassified and 1 arrhythmogenic right ventricular) were enrolled. the cats were randomized to either group a or b according to the presence of hcm or not and whether the cat required hospitalization due to clinical need or not. 9 cats were recruited to group a (sp) and 11 cats recruited to group b (placebo). the only significant difference between the 2 groups at baseline were aortic diameter (p = 0.0077) larger in the sp group, and la:ao ratio (p = 0.012) larger in the placebo group. the survival analysis showed a survival rate at 15 months respectively of 78% and 71% in the intention to treat (itt) and per protocol (pp) populations in the sp group and 12% and 14% in the placebo group. the difference between the 2 groups was significant (log rank test: itt population p = 0.011; pp population p = 0.033). the hazard ratio indicates an 84% (itt) and 80% (pp) reduction in risk of an event occurrence in the sp group. the effect of covariates (age, weight, bcs, systolic blood pressure, ratio la/ao) was not significant. although this is a pilot study with small numbers of cats, this data would suggest that spironolactone is likely to be beneficial in the treatment of cats with congestive heart failure secondary to a cardiomyopathy. disclosures: the study was joint funded by ceva and the university of nottingham. the authors have the right to publish the results. of the study irrespective of outcome. the objectives of this study were to describe pulmonary transit time and myocardial perfusion normalized to heart rate (nptt and nmp, respectively), evaluated by means of contrast echocardiography, in dogs with stable stage c acvim myxomatous mitral valve disease (mmvd), and to assess short-term effects of pimobendan on these parameters. we hypothesized that nptt and nmp are increased in dogs with mmvd compared to normal dogs. additionally, we hypothesized that treatment with pimobendan will decrease both variables in dogs with mmvd. we prospectively enrolled 6 normal dogs and 12 dogs with stable stage c acvim mmvd. all dogs had a standard and contrast echocardiographic examination at the beginning of the study. at this time, mmvd dogs were randomly assigned to receive either pimobendan (0.4 -0.6 mg/kg) or not. all dogs with mmvd were re-evaluated by means of standard and contrast echocardiography after 1 week (t1), by operators blinded to the dog's treatment. our results show that nptt was significantly increased in dogs with mmvd (p = 0.0039), compared to normal dogs. nptt was significantly decreased at t1 in dogs receiving pimobendan (p = 0.0250). nmp was not significantly different in dogs with mmvd, compared to healthy dogs (p = 0.6639), and it was not significantly different at t1 in the treatment group (p = 0.8798). in conclusion, contrast echocardiography is a valid, complementary tool for echocardiographic analysis of dogs with mmvd. pimobendan decreases nptt in dogs affected by mmvd. myocardial perfusion is not different in dogs with mmvd and is not changed by pimobendan treatment. disclosures: michele borgarelli has received research funding by boheringher inghelheim for this study. in human beings, assessment of atrial function using 2-dimensional speckle tracking echocardiography (ste) is useful in several cardiovascular diseases. to date information on the use of ste for the evaluation of canine atrial function is lacking. we assessed the feasibility and reproducibility of ste in the assessment of left atrial (la) function in healthy dogs and dogs with myxomatous mitral valve disease (mmvd) and we compared ste derived indices with other parameters of left atrial and ventricular function and morphology. 150 privately owned dogs including 23 clinically healthy dogs (control, h) and 127 dogs with mmvd subdivided according to heart failure class (b1, b2, c+d) were enrolled. standard echocardiographic examination was carried out in all dogs. furthermore, video clips were acquired from a 4-chamber apical view and ste analysis was done using dedicated software. for the ste analysis a region of interest was drawn including the entire left atrial wall. the software provided a strain/time curve that represents the degree of deformation of the la wall over the entire cardiac cycle. similarly, la areas are provided. the following variables were evaluated: peak atrial longitudinal strain (pals, %), as the point of maximal systolic strain; peak atrial contraction strain (pacs, %) just before atrial contracting phase; contraction strain index (csi, %) calculated from these 2 variables. la areas were recorded during ventricular systole (la maximum area, laamax, cm 2 ) and atrial contraction (la minimum area, laamin, cm 2 ), and the la fractional area change (fac, %) was then calculated. the intra-and inter-observer variability was assessed using the coefficient of variation (cv, %). variability was low for all variables (cvs < 15 left atrial measurements are commonly obtained by cardiologists to assess severity of left heart disease. traditionally, measurements were obtained from m-mode images, however several studies have examined measurements of left atrial dimensions and areas from 2-dimensional (2d) images. studies have demonstrated the interobserver variability of aortic valve measurements, and the effects of timing of the measurements throughout the cardiac cycle. however, few studies have examined interobserver variability of left atrial measurements from 2d images, factors affecting variability, or the consequences of this variability on ascribing degree of disease severity to a patient. 25 images of the right parasternal short-axis view of the left atrium and aorta were provided to 9 cardiologists or cardiology residents. the images depicted left atria of varying size, from both dogs and cats, ranging from normal to markedly enlarged. each participant placed 2 arrows on each image to denote the start and finish points of their left atrial measurements without prior instructions -the first being near the interface with the aorta and the second being along the caudolateral border of the left atrium. thus, 25 sets of 9 images were analyzed. 2d distributions were mapped and analyzed to determine dispersion of the start and finish points. these were compared between images to look for association with severity (estimated as the median la:ao for each image) and image complexity. results: variability of measurements around the origin of the la measurement (interface with aorta) was small, and scaled with increasing heart size. variability at the distal measurement point was complex. in only 8/25 images was interobserver variability <0.3 la:ao, and ranged up to 1 la:ao (8% to 36% variability). a systematic observer effect was noted. variability did not appear to scale with severity of disease or image complexity, although the 2 cases with the greatest variability had severe enlargement and indistinct margins. conclusion: this study demonstrates that highly trained individuals vary considerably in their measurement of left atria from the right parasternal short-axis view. the variability did not increase with increasing disease severity or image complexity. in some instances, the same patient could be classified differently by 2 different observers if relying on la:ao thresholds. the study suggests that standardized methods of measurement should be developed to minimize this variability. disclosures: the study was supported by veterinary information network (salary, imaging software). there is growing evidence that fibrosis plays an important role in the development of remodeling and heart failure during cardiac diseases. at cellular level, fibrotic processes are prior to clinical manifestation of symptoms. fibrosis and extracellular matrix remodeling influences cardiac function in a negative manner. to our knowledge there is no biomarker, which is able to properly detect heart-specific fibrotic processes and remodeling in the peripheral blood. such a biomarker would be of great importance in cardiac diagnostics, risk stratification and therapy monitoring. in a preliminary study, using microarray method and pathway analysis in pooled samples, we were able to identify heart specific gene-expression profile representing fibrotic and inflammatory processes in the peripheral blood of tachypacing-induced cardiomyopathy model dogs. our results were validated by histopathology and quantitative real time rt-pcr (qrt-pcr). based on our microarray results, in this current study we aimed to select and investigate a panel of possible cardiac fibrosis and remodeling specific genes in blood. whole blood and left ventricular samples of tachypaced dogs (n = 13), healthy controls (n = 6) and blood samples of canine clinical patients (n = 10) with different cardiomyopathies were collected in rna-stabilizing solution. rna integrity was confirmed by capillary electrophoresis (rin>7). exon-spanning primers were designed. expression of selected genes were measured by sybr-green based qrt-pcr and normalized to 2 different housekeeping genes (hprt1, rps5) by ddct method. quality controls were made by melting curve analysis and size determination of the pcr products by agarosegel electrophoresis. for data evaluation descriptive statistics, student's t-test and mann whitney u-test were used. the selected gene-expression panel consisted of 13 different mrnas which represent main biological processes related to fibrosis, remodeling and impaired contractility. col1a2, mmp1, timp1, vcan, spp1 are directly related to collagen turnover and remodeling. il8, ccl2 are inflammatory markers. stc1, hsp70, s100a are early stress response genes. tgfb2 has central role in fibrosis while myh6 and myh7 ensure cardiac specificity. we found significant up regulation (p < 0.05) of col1a2, timp1, vcan, spp1, il8, ccl2, stc1, hsp70, s100a9, tgfb2 and down regulation of myh6, myh7 genes in the heart tissue samples. all targets also showed similar changes in the blood samples except mmp1, however not all alterations were significant. based on this selected panel clinical cases could be clearly differentiated from healthy dogs using their gene-expression pattern in blood samples. our findings suggest that the peripheral blood may have a potential to reveal cardiac specific fibrosis in dogs. disclosures: no disclosures to report. within the distal nephron, the enzyme 11-beta hydroxysteroid dehydrogenase 2 (11bhsd2) protects the mineralocorticoid receptor (mr) from activation by cortisol, allowing it to interact with aldosterone. in humans, mutations of 11bhsd2 cause apparent mineralocorticoid excess, characterised by sodium and water retention with resultant hypertension. sodium and water retention is also a hallmark of canine congestive heart failure (chf). this could partly be explained by dysregulation of renal 11bhsd2 activity, exposing mr to activation by cortisol. the aim of this study was to investigate the activity of renal 11bhsd2 in canine chf by measuring the concentration of cortisol and its metabolites in the urine from affected dogs. owners collected urine in a home environment from healthy adult dogs (n = 7), and from dogs prior to presentation with non-cardiac chronic disease (n = 6), and dogs with cardiac disease (isachc ib, n = 4; isachcii or iii, n = 5). levels of cortisol (f) and cortisone (e) excreted in urine were measured by mass spectrometry. urinary cortisol was normalised to creatinine to account for variations in glomerular filtration rate. cortisol was also measured in plasma obtained from all unhealthy dogs. plasma cortisol levels (p = 0.75)and urinary cortisol:creatinine ratio (p = 0.22) did not differ between groups. however, the f/e ratio, was increased in dogs with class ii-iii heart failure (p = 0.048). an increased f/e ratio, in the presence of unchanged plasma cortisol, implies decreased renal 11bhsd2 activity and enhanced mr activation by cortisol in canine chf. this data suggests that changes in renal cortisol metabolism in canine chf cannot be explained by chronicity of disease, that the urinary f/e ratio has potential as a biomarker for canine chf and that renal 11bhsd2 could offer a therapeutic target in its management. further studies investigating 11bhsd2 expression and bioactivity in canine chf are ongoing. disclosures: supported by the fiona and ian russell seed corn grant through the university of edinburgh. in humans endomyocardial biopsy (emb) is highly recommended in case of unexplained left ventricular dysfunction associated to ventricular arrhythmias (va) or high-grade atrioventricular block (avb). despite the frequency of these conditions in dogs, histopathology data are lacking. the aims of this study were to describe the feasibility of emb in dogs and to investigate a possible role of viral myocarditis in case of unexplained dilated cardiomyopathy (dcm) phenotypes, high-grade avbs, supraventricular arrhythmias (sva) and va. twenty-five dogs of different breeds, m/f 1,5, mean age 5.95 + 3.07 years, mean body weight 32.8 + 11.52 kg, presented for third degree avb 9/25, dcm 6/25, va 6/25, sva 2/25, and va+sva 2/25, underwent percutaneous right emb under general anesthesia throughout the jugular vein. for each dog clinical records were analyzed. in all dogs at least one right ventricular sample (range 1-4) was collected for histopathology and immunohistochemistry; in 16/25 dogs at least one sample (range 1-3) for viral pcr was collected. all histopathologic samples were stained with haematoxylin and eosin, masson's trichrome and red elastic picrocirius. in selected cases stains with monoclonal anti-cd3 and anti-cd79 were performed. nucleic acids were obtained after sample storage in rna later solution, disruption with tissue lyser and extraction with trizol; and tested for canine viruses (enteric and respiratory coronavirus, herpes virus, distemper virus, adenovirus 1 and 2, and parvovirus) and for west nile virus and bartonella spp. seven out of 25 dogs had aspecific signs of cardiomyopathy and 2/25 suggestive of arrhythmogenic right ventricular cardiomyopathy (arvc). emb gave normal samples in 6/25 dogs and not diagnostic in 1/25 dog. nine out of 25 samples were suggestive of myocarditis at different stages (3 third degree avb, 5 dcm and 1 sva). two of these dogs resulted positive for virus (1 enteric coronavirus, 1 herpes virus). none of the dogs had positive immunoistochemical stains. two dogs with cardiomyopathy were positive for herpes virus and for herpes virus and parvovirus, respectively. both of these dogs came from a kennel. no complication was noted in 24/25 dogs, one dog had self-limiting pericardial effusion. this study showed, similarly to human cardiology, that emb is a safe and useful technique that allows recognition and classification of unexplained myocardial and rhythm disorders, 25% of which possibly associated with viral myocarditis. further studies are needed to prove the relationship between viruses and myocarditis in a larger cohort of dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right ventricle (rv) is challenging. studies lack quantitative assessment of the rv in dogs. the goal of this study therefore was to evaluate rv morphology and systolic function using different transthoracic echocardiographic (te) views and to compare the results with magnetic resonance imaging (mri) measurements in 10 adult healthy anesthetized beagles. te variables were rv wall thickness (wt) in short axis and from a subcostal view, fractional shortening (fs), rv fractional area change (fac) from 2 different apical views (an optimized view for the rv and a standard 4 chambers view), tricuspid annular plane systolic excursion (tapse) from 2 different apical views, right ventricular outflow tract diameter at proximal (rvot1) and valvular (rvot2) levels both obtained in long and short axis, tissue doppler imaging (tdi) derived tricuspid annulus systolic wave (s¢), isovolumic contraction velocity (ivcvel), and isovolumic contraction acceleration time (ivcat).mri variables were rv wt, rvot1 in short and long axis and rvot2, ejection fraction (ef), and stroke volume (sv) based on flow quantification. there was no difference between rv wt measured with te in both short axis (5.4ae0.7 mm) and subcostal views (5.6ae0.5 mm) and mri (5.2ae0.6 mm). no difference was found between rvot1 or rvot2 when measured in long (22.4ae3.1 mm for the former, 16.6ae1.6 mm for the latter) and short axis (22.8ae2.9 mm for the former, 15.1ae1.1 mm for the latter) with te; however rvot1 in both short and long axis was overestimated by te compared to mri (18.7ae1.4 mm in long axis, and 19.9ae1.4 mm in short axis). both rvot2 in short and long axis obtained by te were lower than mri values (23.6ae3.7 mm). te fs was 12.4ae6%. values of tapse varied significantly when using 2 different apical views, optimized (8.2ae1.2 mm) and standard 4 chambers (7ae0.9 mm); the same was true for fac (optimized view, 36.8ae10.4%; standard 4 chambers, 44.7ae7.8 mm). tdi s¢ was 0.07ae0.01 m/s, ivcvel was 0.05ae0.01 m/s, and ivcat was 23.3ae2.1 msec. the only te correlations found were tapse with fac and s¢. the only 2 echocardiographic variables correlating with the mri based sv (18.4ae6.2 ml) were fac and s¢. mri based ef (24.5ae6.2%) did not correlate with any echocardiographic variable. this new approach to assess rv function revealed problems similar to earlier attempts. without a reliable standard for comparison of quantitative results, the value of any te parameter is questionable. furthermore, te parameters obtained from different views produced different results, indicating that standardization maybe difficult, respectively increasing the risk of variability. disclosures: no disclosures to report. spontaneous echo contrast (sec) or 'smoke' is caused by low blood velocity and appears on ultrasound as a swirling blood flow pattern. it is associated with increased risk of thromboembolism in small animals. detection of sec is entirely subjective and there is limited consensus in veterinary medicine regarding the echocardiographic technique that is best for detection of sec. the main hypothesis of this study was that 2 dimensional (2d) colour tissue doppler imaging (tdi) would significantly outperform 2d colour and grey scale as the best echocardiographic technique to view sec. a further hypothesis was that colour blindness would have no influence on results. echocardiographic data was obtained retrospectively from 10 small animal cases that presented to the university of glasgow small animal hospital. all cases had evidence of sec. using a ge vivid 7 echocardiography machine each of the 10 cases had one video loop recorded with 2d colour tdi. colour tdi was then replaced by 3 different 2d colours (gold, sepia and aqua) and 2 different grey scale colours (machine presets 2 and 3) and video loops recorded. for each case the order of the 6 recorded video loops was randomised. the video loops from the 10 cases were viewed in standardised conditions by 18 observers (veterinary cardiologists, residents, interns and students); 12 observers had full colour spectrum vision (fcsv) and 6 were red-green colour blind (deuteranopia). each observer ranked their ability to see sec with each colour, with 1 being the least able to visualise sec and 6 being the best able to visualise sec. binary logistic regression using minitab (version 17.1) was used to identify factors associated with whether tdi was ranked best or not. potential explanatory variables that were examined were view and colour blindness. observer and case were also fitted as fixed and random effects to account for clustering within these variables. tdi was chosen by the observers 118/180 (66%) occasions as the best technique to diagnose sec, which was significantly more frequently than all other 2d colour views together (p < 0.001). there was no significant difference between colour blind observers and those with fcsv (p = 1.0) and there was no influence of observer or case on the final model. in conclusion, 2d colour tdi may assist in easier diagnosis of spontaneous echo contrast in veterinary medicine regardless of colour visual spectrum of the observer. easier detection of sec would allow earlier implementation of preventative measures. disclosures: no disclosures to report. the foramen ovale (fo) is a slit-like passageway between septum secundum and primum that typically closes after birth by fusion of these septa. in 25-30% of humans, a patent foramen ovale (pfo) persists into adulthood. the prevalence of pfo in small animals is unknown. this interatrial channel may serve as a bypass to the pulmonary circulation and is an important cause of paradoxical embolism (pe) and stroke in people. the primary aim of the study was to evaluate the prevalence of pfo in a large population of dogs and cats. secondary aims were to gather data on the prevalence of atrial septal defects (asd) and on the potential association between pfo and a) clinical/pathological signs of stroke or thromboembolism and b) the presence of right-sided heart disease. hearts of all dogs and cats that underwent a full diagnostic post mortem examination were prospectively evaluated for a pfo in a blinded fashion (to clinical history and cause of death). in selected cases with patent and closed fo respectively, a histological examination of the interatrial septum was undertaken. clinical information and the results of the post mortem examination were only evaluated after all hearts had been examined. a total of 198 hearts (113 cats, 85 dogs) were examined, of which 18 cats (16%) and 25 dogs (29%) with a median age of 7 [1 day-21 years] and 8 years [1 day-16 years] respectively, exhibited a probe-patent pfo. in adult animals with presumed normal right atrial pressure the prevalence of pfo was 15% (cats) and 22% (dogs). none of the animals had an asd. one dog with a pfo also exhibited an aortic thrombus; otherwise there was no evidence of pe in this population. in 82% (dogs) and 60% (cats) with closed fo the left side of the interatrial septum (septum primum) was still partially probe-patent through a channel extending from the left atrial crescentic ridge (ostium secundum) to the limbus, but closed at this level by a thin, easily rupturable membrane. in conclusion, pfos are common in dogs and cats, but less prevalent than in humans. in contrast, asds appear to be rare in either. despite the high prevalence of pfo, clinical complications seem to be very rare. the majority of dogs with a closed fo have a fossa ovalis that is weakly fused to the limbus, which may facilitate blunt trans-septal atrial catheterisation and provide an easier access to the left atrium. disclosures: jose novo matos and tony glaus have performed consultancy work for boehringer ingelheim and vetoquinol. in human medicine diabetes mellitus (dm) is known to lead to cardiovascular dysfunction and heart failure, characterized by early diastolic and late systolic dysfunction. diabetic cardiomyopathy has been defined as the existence of left ventricular dysfunction in diabetics without coronary artery disease, hypertension or other potential etiological conditions. the prevalence of a diabetic cardiomyopathy in cats has not been previously studied. we sought to prospectively identify if cardiac diastolic dysfunction was present or would develop in a population of cats with newly diagnosed dm. cats were recruited based on a diagnosis of primary dm. patients received physical examination, biochemical and hematologic profiles including thyroxin and insulin-like growth factor 1, urinalysis, blood pressure measurement, thoracic and abdominal radiographs and abdominal ultrasound. echocardiography was performed at both diagnosis and 6 months post diagnosis. echocardiographic assessment included conventional 2d, m-mode, spectral and tissue doppler measurements. patients with relevant concomitant systemic illness or secondary dm were excluded from the study. healthy age matched control cats were retrospectively enrolled. thirty-two diabetics (d0) were enrolled in the study. eighteen were females and 14 were males. mean age was 10.8 years. on march 2015, 15 cats had received a 6 month echocardiographic control (d6). ten control cats were enrolled (c). eight were males and 2 females. mean age was 9.2 years. results ( fluoroscopically guided pacemaker implantation imparts a risk of radiation exposure. ideally exposure risk should be minimized or avoided. we previously reported using transthoracic (tte) and transesophageal echocardiography to guide pda occlusion and balloon valvuloplasty. therefore, we hypothesized that tte could be used to minimize fluoroscopy time during pacemaker implantation in dogs. we implanted single bipolar lead pacemakers (vvir) in 10 dogs, using either active or passive fixation, as determined by tte assessment of the right ventricular apical myocardium thickness: in dogs with an apical rv thickness < 2.2 mm we implanted passive fixation leads. dogs were anesthetized, positioned in right lateral recumbency on a standard echocardiography table and a left jugular vein exteriorization and venotomy were performed. in all dogs, a permanent pacing lead was advanced through the left jugular venotomy and was directed from cranial vena cava through the right atrium into the rv with tte guidance. echocardiographic right parasternal views optimized to visualize the pacing lead were used, starting with a short axis image of the right atrium and ending with a long axis view of the rv optimized to image the ventricular apex. after placing the pacing lead in the rv apex with tte guidance, and after acceptable measures of the capture threshold and impedance had been obtained, fluoroscopy was used to confirm lead placement. the pulse generator was connected to the pacing lead and secured within a right dorsal cervical pocket. incisions were closed routinely and post-implantation thoracic radiographs were performed. the pacing lead appeared hyperechoic on tte images and tte guidance provided images of a quality sufficient to clearly monitor implantation in real-time. real-time monitoring allowed for immediate corrections to pacing lead malpositioning or excessive looping. with active-fixation leads, tte allowed visualization of the fixation helix being implanted into the myocardium in some cases. the right parasternal echocardiographic window allowed imaging of the different positions of the lead in the cardiac chambers during the entire procedure. the implantations were successful in all dogs but in the first 3 cases we required fluoroscopic guidance to follow the intraventricular progression of the lead. in the last 7 dogs we only used the fluoroscope as an intraoperative x-ray machine (no cine mode). we have demonstrated that tte monitoring can guide pacemaker lead implantation and that fluoroscopy is only required to confirm the correct placement of the lead just before the end of the procedure. disclosures: no disclosures to report. deciding whether a cardiac murmur is innocent or the result of a congenital cardiac anomaly could be challenging. the gold standard method to differentiate innocent murmurs from congenital cardiac anomalies is echocardiogram, performed by a skilled operator. the present study investigated whether objective auscultation-criteria can differentiate innocent from pathologic murmurs in asymptomatic puppies between 1.5-9 months of age. the null-hypothesis was that a systolic murmur with an intensity of 1-2 out of 6 with a musical character is innocent. a total of 50 puppies were included between july 2014 and january 2015. these puppies originated either from breeders who brought their puppies to the clinic for screening for congenital porto-systemic shunts, or were referred to the cardiology service for evaluation of a murmur. of the 210 puppies that were brought for shunt-screening 28 puppies (age 45-92 days) had a murmur that was audible on every beat. all these dogs were small terrier breeds. dogs with intermittently audible murmurs were excluded. the remaining 22 puppies (age 49-210 days) were referrals to the cardiology service. based on the above described auscultation criteria the murmur was classified as innocent in 28 dogs and pathologic in 22 dogs. on each of the 50 dogs an echocardiogram was performed. no abnormalities were seen in 27 of the 28 dogs whose murmur was classified as innocent. echocardiography revealed one or more congenital cardiac anomaly in 21 of the 22 dogs whose murmur was classified as pathologic. the congenital anomalies were 8 patent ductus arteriosus, 6 pulmonic stenosis (3 severe, 1 moderate and 1 mild; 2 in combination with a small ventricular septal defect), 3 aortic stenosis (2 severe and 1 moderate), 2 ventricular septal defects, 1 mild mitral valve dysplasia and 2 double-chambered right ventricle (both moderate, 1 isolated and 1 combined with a mild aortic stenosis, small ventricular septal defect and mild mitral valve dysplasia). the puppy with the isolated mild mitral dysplasia had a soft systolic murmur with a musical character. on every puppy with a murmur of the shunt-screening group also a phonocardiogram was performed. phonocardiograms of the innocent murmurs revealed early systolic murmurs with low amplitude. auscultation turned out to be a sensitive and specific method to differentiate innocent murmurs from pathologic ones. phonocardiogram did not have an additional value. limitation: the above described findings may not be used in breeds predisposed to aortic stenosis, such as boxer. disclosures: no disclosures to report. since the first description of feline hyperthyroidism (feht4) several epidemiological studies have suggested diet as a causal factor of feht4. the aim was to critically assess the evidence presented in epidemiological studies suggesting food-associated factors in etiology of feht4. scientific literature was screened for peer reviewed publications investigating food-associated factors in feht4 since it was first described in 1979. study designs were checked against classical epidemiology biases. food-associated factors showing an increased risk for feht4 were assessed for compatibility with the 9 bradford-hill (b-h) criteria, which are used to evaluate whether an association involves a causal component. evidence for a causal component is higher when more b-h criteria are met. a total of 9 publications investigating food-associated factors in feht4 were identified, all retrospective. three publications investigated qualitative factors only (e.g. preferred food flavors) but not quantitative. feeding canned food was not found to be a significant risk factor for feht4 in one study, while it was found to be a significant and quantitative risk factor in 5 publications. however there were important limitations in their design: controls included sick cats (3 studies), cases outnumbered controls (2 studies), cases and controls differed in age (3 studies), or diet information did not reflect diet fed at time feht4 developed (2 studies). three out of 9 b-h criteria were met when considering canned diet feeding as an increased risk for feht4 development. from the available literature there is insufficient evidence to conclude that canned diet is a food-associated factor in the etiology of feht4. retrospective studies only describe an association and not a cause to effect relationship, are sensitive to bias from host and environment, and are less likely to identify etiologic factors when prevalence is below 10% as is the case in feht4. hypotheses linking food-associated factors to feht4 (such as bpa, pbdes, flavonoids and iodine content) have never been proven to date, and lifelong prospective studies are required to investigate food-associated factors in etiology of feht4. an iodine-restricted food has been recently introduced as a potential therapeutic option for feline hyperthyroidism. no controlled studies have been published on this treatment. the aim of this non-randomised controlled trial was to evaluate the effects of the iodine-restricted food on serum tt4 concentrations, clinical and clinicopathological parameters in client-owned cats with hyperthyroidism. indoor cats with newly diagnosed hyperthyroidism (consistent clinical signs and serum tt4 concentration >50 nmol/l), with or without mild to moderate azotemia (iris stage≤2), were included. cats with severe concurrent disorders (i.e. lymphoma, neoplasia, malabsorption or severe azotemia [iris>2]) were excluded. cats were allocated on owner preference into 2 groups: group a received an iodine-restricted food as a single therapy; the control group (group b) received transdermal methimazole in pluronic ò lecithin organogel (plo, 25 mg/ml) at the starting dose of 2.5 mg/cat q 12 h. in both groups clinical parameters, biochemistry and serum tt4 were evaluated at baseline (t0), 10 (t10), 30 (t30), 60 (t60), and 90 (t90) days after treatment began. twenty-five cats were enrolled in the study, 14 were included in group a, and 11 in group b. no statistical differences were present between group at t0 for signalment, clinical and laboratory findings, including tt4 concentrations. in group a, only 6/14 cats (42.9%) completed the study. the causes of interruption were: food refusal in 4/14 (28.6%), loss to follow-up in 2/14 (14.3%), death of unrelated cause in 1/14 (7.1%), and poor owner compliance in 1/14 (7.1%). in group b, 9/11 cats (81.8%) completed the study. the causes of interruption were: suspected methimazole induced hepatotoxicity in 1/11 (9.1%) and loss to follow-up in 1/ 11 (9.1%). median serum tt4 concentration at t90 was 36 nmol/l (range 18-55 nmol/l) in group a, and 19 nmol/l (range 5-73 nmol/l) in group b. no significant differences were found in serum tt4 concentrations between group a and group b in any of the evaluated timing. bodyweight and serum creatinine significantly increased only in group b between t0 and t90 (p = 0.0027 and p = 0.008, respectively); nevertheless, urea did not significantly change in both groups. ast, alt, and alp significantly decreased only in group b between t0 and t90 (p = 0.0027, p = 0.008 and p = 0.0047, respectively). these results suggest that iodine-restricted food is effective in reducing serum tt4 concentration in hyperthyroid cats. compared to transdermal methimazole, food does not produce an increase in serum creatinine, but apparently is less effective in improving bodyweight and liver parameters. disclosures: no disclosures to report. the objective of this study was to identify whether pre-treatment plasma ionized calcium (ica) concentrations are predictive of hypocalcemia following parathyroid removal or heat ablation in dogs with primary hyperparathyroidism. fifty-five dogs seen between january 1, 2004 and february 28, 2015 met the inclusion criteria of having persistent hypercalcemia (defined as ica > 1.45 mmol/l) due to primary hyperparathyroidism, absence of pre-emptive calcitriol therapy, and ica monitoring post-operatively. each dog was treated with surgery (n = 37) or ultrasound-guided percutaneous heat ablation (n = 18). hypercalcemia resolved for all dogs within 48 hours of the procedure. dogs were split into pretreatment ica groups of 1.45-1.60 mmol/l, 1.61-1.80 mmol/l and >1.80 mmol/l. there was a significant association between higher pretreatment hypercalcemia and lower post treatment hypocalcemia. in addition, there was a significant dose-response relationship between pretreatment calcium concentration and the absolute decline in calcium following treatment. sixteen dogs became notably hypocalcemic (ica < 1.10 mmol/l). four out of 20 dogs with pretreatment ica ≤1.60 mmol/l, 6 out of 21 dogs with ica between 1.61-1.80 mmol/l and 6 out of 14 dogs with pretreatment ica ≥1.80 mmol/l became hypocalcemic (ica <1.10 mmol/l). adverse effects of hypocalcemia were observed in 5 dogs, 4 of which had pretreatment ica >1.60 mmol/l. given the risk of significant hypocalcemia following parathyroid removal in dogs with pretreatment ica >1.80 mmol/l, these patients should be treated to prevent rapid decline and development of clinical hypocalcemia. disclosures: no disclosures to report. diabetes mellitus is estimated to affect 0.32% of pet dogs in the uk. most cases are thought to result from insulin deficiency resulting from loss of pancreatic beta cells, similar to human type 1 diabetes. juvenile-onset diabetes is rare (catchpole, et al., 2005) . seven labrador retriever puppies were diagnosed with diabetes mellitus at between 8 and 24 weeks of age on the basis of the presence of hyperglycaemia, glucosuria and compatible clinical signs including polyuria, polydipsia and polyphagia. two of the dogs were known to be related (full siblings); of the remaining cases, other family members (full or half siblings or one sire) were also reported to be affected, though clinical details were not available. pancreatic histopathology at post mortem in 2 cases showed islet hypoplasia. in the only puppy tested, hypoinsulinaemia was identified. two puppies were euthanased soon after diagnosis, 3 were lost to follow up and 2 survived to >8 years of age with well controlled diabetes. due to the early age of onset and occurrence within a single breed, it was hypothesised that diabetes in these cases might be due to mutation(s) in a single gene. three candidate genes were selected on the basis that they are the more common genetic causes of monogenic diabetes in human neonates or young children (kcnj11, hnf1a, hnf4a). the coding regions of the canine orthologues of those genes (q5bmm8_canfa, hnf1a, hnf4a, respectively) were amplified and sequenced from genomic dna from all 7 affected puppies and unaffected siblings and control labradors. no variants were identified in the coding sequences of hnf1a or hnf4a. in q5bmm8_canfa, 2 novel, synonymous coding, single nucleotide base substitution variants were identified in 2 of the 7 affected dogs. however, these variants were considered unlikely to be the cause of the clinical syndrome, as their presence did not co-segregate with disease within families or across the cohort and did not change the protein coding sequence. in conclusion, we report a case series of 7 labrador puppies suspected to be affected with monogenic diabetes mellitus, and results of candidate gene screening which did not identify a causa-tive mutation. further investigation of a possible genetic cause would be required to elucidate the cause of early onset diabetes in this breed. disclosures: er's salary and laboratory costs were paid using a grant from the wellcome trust. samples were submitted to the canine diabetes register at the rvc which has received funding from masterfoods, kennel club charitable trust, msd animal health and eu grant fp7 'lupa'. meh is a trustee of the kennel club charitable trust. he was not part of discussions to award funding to the canine diabetes register. some samples were submitted as part of ljd's phd which was funded by the rvc (clinical research fellowship) with additional funding from intervet pharma r&d (currently msd animal health). diabetes mellitus (dm) is a common feline endocrinopathy and pathophysiologically similar to human type 2 diabetes (t2dm). t2dm occurs due to a combination of insulin resistance and b-cell dysfunction. several studies have identified environmental and genetic susceptibility factors for t2dm. in cats, environmental factors such as obesity and physical inactivity have been linked with dm; however, identification of genetic factors has been challenging. to date, mc4r is the only gene shown to be associated with increased susceptibility to dm in overweight domestic short hair (dsh) cats. the aim of the present study was to perform a genome-wide association study (gwas) to identify loci associated with dm in lean dsh cats. illumina infinium 63k iselect dna arrays were used to interrogate genomic dna samples from 200 lean diabetic dsh cats from the royal veterinary college feline dm archive and 400 control dsh cats. the data was analysed using plink whole genome data analysis toolset. significance was established at p <1 9 10 à5 . snps with a minor allele frequency below 0.05 and a call rate below 95% and individuals with a genotyping rate <90% were excluded from analysis. a total of 49,930 snps were available for analysis. after excluding cats with low genotypic rate, 389 control dsh and 192 lean diabetic dsh cats were evaluated. diabetic cats had a mean (sd) age of 11.62 (3.44) years; 123 (63%) were male, 71 (37%) female. non-diabetic cats had a mean (sd) age of 14.83 (2.06) years; 216 (54%) were female, 183 (46%) male. control cats were significantly older than diabetic cats (p < 0.0001; t-test). five significant snps were identified: chra2.4150731 (p = 1.4 9 10 à7 ); chrun17.115508 (p = 7 9 10 à7 ); chrun17.394136 (p = 3 9 10 à7 ); chrun17.314128 (p = 3 9 10 à7 ) and chrun17.7283 (p = 9 9 10 à6 ). the first snp is located within chromosome a2; the others are located within a 0.8 mb region towards the end of chromosome a3. the snp in chromosome a2 is located 3 kb upstream of dipeptidyl-peptidase-9 (dpp9), a peptidase similar to dpp-4, involved in incretin inactivation. within the identified region of chromosome a3, genes of interest include tmem18 and acp1; both have been associated with t2dm in humans, most likely causing insulin resistance. this is the first gwas of dm in cats. a number of significant snps have been identified; some of which are located in proximity to genes that have been associated with t2dm in humans others could be involved in pathophysiology related to dm. further investigation of these candidate genes is warranted. disclosures: snp chips for the gwas were provided by the morris animal foundation. diabetes mellitus (dm) and its treatment have been documented to exert a negative psychosocial impact on cats and their owners. common owner concerns include hypoglycaemia worry, socialand work-life impact and a desire for more control over their cat's treatment. home blood glucose monitoring (hbgm) has been suggested to enable superior glycaemic control and could address some of the above-mentioned quality-of-life (qol) issues. conversely, hbgm could also exert negative psychosocial effects such as disturbance of the cat-owner bond. this prospective 3-month trial aimed to document the acceptance rate of hbgm among owners of diabetic cats, reasons for declining hbgm, possible impact on glycaemic control and whether acceptance altered pet and owner qol measured through the use of a validated psychometric diabetic pet qol-tool (diaqol-pet). at baseline (m0) all owners of recently diagnosed cats received a veterinary glucometer (alphatrak ò 2, zoetis) and a standardised demonstration of its use on their cat. diabetic management was standardised and included a low carbohydrate diet, twice-daily insulin following a standardised dose-adjustment-protocol according to, initially, weekly blood glucose curves (bgcs) carried out through hbgm (if adopted; hbgm-group) or in-hospital (if not adopted; non-hbgm-group). mann-whitney u-tests assessed for significant differences in fructosamine, average bgc-value, insulin dose and diaqol-pet-scores at m0 and month 3 (m3) between the hbgmand the non-hbgm-group. fisher's exact test was used to compare remission rates; average values are given as median (range). hbgm was introduced to 21 owners and was successfully adopted by 15 (71.4%). reasons for failure were patient aggression (n = 1), owner concerns about patient distress (n = 4) and lack of available assistance (n = 1). at m3, there was no significant difference in fructosamine (hbgm: 347(215-606)lmol/l, non-hbgm: 414(344-560)lmol/l; p = 0.20), insulin dose (hbgm: 0.35(0-1.01)u/kg/dose, non-hbgm: 0.44(0.27-0.68)u/ kg/dose; p = 0.41), average bgc-value (hbgm: 8.7(4.1-22.0) mmol/l, non-hbgm: 12.6(7.7-12.6)mmol/l; p = 0.3) or overall diaqol-pet-score (hbgm: à0.69(0.38 to à4.83), non-hbgm: à0.97(à0.31 to à3.34). on examination of individual diaqolpet-categories hbgm-cat owners reported no significant difference in the bond they felt with their cat (p = 0.56), degree of worry about hypoglycaemia (p = 0.72) or restriction to their work-(p = 0.33) or social-life (p = 0.23) compared to the non-hbgmgroup. four hbgm-cats (26.7%) achieved diabetic remission; none of the non-hbgm-group did (p = 0.23). hbgm can be successfully adopted in a majority of cat-owner combinations without a demonstrable extra burden on cat and owner's qol. hbgm also did not appear to compromise owners' relationships with their cat. a larger sample size is likely needed to assess whether hbgm promotes superior glycaemic control and remission. disclosures: the research presented in this abstract was supported by zoetis. the clinic at which this research was conducted is also supported by boehringer ingelheim and nestle purina pet-care. ruth gostelow and christopher scudder both receive phd funding from the evetts-luff animal welfare trust. stijn neissen acts as a consultant for the veterinary information network (vin). a major difficulty in the management of diabetes mellitus (dm) is our inability to predict blood glucose values in response to an insulin dose. this is linked to the existence of numerous factors impacting on blood glucose in the diabetic patient (e.g. caloric intake, type of food, exercise, insulin type and dose, stress). artificial neural networks (anns), which are statistical learning algorithms, have shown potential to aid in this prediction process in human dm. their development has been hugely aided by the introduction of continuous glucose monitoring systems (cgms), enabling generation of sufficient data-points. the goal of the current study was to develop an ann for feline dm. algorithms were developed with matlab ò (mathworks, uk) using data on exogenous insulin dose, serial blood glucoses (obtained through traditional blood glucose curves and cgms) and caloric intake over 24 hours of 46 diabetic cats. all cats were maintained in an environment that enabled normal activities, limiting stress. the neural network toolbox tm (mathworks, uk) was used to construct and test 2 types of anns: multi-layer perceptron (mlp) and radial basis function (rbf). both anns were trained using the diabetic cat data, followed by so-called detrending, elimination of outliers and configuration of external delays. in order to increase accuracy, neural architecture was set as a single hidden layer with a maximum of 12 neurons; inclusion of saturated neurons was forbidden. the accuracy of the resulting mlp and rbf models was assessed by calculating the mean squared normalised error (threshold: 0.01), generated through comparison of predicted data with actually registered data in recruited diabetic cats. in total, 46 diabetic cats were recruited for this study (25 males, 21 females, 4.3ae1.2 [sd]kg, age 10.2ae3.7; 19 burmese, 20 dsh, 7 other breeds). all had a 24-hour blood glucose curve performed (n = 10 with cgms) and response to insulin was predicted by mlp and rbf. calculation of the mean squared normalized error revealed that in 39/46 diabetic cats (85%; mlp) and 38/46 (83%; rbf), the dynamics of the blood glucose curve were correctly predicted by the ann. in conclusion, our study is the first to describe the successful development of an ann to predict blood glucose dynamics in insulin-treated diabetic cats. further evaluation is indicated, though ann-model-based prediction of glucose concentration may allow clinicians in future to optimise insulin management protocols or may allow the development of an artificial pancreas for the diabetic cat. disclosures: no disclosures to report. glucagon-like peptide (glp) -1 analogues induce significant weight loss in humans; presumably by slowing gastric emptying and increasing satiety. in lean purpose-bred cats, short-term glp-1 analogue treatment also induced weight loss. we evaluated the effect of 12 weeks exenatide, a glp-1 analogue approved for treatment of human type 2 diabetes, or placebo treatment on body composition and adipokines in obese, client-owned cats. cats were randomized to subcutaneous saline (n = 6) or exenatide (n = 6) injections; 0.5 lg/kg q12 h during the initial 4 weeks and 1.0 lg/ kg q12 h during the following 8 weeks. body weight, body composition using dual-energy x-ray absorptiometry and adipokine levels were measured before and after treatment. all cats were obese (body condition score ≥ 7 out of 9). mean body weight was 7.71 kg (range 5.04-10.80 kg) and mean % body fat was 47.5% (32.6-61.1%). median percent loss of baseline body weight was 5.1% (range 1.7-8.4%) for exenatide and 3.2% (range à5.3 to 5.7%) for placebo. only the exenatide group had a significant absolute weight loss; however the difference in median percent loss between groups was not significant. change in total amount or % body fat were not different between groups. correspondingly, plasma leptin and total adiponectin were unaltered by treatment. complications were limited to a single, mild hypoglycemic episode and 2 cases of self-limiting gastrointestinal signs. we conclude that the appointed dose of exenatide was well tolerated and safe in obese healthy cats. a larger study population may be required to fully elucidate the effect of exenatide in obese cats. disclosures: no disclosures to report. feline diabetes mellitus (dm) is recommended to be treated through addressing underlying diseases, bid insulin injections and low carbohydrate diets. good glycaemic control is suggested to promote diabetic remission. a recent systematic review of feline dm literature identified studies on glargine and lente insulin to be proportionately overrepresented compared to other insulin types. additionally, most insulin studies suffered from lack of screening for concurrent disease, homogeneity in management and assessment of quality of life (qol). until recently, only porcine lente insulin was available as a veterinary-licensed product. this prospective trial evaluated the impact of newly veterinary-licensed human-recombinant protamine zinc insulin (prozinc tm , boehringer ingelheim) on clinical signs, glycaemic control and qol in diabetic cats. recently (<5 months) diagnosed diabetic cats, treated with caninsulin ò (msd) bid for at least 6 weeks and receiving a specific low carbohydrate diet were recruited. a full history, physical examination, diabetic clinical score (dcs; range 0 [no diabetic signs] -12 [many diabetic signs]), fructosamine concentration, 24hour blood-glucose-curve (bgc) and qol-assessment (diaqolpet-score) were performed before and 3 months after transition to prozinc tm (start dose: 0.2-0.7 unit/kg bid), following a set protocol of weekly bgcs and dose adjustments (0.5-1 unit change/injection/week guided by nadir). cats were excluded if screening (biochemistry, urinalysis, fpli, tt4, igf-1, abdominal ultrasound) identified: ketoacidosis, clinical pancreatitis, glucocorticoid/ progestogen administration, hyperthyroidism, acromegaly, other conditions impairing treatment response. data were assessed for normality and reported as meanae-standard deviation; changes were assessed using paired t-tests (p < 0.05; without multiple comparisons correction following latest statistical guidelines). sixteen cats were recruited (10 male neutered, 6 female neutered; age 128ae22 months); all completed the trial. at time of entry cats received 0.50ae0.3unit/kg caninsulin bid, had a dcs of 3.8ae3.4; diaqol-pet-score of -2.28ae1.5; bgc-value of 13.0ae5.4 mmol/l; and fructosamine of 449ae105 lmol/l. three months after transitioning to prozinc tm , cats were receiving 0.37ae0.2unit/kg bid; had a significantly lower mean dcs (0.8ae1.3, p = 0.001) and diaqol-pet-score (à1.76ae1.4, p = 0.04); bgc-value (10.7ae4.2 mmol/l) and fructosamine (388ae113 lmol/l) were also lower, though not significantly (p = 0.15 and p = 0.08, respectively). three cats entered diabetic remission (19%). these results show that transitioning cats from caninsulin to prozinc tm produced a significant improvement in clinical signs and qol. more cases are likely needed to document any additional significant glycaemic impact after transition. finally, in veterinary dm research, this represents the first clinical trial to include validated quantitative qol-assessment as an outcome parameter. disclosures: the study described in this abstract received financial support from boehringer ingelheim. the clinic in which this research was produced also receives support from nestle purina petcare and zoetis. ruth gostelow and christopher scudder both receive phd funding from the evetts-luff animal welfare trust. stijn niessen is a consultant for the veterinary information network (vin). a. hope, s. spence, i.k. ramsey. university of glasgow small animal hospital, bearsden, uk serial blood glucose measurements are currently used as an accepted method to assess diabetic control, however extended curves are expensive and can be technically challenging. a new 7 day continuous glucose monitoring system (cgms) called the dexcom g4 platinum ò that incorporates a glucose oxidase-based sensor to measure interstitial blood glucose was evaluated by comparing the results to the blood glucose measured contemporaneously on a glucometer (alphatrak ò ). these measurements were made at least twice daily at the time of calibration of the cgms, which was a variable period (but not more than 12 hours) after the previous calibration. a total of 77 measurements from eight dogs' glucose curves (blood glucose range 3-33.3 mmol/l, with a median of 16.43 mmol/l) were compared to a paired measurement of interstitial glucose by calculation of the pearson correlation coefficient (r). a minimum of 4 measurements were obtained from each dog. the device only provides a specific measurement of interstitial glucose in the range 2.2 to 22.2 mmol/l. values above and below this range were not included in the study. subjectively, the device was easy to use with an intuitive user interface that provided wireless real time measurements and was much smaller than older cgms systems. overall, there was excellent correlation between the glucometer and the cgms readings (r = 0.9), which was statistically significant (p = <0.0001). the range of differences between the blood and interstitial glucose concentrations was 0-14.6 mmol/l with a median of 1.5 mmol/l. problems encountered with the system included detachment of the system from the dog's skin, as well as variably correlated glucometer and cgms readings in individual dogs (r range 0.14 -0.95, median 0.53). in conclusion, the dexcom ò g4 cgms can be used to assess interstitial glucose concentrations in dogs, and these are generally well correlated with blood glucose concentrations. more work is needed (with larger numbers of patients) to determine the relationship of the correlation to the timing of calibration, and to determine why some dogs seem to have poorer correlation than others. disclosures: no disclosures to report. several continuous glucose monitoring systems that measure interstitial glucose (ig) are currently available. however, they require multiple calibrations and therefore multiple blood sampling; moreover, the sensors are quite expensive and can be used only for a few days. a new human flash glucose monitoring system (fgm) (freestyle libre, abbott, uk) measures ig, does not require calibration, is rather inexpensive and the sensor can be used as many as 14 days. it is composed by a small sensor applied subcutaneously that has to be "scanned"with a reader to obtain real time glucose values. the aim of this study was to assess the accuracy of this fgm in diabetic dogs. in all dogs the sensor was placed on the neck area and fixed with an adhesive patch. during the 1 st -2nd, 6 th -7th, 13 th -14th days from the application, the ig measurements were compared with the peripheral blood (edta plasma) glucose (pg) concentrations analysed by a reference hexochinase based method (olympus/beckman coulter au400). linear regression, bland altman plots and the clarke error grid analysis were used to assess the accuracy. ten client-owned diabetic dogs on insulin treatment were included. median age was 9.5 years (range 2-13), 7 were female (spayed), 3 were male (2 neu-tered). median body weight was 17.9 kg (range 5.4-43.1). four hundred and sixty four simultaneous measurements were taken with fgm (ig) and with the reference method (pg): 29 samples were in the hypoglycemic range (<70 mg/dl), 175 in the euglycemic range (70-180 mg/dl) and 260 in the hyperglycemic range (>180 mg/dl). considering all the measurements together a positive significant correlation between ig and pg concentrations (r 2 = 0.86) was found. meanaestandard deviation difference from the reference method was à1.8ae22 mg/dl in the hypoglycemic range, à9.8ae47 mg/dl in the euglycemic range, à1.25ae63 mg/dl in the hyperglycemic range. ig values differed >50 mg/dl from the reference method in 0%, 11% and 32% and >25 mg/dl in 13%, 27% and 70% in the hypoglycemic, euglycemic and hyperglycemic range, respectively. underestimation-overestimation of ig compared to pg was observed in 31-69%, 60-38% and 48-39% of hypoglycemic, euglycemic and hyperglycemic measurements, respectively; 78.6% and 97.8% of glucose values measured by fgm fall in zone a and zones a+b of the error grid analysis, respectively. the application of the sensor was easy and apparently painless; a mild local erythema after sensor removal was found in 5/10 dogs. fgm is a simple and promising glucose monitoring system that seems accurate for the clinical use in diabetic dogs. disclosures: no disclosures to report. the cornerstone of treatment in diabetic cats is insulin. among other issues, insufficient duration of insulin action may lead to poor metabolic control and persistence of clinical signs. with the aim to improve current therapeutic options, the present study evaluated pharmacodynamics parameters, such as onset of action, time to glucose nadir and duration of action, of protamine zinc insulin (prozinc ò , boehringer ingelheim) and insulin degludec (tresiba ò , novo nordisk) in healthy cats. additionally, the accuracy of 2 different sensors, sof-and enlite-sensor, of the continuous glucose monitoring system (cgms) ipro2 ò (medtronic) was determined with particular attention to the low glycemic range, since reliability of cgms in case of hypoglycemia is crucial. three different doses (0.1, 0.2 and 0.3 iu/kg) of each insulin and both ipro2 ò sensors were tested in 6 healthy purpose bred cats in a randomized crossover trial. the sensors were placed in the neck area for 7 days. paired glucose measurements were obtained every 8-12 hours with a validated portable blood glucose meter (alphatrak ò , abbot) set as standard and accuracy was assessed by using iso 15197 2013 criteria. additionally, to determine onset of insulin action, time to glucose nadir and duration of action, glucose concentrations were measured 30 and 5 minutes before and 30, 60, 90, 120, 180, 240, 300 and 360 minutes after each insulin administration, then every 2 hours for 18 hours. median (range) onset of action was 1.5 (1.5-3) hours for pro-zinc ò and 1.5 (1.5-4) hours for tresiba ò . median (range) time to glucose nadir and duration of action were 4 (1.5-6) hours and 7 (5-10) hours for prozinc ò and 5.5 (3) (4) (5) (6) (7) (8) hours and 11 (8->24) hours for tresiba ò , respectively. with regard to ipro2 ò , 100% of paired glucose measurements with both sensor types were in zone a and b of the consensus error grid. at glucose concentrations <5.55 mmol/l 90% (160/177) of sof-sensor measurements and 87% (269/309) of enlite-sensor measurements were within ae0.83 mmol/l of the standard; at glucose concentrations >5.55 mmol/l 43% (6/14) of sof-sensor measurements and 40% (6/15) of enlite-sensor measurements were within ae15% of the standard. in conclusion, healthy cats injected with prozinc ò and tresiba ò showed similar onset of action. later glucose nadir and longer duration of action was seen in cats treated with tresiba ò compared to those treated with prozinc ò . both ipro2 ò sensors revealed good clinical accuracy and performed similarly in the low glycemic range. disclosures: no disclosures to report. the canine adrenal cortex consists of 3 concentric zones: the zona glomerulosa (zg), the zona fasciculata (zf) and the zona reticularis (zr), which produce mineralocorticoids, glucocorticoids and androgens, respectively. in humans, critical step for the production of either aldosterone or cortisol are the zg-specific aldosterone synthase (cyp11b2) and the zf-specific 11b-hydroxylase cytochrome p450 (cyp11b1). the fact that humans and dogs have the same adrenocortical end products, i.e. aldosterone and cortisol, has led to the assumption that canine steroidogenesis is identical to that of humans. however, in dogs, the zonal expression of steroidogenic enzymes has not been studied previously. moreover, in dogs the expression of cyp11b1/2 is unclear, as only one coding gene sequence (cyp11b2) has been published in the ncbi database and, adjacent to this, a large non-sequenced gap is present. we hypothesized that canine adrenals possess only one cyp11b gene, similar to sheep and pigs. zg and zf tissue was isolated separately by use of laser-guided microdissection of 5 adrenals of healthy dogs. the zone-specificity of the tissues was confirmed by specific markers, with mrna relative expression of wnt4, angiotensin ii receptor and disabled-2 being significantly higher (p = 0.05, p = 0.014, p = 0.014, respectively) in the zg compared to the zf. rt-qpcr analysis of mrna relative expression of steroidogenic enzymes demonstrated a significantly higher fold change of steroidogenic acute regulatory protein (star), cytochrome p450 side chain cleavage (cyp11a1) and 17a-hydroxylase/17,20-lyase (cyp17) (p = 0.014, p = 0.014, p = 0.05, respectively) in the zf compared to the zg. the zfspecific presence of cyp17 was also demonstrated by immunohistochemistry. no significant difference (p = 0.62) in the mrna relative expression of cyp11b2 mentioned in the database was found, and southern blot analysis showed that the non-sequenced gap does not contain another cyp11b gene. we conclude that there is only one functional cyp11b enzyme in canine adrenals. the zone-specific production of aldosterone and cortisol is probably due to zone-specific cyp17 expression. its presence in the zf is crucial for cortisol synthesis, while lack of cyp17 in the zg conducts steroidogenesis to mineralocorticoid production. this is the first report providing insights in one of the most important physiological mechanisms of the canine adrenal cortex, its zone-dependent steroidogenesis. disclosures: no disclosures to report. the pathogenesis of cortisol-secreting adrenocortical tumors (ats) has become more clear recently. mutations of the gnas gene provide an explanation for acth-independent hormonal activity of ats, but the autonomous growth remains greatly undisclosed. an approach to elucidate the proliferative capacity of ats is to learn from the current understanding of adrenal growth biology. the sonic hedgehog (shh) signaling pathway plays an essential role in the development of the adrenal gland and in regulating adrenocortical cell proliferation. the members of the shh signaling pathway are present in progenitors of the steroidogenic cells of the normal adrenal gland and dysregulation of shh signaling has been implicated in adrenal cancer in humans. we hypothesized that shh signaling is also enhanced in canine ats, predominantly in carcinomas. we examined the relative expression of shh pathway components (shh, ptch1, smo, gli1, gli2 and gli3) by rt-qpcr analysis in cortisol-secreting adenomas (n = 15) and carcinomas (n = 21) and normal canine adrenals (n = 7). the relative expression of members of the shh pathway was detected in both ats and normal adrenal tissue. a significant (p < 0.05) lower mrna expression of gli3 was detected in carci-nomas when compared to normal tissue. amongst the other genes, no significant differences were found. since gli3 is mainly a repressor of genes activated by the shh pathway, a down regulation of gli3 in carcinomas could point to enhanced shh signaling in adrenocortical carcinomas and could theoretically be responsible for their expansive growth. in conclusion, dysregulation of shh pathway might be involved in the pathogenesis of canine cortisol-secreting carcinomas. modulating shh expression might provide a new target therapy for adrenocortical carcinomas and will need to be explored in the future. disclosures: no disclosures to report. feline hypersomatotropism (acromegaly) has been suggested to be an underdiagnosed endocrinopathy among diabetic cats. treatment options include management of the secondary diabetes mellitus alone, medical pituitary inhibition, radiotherapy (rt) and hypophysectomy (hpx). tools to diagnose the disease and monitor treatment effect are limited, with insulin-like growth factor-1 (igf-1) currently being the only easily accessible blood test. serum igf-1 has previously been shown to be insensitive when assessing rt effect. development of additional serological diagnostic tools that can be measured alongside serum igf-1 is therefore desirable. a pilot study previously validated an n-terminal type iii pro-collagen propeptide (piiinp) elisa for use in cats and found this peripheral biomarker of collagen turnover to be significantly elevated in a small number of hypersomatotropic diabetic (hsdm) cats. this study therefore aimed to: 1. further evaluate the use of serum piiinp to differentiate hsdm from dm; and 2. to evaluate piiinp as a marker for treatment success. piiinp concentrations were measured in 30 cats with uncomplicated diabetes mellitus (dm) (igf-1 <600 ng/ml [radioimmunoassay], <1.5iu/kg/injection exogenous insulin requirement) and 30 with confirmed hsdm (igf-1 >1000 ng/ml, pituitary mass on computed tomography) using the previously validated elisa. additionally, piiinp and igf-1 were measured in preand post-treatment (1-18 months) samples of hsdm cats that responded favourably (decreased insulin requirement) to radiotherapy (rt; n = 5) or hypophysectomy (hp; n = 9, of which 8 had achieved diabetic remission at time of sampling). serum piiinp concentrations were significantly higher in hsdm cats (median 19.77 ng/ml; range: 1.69-27.93) compared to dm cats (median 5.03 ng/ml; 2.07-10.44; p < 0.001, mann whitney u-test). receiver-operator-curve-analysis revealed a 10.5 ng/ml cut-off to differentiate between dm and hsdm cats with 87% sensitivity and 100% specificity (auc: 0.91; 95% confidence interval: 0.82-1.0). after rt, piiinp increased significantly (median pre-rt 13.53 ng/ml, 10.52-19.77; post-rt 14.96 ng/ml, 12.69-21.51; p = 0.043, wilcoxon signed rank-test) despite absence of significant change in igf-1 concentrations (median pre-rt 1915 ng/ml, 1087-2000; post-rt 1263 ng/ml, 645-2000; p = 0.068, wilcoxon signed rank-test). following hpx, serum piiinp concentrations did not change significantly (median pre-hpx 20.5 ng/ml, 14.59-27.93; post-hpx 18.87 ng/ml, 8.7-28.43; p = 0.441, wilcoxon signed rank-test) despite significant serum igf-1 decreases (median pre-hpx 1875 ng/ml, 590-2000; post-hpx 44 ng/ml, 15-1819; p = 0.008, wilcoxon signed rank-test). in conclusion, serum piiinp concentration was confirmed to be a useful additional parameter when differentiating hsdm from dm in cats. however, the current data do not suggest piiinp to be a reliable marker of treatment success following rt or hpx. disclosures: no disclosures to report. decreased frequency and function of peripheral regulatory t cells (tregs) have been documented in people with immune-mediated haemolytic anaemia (imha), suggesting that defects in peripheral tolerance may play a role in the pathogenesis of this disease. the aim of the current study was to test the hypothesis that the frequency of peripheral tregs is decreased in dogs with primary imha, accompanied by increases in t helper (th) cells, cytotoxic t (tc) cells and b cells. residual edta-anticoagulated blood samples were obtained from dogs with primary imha (n = 11), dogs with inflammatory diseases (n = 10) and healthy dogs (n = 12). primary imha was diagnosed in dogs with regenerative anaemia (packed cell volume [pcv] <35%) and either persistent agglutination of erythrocytes after saline dilution or detection of spherocytosis on a fresh blood smear. the study was approved by an institutional ethics and welfare committee. after erythrocyte lysis, peripheral blood mononuclear cells were stained with fluorophore-conjugated antibodies specific for extracellular (cd4, cd5, cd8) and intracellular (cd79b, foxp3) antigens. multicolour flow cytometry was undertaken to determine the proportion of lymphocytes that were b cells (cd79b hi ), th cells (cd5 hi cd4 + ), tc cells (cd5 hi cd8 + ) and tregs (cd5 hi cd4 +-foxp3 + ); the kruskal-wallis test was used to compare proportions between groups. correlations between the proportions of tregs and pcv and serum total bilirubin concentration (tbil) in dogs with imha at presentation were assessed using spearman's rho coefficients. the median proportion of cd4 + t cells that expressed foxp3 was 4.29% (inter-quartile range [iqr]: 1.55-5.56) in dogs with imha, 2.72% (iqr: 2.38-4.21) in dogs with inflammatory diseases and 5.1% (iqr: 4.10-6.81) in healthy dogs, with no difference between groups (p = 0.120). there was no difference in proportions of t cells that were cd4 + (p = 0.517) or cd8 + (p = 0.332) between groups, nor in the proportion of b cells (p = 0.801). the proportion of cd5 hi cd4 + foxp3 + tregs was positively correlated with tbil in dogs with imha (spearman's rho 0.686, p = 0.041), but not pcv (rho à0.613, p = 0.079). though limited by its size, the results of this pilot study suggest that the frequency of tregs is not decreased in dogs with imha; proportions of th, tc and b cells were also comparable to those in control dogs. further work is required to determine whether the function of tregs is altered, or whether other defects in peripheral tolerance contribute to development of this disease. disclosures: no disclosures to report. xenotransfusion of canine blood to cats may be a life-saving procedure when treating an acute anaemic syndrome and compatible feline blood cannot be obtained. published evidence in a limited number of cases dating from the 1960s indicates that cats do not appear to have naturally-occurring antibodies against canine red blood cell antigens. in fact compatibility tests before the first transfusion did not demonstrate evidence of agglutination or haemolysis of canine erythrocytes in feline serum and no severe acute adverse reactions have been reported in cats receiving a single transfusion with canine blood. severe acute reactions not reported so far cannot however be excluded and we decided to perform a pilot study to evaluate the presence of naturally occurring antibodies against canine red blood cell antigens in cats and viceversa. surplus material from diagnostic samples (blood edta and blood serum samples) of 13 cats and 24 dogs was used to perform test-tube major and minor cross-match tests (at 37°c, 4°c and room temperature (rt)) and blood typing, after obtaining the informed consent from owners. hemolysis, macro-and micro-ag-glutination were investigated in each test tube and were considered markers of a positive matching. blood from each cat was tested with blood from 2 to 6 different dogs for a total of 49 major and minor cross-matchings each one performed at the 3 different temperatures of incubation. thirty-eight overall major cross-matchings proved positive at 37°c, 33 at rt and 39 at 4°c respectively. the minor cross-matching was positive in all but 2 tests performed at 37°c. no cat tested totally negative (37°c, 4°c, rt) at both major and minor cross-matching procedures performed towards any single dog. ten cats experienced positive major and minor cross-matching at 37°c, rt and 4°c towards 1-3 different dogs. five cats were positive in the major cross-match, at least at 37°c, towards 1-3 different different dogs. seven cats obtained a positive major cross-match at rt and/or at 4°c towards 1-5 dogs. only 2 cats tested completely negative at 37°c, rt and 4°c, in one out of the 4 different major cross-matchings performed. in conclusion, naturally occurring antibodies against canine red blood cell antigens appear to be frequently detected in cats as well as those against feline red blood cell antigens in dogs. xenotrasfusion of canine blood to cats should only be performed after the selection of a compatible donor by means of at least a negative major cross-match test result. disclosures: no disclosures to report. anti-thymocyte serum (ats), a potent immunosuppressive agent, is commonly used perioperatively in human patients to increase graft survivaland decrease rejection of transplanted tissue. ats has been reported as part of an immunosuppressive protocol to treat immune-mediated diseases including aplastic anemiaand myelodysplastic syndromes (mds). rabbit anti-dog thymocyte serum (radts) has been used in veterinary medicine for perioperative immunosuppression in canine renal transplants. however, there are no reports regarding the use of radts in the treatment of dogs with immune-mediated disorders. the medical records of 5 dogs diagnosed with imha, 3 dogs with itp and 1 dog with mds were reviewed. median age was 6.3 years (8.5 months to 11 years). all dogs failed to respond to traditional immunosuppressive therapy and received radts. none of the dogs experienced any adverse reaction. lymphocyte counts were used to monitor the response to therapy. all dogs, except 1, had a significant decrease in their lymphocyte count; 6/9 had a decrease to <10% of the initial lymphocyte count, which was the aim in previous studies on radts. all dogs were discharged, however, the same dog with no changes in his lymphocyte count experienced a relapse of his imha after 1 week and was euthanized. all other cases achieved clinical remission with immunosuppressants being tapered or discontinued. radts appeared to be a safe immunosuppressant agent of interest in refractory immune mediated diseases. disclosures: no disclosures to report. in cats with upper respiratory tract aspergillosis (urta), invasive disease is common. in other species, invasive mycoses are associated with immunodeficiency. characterisation of the humoral immune response in feline urta serves to identify whether selective immunodeficiency underlies susceptibility and to determine the utility of class-specific antibody detection for early diagnosis. we have shown that serum anti-aspergillus igg has high sensitivity and specificity for diagnosis. the aims of the study were to (1) determine whether serum anti-aspergillus iga can be detected in cats with urta, and (2) evaluate the sensitivity and specificity of iga detection for diagnosis. sera were collected from 3 groups of cats; group 1 -confirmed urta (n = 23), group 2 -upper respiratory disease without aspergillosis (n = 25), group 3 -healthy cats (n = 36) and cats with non-fungal, non-respiratory illness (n = 48). an indirect elisa to detect anti-aspergillus iga was developed. inter-assay and intraassay coefficients of variation were 4.50% and 6.17%, respectively. serum iga was detected in 91.3%, 44% and 50% of group 1, 2, and 3 cats, respectively. using an optimal elisa cut-off value for diagnosis (71.9 elisa units/ml), determined by receiver-operating curve analysis, assay sensitivity for group 1 cats was 78.3%. specificity was highest (96.0%) when group 2 was used as the control, compared to group 3 (85.7%) or group 2 and 3 combined (88.1%). we found no evidence of a role for primary iga deficiency in the pathogenesis of feline urta. serum anti-aspergillus iga detection has moderate sensitivity and moderate specificity for diagnosis of urta. disclosures: there is no conflict of interest. the study was partially funded by a australian companion animal health foundation grant 2014. non-invasive topical infusion therapies are widely used in canine sinonasal aspergillosis (sna) but are time-consuming and associated with prolonged recovery and increased costs. therefore, the main goal of the present study was to compare the efficacy of a simplified infusion protocol (d15e) with a 1-hour infusion protocol (d60eb). d60eb consisted in endoscopic debridement followed by 60 minutes 2% enilconazole infusion and 1% bifonazole cream depot into the affected frontal sinus through endoscopically placed catheter. for d15e protocol, after debridement, enilconazole infusion was shortened to 15 minutes, with the dog remaining in dorsal recumbency, head flexed at 90°, during the whole procedure. adjunctive oral itraconazole therapy was prescribed in both protocols. effective debridement of fungal plaques is considered as an essential therapeutic step. unfortunately, it is not always possible to achieve perfect debridement of the sinonasal cavities, due to incomplete accessibility of the whole sinusal area with the endoscope; however, its effect has never been assessed as such. therefore, the second aim of this study was to evaluate the effectiveness of debridement on success rate after the first treatment. fisher's exact test was used to assess the difference in success rate between both protocols and in function of full debridement. twenty-eight dogs with sna were treated with d15e and 25 dogs with d60eb. the median (range) duration of d15e was only 92 minutes (40-140) compared to 176 minutes (135-225) for d60eb. first treatment success rate did not differ between both protocols and were 68% for d15e and 60% for d60eb. both protocols had an overall success rate of 96% after 2 procedures. in contrast to the majority of dogs with d60eb, all dogs receiving d15e recovered quickly and were discharged the same day. completeness of debridement was assessed endoscopically in 48 dogs (28 treated with d15e and 20 with d60eb). debridement was judged complete in 28/48 dogs and had a significant effect on first treatment success rate (p = 0.01). when debridement was complete, 79% of the dogs (d15e: 15/19 dogs; d60eb: 7/9 dogs) were cured after the first procedure, compared to 40% (d15e: 4/9 dogs; d60eb: 4/11 dogs) of the dogs with incomplete debridement. we concluded that (1) the simplified infusion protocol is quick, safe, easy and effective, and offers a favourable alternative to 1hour infusion protocols for treatment of canine sna; (2) completeness of the debridement undoubtfully is an important step for treatment success of infusion protocols. disclosures: no disclosures to report. gastroesophageal (ge) symptoms are commonly reported in dogs with brachycephalic upper airway obstructive syndrome (bs). since ge symptoms frequently occur during situations of increased inspiratory effort (excitement, respiratory distress), dynamic disorders of the ge junction (gej) are probably involved, due to transient increased negative intrathoracic pressure. however, according to a previous study, only few dynamic abnormalities of the gej are observed during gastroscopy. we hypothesized that both anaesthesia and endotracheal intubation during gastroscopy lead to underestimation of gej abnormalities. the aim of this study was to improve detection of dynamic gej abnormalities during gastroscopy using obstructive manoeuvers mimicking and reproducing upper airway obstruction of variable severity. twenty-six dogs presented with bs were prospectively included. respiratory and digestive symptoms as well as endoscopic abnormalities were scored at initial diagnosis and at control 1 month after surgery. during each endoscopy, gej was assessed and scored (based on esophagitis, gej atony, ge reflux, cranial displacement of the gej) in the 3 consecutive situations: (1) absence of obstruction with the dog intubated (ob-0), (2) presence of natural obstruction with the dog extubated (ob-nat) and (3) during complete manual obstruction of the endotracheal tube during up to 3 spontaneous breathings (ob-compl). spearman's rank test was used to assess correlations between the different clinical and endoscopic scores. taking all endoscopic procedures together, the severity of respiratory symptoms correlated significantly with the severity of respiratory endoscopic abnormalities (p < 0.001, r = 0.6) and the severity of digestive symptoms (p = 0.039, r = 0.24). at diagnosis, 23 dogs (89%) presented digestive symptoms while endoscopic gej abnormalities were observed in 17 (65%), 24 (92%) and 26 (100%) dogs during ob-0, ob-nat and ob-compl respectively. gej atony, ge reflux, cranial displacement of the gej and sliding hiatal hernia were present in 9 (34.6%), 2 (7.7%), 9 (34.6%) and 0 dogs during ob-0, in 19 (73.1%), 5 (19.2%), 23 (88.5%) and 4 (15.5%) dogs during ob-nat and in 21 (80.8%), 6 (23.1%), 26 (100%) and 10 (38.5%) dogs during ob-compl, respectively. a significant correlation was found between digestive and endoscopic gej scores during ob-compl (r = 0.55, p = 0.003) as well as during ob-nat (r = 0.41, p = 0.03) but not during ob-0. it can be concluded that in dogs with bs (1) gej abnormalities are dynamic and related to the degree of upper airway obstruction; (2) the use of obstructive manoeuvres during gastroscopy improves the detection of gej abnormalities. disclosures: no disclosures to report. tracheobronchial foreign bodies are common causes of respiratory disease in children but they are rare in veterinary medicine. particularly in cats, reports of tracheobronchial foreign bodies are scarce. this study aimed to describe clinical presentation, diagnostic findings and treatment modalities in confirmed cases of tracheobronchial foreign bodies in cats. we hypothesize that bronchoscopy is highly effective in their extraction in cats. cases of confirmed tracheobronchial foreign bodies in cats admitted to 3 referral centers in france, from may 2009 to november 2014, were included. files were retrospectively analyzed for age, sex, breed, clinical signs, delay between onset of signs and presentation, diagnostic procedure, method of extraction, location and nature of foreign bodies. twelve cats were included (6 males, 6 females). mean age at presentation was 4 years old (3.75 years ae 2.5). cough was the main chief-complaint, being present in 9/12 (75%) cats. while 4/12 (33%) cats presented to consultation in the first week after the beginning of respiratory signs, 8/12 (67%) cats exhibited clinical signs for more than 1 week. chest radiographs were done in 12/12 cats. bronchoscopy was performed in 12/12 cats, confirming the presence of foreign body material and allowing their extraction in 10/12 animals (83%). in 2/12 cats (17%), bronchoscopic extraction was unsuccessful and a pulmonary lobectomy was required. the foreign body was located in the trachea in 6/12 cats (50%) and in the bronchial tree in the remaining 6 cats (4/6 in the right caudal bronchus, 1/6 in the left caudal bronchus and 1/6 in the main left bronchus). 7/12 (58%) were vegetal foreign bodies (grain seeds and foxtail awns), 3/12 (25%) were mineral (a bone fragment, a teeth and a small stone) and 2/12 (17%) of undetermined origin. all the mineral foreign bodies were extracted from the trachea, whilst the majority of the vegetal ones (5/7 -71%) were removed from the bronchial tree. in this case series, bronchial foreign bodies were as frequent as tracheal foreign bodies in cats. this finding differs from previous data reporting that trachea is the preferential location for feline respiratory foreign bodies. vegetal foreign bodies are more common. due to their nature and shape, they may be more prone to lodge in the bronchial tree, while mineral foreign bodies remain in the trachea. according to our results, bronchoscopy is highly effective for identification and extraction of tracheobronchial foreign bodies in cats. disclosures: no disclosures to report. mycophenolate mofetil is the prodrug of mycophenolic acid (mpa). it is a selective non-competitive inhibitor of inosine-5 0monophosphate dehydrogenase, which is expressed in many cell types. mpa's ability to induce lymphocyte cytotoxicity, reduce monocyte recruitment, and suppress dendritic cell maturation, are useful targets in treating immune mediated and inflammatory diseases. it has been used extensively in human medicine for transplant recipients and more recently in veterinary medicine in dogs with various immune mediated diseases. however, its use in cats is limited in part because mpa is primarily metabolized by glucuronidation and cats inherently have a decreased ability to glucuronidate many drugs. we proposed that cats may glucuronidate mpa more slowly than humans and dogs and conducted a series of in vitro studies to explore this hypothesis. we used liver microsomes from cats (16 individual and pooled), dogs (pooled), and humans (pooled). these liver samples were incubated at 37°c in a water bath with mpa and udp-glucuronic acid or udp-glucose. udp-glucose was studied since mpa glucoside is a minor metabolite in humans but may be a major metabolite in other species. hplc-ms was used to determine concentrations of mpa-glucuronide (phenol and acyl) and mpa-glucoside (phenol) formed by incubation. cats formed much less mpa phenol glucuronide (0.7 nmoles/ min/mg) than dogs (2.2 nmoles/min/mg), or humans (3.7 nmoles/ min/mg). cats formed similar amounts of mpa acyl glucuronide (0.3 nmoles/min/mg) than humans (0.3 nmoles/min/mg), but less than dogs (0.9 nmoles/min/mg). in contrast, cats (0.5 nmoles/min/ mg) formed more mpa phenol glucoside than humans (0.3 nmoles/min/mg) but less than dogs (1.3 nmoles/min/mg). when the 3 pathways of metabolism were summed for each species, cats (1 nmoles/min/mg) metabolized mpa much less efficiently than dogs (3 nmoles/min/mg) or humans (4 nmoles/min/mg). variability in metabolite formation between the 16 individual cats was high ranging from 6 fold for mpa glucoside, to 8 fold for mpa phenol glucuronide, to 14 fold for mpa acyl glucuronide. our preliminary results confirm that cats glucuronidate mpa less rapidly than dogs and humans, however cats and dogs were found to glucosidate mpa more efficiently than humans. in addition, individual cats are variable in their ability to glucuronidate and glucosidate mpa. this preliminary in vitro data will be compared to in vivo studies of mpa pharmacokinetics to elucidate proper dosing of mpa in cats. disclosures: no disclosures to report. cystocentesis is the gold standard sampling method for urine microbiology in dogs, as voided samples are associated with a higher risk of contamination. however, the accuracy of the veterinary cut-off values currently recommended for detection of clinically significant bacteriuria in voided urine has been poorly investigated. the aim of this study was to evaluate the accuracy of veterinary and human criteria for diagnosis of urinary tract infection (uti) in dogs using voided urine samples. dogs with suspected uti were prospectively enrolled. paired urine samples collected by cystocentesis and voiding, respectively, were stored at 5 • c and cultured within 4 hours. bacterial counts in voided urine were interpreted using both the veterinary (≥100.000 colony forming units (cfu) per ml) and the human (≥1.000 cfu/ml plus presence of clinical signs) criteria for diagnosing uti, and compared to those obtained in urine collected by cystocentesis (gold standard). significant bacteriuria in cystocentesis samples was defined as ≥1.000 cfu/ml. sixty-five dogs were included in the study. when applying the veterinary criteria for diagnosing uti in voided samples, the diagnostic accuracy was 97% (sensitivity 100%, specificity 96%, positive predictive value (ppv) 90% and negative predictive value (npv) 100%). when applying the human criteria the accuracy fell to 75% (sensitivity 68%, specificity 78%, ppv 57% and npv 86%). the results indicate that, in most dogs with suspected uti, an accurate diagnosis can be obtained using voided urine, if applying the current veterinary cut-off values to samples stored at refrigeration temperature and cultured shortly after collection. disclosures: the study was supported financially by the uc-care research centre, university of copenhagen, and 2 minor external foundations. antimicrobial resistance (amr) is a major public health concern and will likely be the first cause of mortality in human medicine in 2050. canine bacterial urinary infection is a frequent condition and might be implicated in interspecies transmission of resistance mechanisms. this study aimed to retrospectively describe the evolution of the amr of uropathogens over a 10-year period in a veterinary teaching hospital. positive urinary cultures obtained by cystocentesis (>1000 cfu/ ml) from dogs treated at the national veterinary school of toulouse (envt) between 2005 and 2014, were reviewed. annual prevalence of amr for several enterobacteriaceae, staphylococcus spp, enterococcus spp and streptococcus spp were recorded for various veterinary and human antimicrobials. frequency of extended spectrum beta-lactamase-producing enterobacteriaceae (esbl) and multidrug resistant (mdr) bacteria were noted. logistic regression was performed to analyze the evolution of amrs. a p-value <0.5 was considered significant. over the study period, 751 isolates with stable annual distribution were identified. considering enterobacteriaceae, amr for several antimicrobials significantly evolved over time: despite a possible increase in 2014 for some antimicrobials, a general decrease of amr was observed. prevalence of esbl and mdr bacteria remained stable with mean prevalence of 5% and 26%, respectively. trends of amr of enterobacteriaceae over the study period in envt are not in accordance with the worrisome general tendency and could be consistent with a rationalized antimicrobials use. however, the persistently elevated prevalence of esbl and mdr bacteria, and the possible increase of amr during the last studied year warrant further investigation and surveillance. disclosures: no disclosures to report. klebsiella pneumoniae are important pathogens that cause urinary tract infections (uti). antibiotic-resistant and virulent bacterial clones with high interhost transmission may play an important role in the spread of antimicrobial resistance. this study aimed to characterize the antimicrobial resistance and virulence of clinical klebsiella isolated from animals and humans with uti and to determine the lineages of companion animal klebsiella pneumoniae resistant to third-generation cephalosporins (3gc). thirty-five companion animal clinical klebsiella spp., obtained between 2002 and january 2015, and 61 human clinical strains isolated in 2014 were included. antimicrobial susceptibility testing was performed using disk diffusion and clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) and plasmid-mediated ampc genes were detected by pcr whenever resistance to 3gc was detected. furthermore, 3gc-resistant klebsiella were characterized by multi-locus sequence typing. regarding virulence, pcr for detection of fimh (adhesin type-1 fimbriae), mrkd (adhesin type-3 fimbriae), entb (enterobactin), ybts (yersiniabactin) and rpma (regulator of mucoid phenotype-a) genes was conducted on 27 and 61 companion animal and human strains, respectively. k. pneumoniae was the main species isolated in companion animals (85.7% n = 30/35) and in humans (98.4%, n = 60/61) but klebsiella oxytoca was also identified. resistance to 3gc (cefotaxime or ceftazidime) was present in 51.4% (n = 18/35) and 21.3% (n = 13/61) of companion animal and human strains, respectively. overall, 3cg-resistant k. pneumoniae were found to be ctx-m group-1 (88.9%, n = 24/27), cmy (11.1%, n = 3/27) and dha (3.7%, n = 1/27) producers. both companion animal k. oxytoca were dha producers. companion animal 3cg resistant klebsiella were frequently (66.7%, n = 12/18) co-resistant to fluoroquinoles and trimethoprim/sulphamethoxazole rendering them as multidrug resistant. moreover most companion animal 3cg resistant strains were also resistant/intermediate to amoxicillin/clavulanate (83.3%, n = 15/18). overall, companion animal klebsiella resistance to amoxicillin/clavulanate (40.0%, n = 14/35), fluoroquinolones (65.7%, n = 23/35) and trimethoprim/sulphamethoxazole (58.8%, n = 20/34) was high. companion animal k. pneumoniae resistant to 3gc belonged to st15 (n = 10), st348 (n = 1), st147 (n = 1) and st11 (n = 1) lineages. concerning virulence, all k. pneumoniae were positive for fimh, mrkd and entb. yersiniabactin was present in 16.0% (n = 4/25) and 43.3% (n = 26/60) of companion animal and human k. pneumoniae, respectively. k. oxytoca (n = 3) were positive for entb and ybts. all tested strains were negative for rpma. the detection of k. pneumoniae lineages highly important for humans in companion animals with uti raises great concerns regarding their role as reservoirs. moreover, the fact they were also found to share 3gc resistance genes and common virulence factors with humans further extends the risk of transfer. disclosures: conflicts of interest: the first author currently receives a phd grant funded by the portuguese foundation for science and technology. feline lower urinary tract disease (flutd) refers to a heterogeneous group of disorders with similar clinical signs. some diets are designed to manage flutd by promoting struvite stone dissolution and addressing key risk factors (overweight, low water intake, stress. . .). the goal of this study is to compare the struvite dissolution potential of 2 diets marketed for flutd, in standardized in vitro conditions. six adult healthy cats were fed successively 2 dry diets (a = royal canin s/o-biopeptide; b = hill's c/d-urinary-stress) for 12 days, with urine collection on the last 5 days. urines collected for each diet were pooled and distributed in bottles containing the mean urine volume produced daily per cat. urine ph and struvite relative supersaturation (rss) were measured for each pool. two feline struvite uroliths homogenous in shape and weight were immersed separately in a urine bottle of each diet, and put in a stove at 38°c. twenty-four hours later, the urine was filtered to collect the stones, which were dried and weighed. every day, the stones were placed in new bottles of the corresponding urine until the first complete stone dissolution. diet effect on urine ph, volume and rss was analysed non parametrically (wilcoxon paired rank test). diet effect was combined with a period effect (period = 5 days dissolution), in a 2 9 5 complete factorial design to analyse struvite stones weight evolution at each period for each diet (mixed model with diet, period, diet x period as fixed effects). fdr method was applied to compare diets at each period. diet a induced a higher mean urine volume (14.2 versus 10.2 ml/kg/day), and a lower rss than diet b (0.15 versus 0.81) (p < 0.05). the urine ph of the 2 diets were not significantly different (6.06 and 6.13) (p > 0.05). after 25 days, the struvite stone immersed in urine from diet a was totally dissolved, versus 62% dissolution for the stone in urine from diet b. when considering periods of 5 days, the struvite weight diminution was significantly higher when struvite stone was immersed in urine from diet a than from diet b (diet effect: p < 0.001). the interaction between diet and period effect revealed that the difference in dissolution rate between the 2 diets was significant as soon as the first 5 day period (p < 0.01), and increased over the other periods (p < 0.001). a diet inducing a lower struvite rss and a greater urine dilution allows faster struvite stone dissolution. disclosures: the author and co-authors work for royal canin, the company commercializing one of the diets evaluated. serum cystatin c (scysc) and urinary cystatin c (ucysc) are potential markers for detection of feline chronic kidney disease (ckd). our aims were twofold. firstly, we evaluated cysc as marker for ckd. we compared scysc and ucysc between ckd and healthy cats, correlated scysc and scr with glomerular filtration rate (gfr) and calculated sensitivity, specificity for detecting decreased gfr. secondly, we compared assay performance of the turbidimetric assay (petia) with the previously validated nephelometric assay (penia). forty-nine ckd (iris stage 1-4) and 41 healthy cats were included. gfr was measured with plasma exogenous creatinine (pect), endo-(penict) and exo-iohexol (pexict) clearance test in 17 ckd and 15 healthy cats. based on pexict, scysc was evaluated to distinguish normal, borderline and low gfr. sensi-tivity and specificity to detect pexict<1.7 ml/min/kg were calculated. validation of petia was performed and scysc results of penia and petia were correlated with gfr. statistical analysis was performed using general linear modelling. serum cysc and ucysc were significantly higher (p < 0.001) in ckd cats. however, ucysc was detected only in 35/49 ckd cats. r 2 values between gfr and scr or scysc were 0.71 and 0.39 respectively. sensitivity and specificity were 22% and 100% for scysc and 83% and 93% for scr. serum cysc could not distinguish healthy from ckd cats, nor normal from borderline or low gfr, in contrast to scr. penia appeared superior to petia. in conclusion, scysc is not a reliable marker for gfr in cats and ucysc could not be detected in all ckd cats. disclosures: for this work support was received from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l.f.e. ghys. soblechero, f.j. duque, p. ruiz, r. barrera. university of extremadura, c aceres, spain serum cystatin c (scys c) is a marker of glomerular filtration rate with advantages over serum creatinine. in human medicine, some authors observed that scys c is influenced by methylprednisolone or prednisone administration. with the aim to follow the course of this maker of renal function in acutely diseased patients with a receiving glucocorticoid medication, we followed at dog's whit steroid responsive meningitis. the study was carried out on 30 patients that where divided in 3 groups: control group (10 clinical healthy dogs), group b, 10 dogs treated with prednisone due to steroid responsive meningitis (treated with 4 mg/kg prednisone during 7 days, followed to 2 mg/kg prednisone for another 7 days). dogs had no known preexisting renal disease, and have no previous glucocorticoid medication. group c was established to test the effects of endogenous steroids: 10 dogs with hyperadrenocorticism were evaluated. serum cys -c was measured by turbidimetric latex and creatinine by jaffe reaction (spinreac ò ) and were determined at time of diagnosis for group b, and on days 0,7 and 14 in the meningitis dogs. a statistically significant increase of scys-c was observed in group b, with doses of 2 mg/kg of prednisone (0.18 ae 0.03 mg/l; p < 0.01), and doses of 4 mg/kg of prednisone (0.28 ae 0.15 mg/l; p < 0.001) respect to these same dogs before treatment (0.05 ae 0.04 mg/l) and compared to the control group (0.07 ae 0.04 mg/l). however, the serum concentration observed in hyperadrenocorticism (0.09ae 0.06 mg/l) was similar to the one find in the healthy animals group. the creatinine concentration was not increased either, during the prednisone treatment, or in the case of hiperadrenocorticism. in conclusion, the present study agrees with that is described in human medicine, and confirms the effects of glucocorticoids on scys c concentrations in dogs. the administration of high doses of prednisone is associated with a scys c increase. on the other hand endogenous cortisol increase (hyperadrenocorticism) in dogs is not seen to modificate the scys c. disclosures: no disclosures to report. chronic kidney disease (ckd) produces progressive reduction in the number of functional nephrons and directly affects the homeostasis of the solutes excreted in the urine, including phosphorus. hyperphosphatemia is considered a factor directly related to the increased mortality in humans, cats and dogs. in order to provide data from controlled clinical studies to examine the effects of hyperphosphatemia on the progression and survival of naturally occurring canine ckd, the following study was conducted. for the present study 85 dogs, which were followed up by the veterinary teaching hospital of the university of extremadura (spain), were used for the study. distributed in the following groups: group i (25 healthy adult dogs) and group ii (60 adult dogs with ckd). this second group had a subclasification attending to different factors: phosphatemia: iia (20 dogs with phosphatemia < 5.5 mg/dl) and iib (40 dogs with phosphatemia > 5.5 mg/dl). leishmaniasis: iic (20 dogs with ckd due to leishmaniasis) and iid (40 dogs with ckd not due to leishmaniasis). iris classification: iris1 (10 dogs), iris2 (10 dogs), iris3 (20 dogs) and iris4 (20 dogs). the results of survival were as followed: statistically lower survival was found between the groups iia and iib (p < 0.01) also between the iris grades 2, 3 and 4 and the iris grade 1 (p < 0.001), iris 3 and 4 with iris 2 (p < 0.001) and iris 4 with iris 3 (p < 0.001). no significant differences between positive and negative leishmaniasis. in conclusion, plasma phosphate concentration in dogs increases as chronic kidney disease develops. and an inverse relationship to survival in dogs with phosphorus concentrations above 5 mg/dl, and as it progresses the iris scale was observed. disclosures: no disclosures to report. studies analyzing to which extent upc in dogs is influenced by pyuria have yielded conflicting results. moreover, there is no data on the effect of proteinuria on plasma acute phase proteins in dogs. in 315 dogs upc was prospectively measured. upc and if available, results of plasma biochemistry including measurement of c-reactive protein (crp) from the same day were analyzed using the mann-whitney-u-test. samples without sediment analysis (n = 7) were excluded resulting in 308 urine samples for analysis. hematuria (>5 erythrocytes/hpf), pyuria (>5 leucocytes/hpf), and bacteriuria were present in 86, 53 and 27 samples, respectively. upc in samples with hematuria was significantly (p = 0.001) higher (median 0.32; 25 th -75 th percentile 0.13-1.76) compared to samples without hematuria (0.17; 0.1-0.64). in dogs with pyuria upc was significantly (p < 0.001) higher (1.14; 0.19-1.96) compared to samples without pyuria (0.17; 0.1-0.62). 53% of the samples with pyuria had an upc >1. this is in contrast to data reported previously where only 6% of pyuric urine samples had an upc >1 (vet clin path 2008;33:14). bacteriuria did not influence upc (p = 0.26). samples of 3 dogs with negative protein dipstick results had an upc >0.4 (0.42, 0.6, and 1.14, respectively). all 3 samples had low urine specific gravity (1.004-1.012) and alkaline ph. for a total of 155 samples corresponding plasma data on albumin (reference interval ri: 29.6-37.0 g/dl) and crp (ri: 0-14.9 mg/l) were available. crp was significantly (p = 0.003) higher in dogs with upc >0.4 (5.0 mg/l; 1.7-54.3) compared to dogs with upc ≤0.4 (2.4 mg/l; 0.7-7.0). albumin was significantly (p < 0.001) lower (26.0 g/dl; 22. 6-29.6) in dogs with upc >0.4 compared to dogs with upc ≤0.4 (29.4 g/dl; 26.5-31.7). naturally occurring pyuria has a more profound effect on upc results than previously reported. proteinuria is associated with changes of acute phase proteins such as hypoalbuminemia and increased crp. whether this is consequence or cause of the proteinuria needs further investigation. furthermore, animals with low urine specific gravity may have clinically relevant proteinuria even in the light of a negative dipstick result. therefore measurement of upc is recommended to exclude renal protein loss in hypo-and isosthenuric dogs. disclosures: no disclosures to report. high blood pressure causes an increase in vascular endothelial growth factor (vegf) secretion. feline hypertension is commonly associated with chronic kidney disease (ckd) amlodipine is the first choice antihypertensive treatment in cats but could have a negative effect on the kidney by increasing glomerular pressure through afferent arteriolar dilatation. the aims of this study were to: (1) validate a method for the quantification of vegf in feline serum samples; (2) assess the association between urinary vegf, serum vegf (svegf) and biochemical and clinical variables in hypertensive cats and (3) investigate changes in urinary vegf with amlodipine treatment. a randomised, double blinded, placebo controlled parallel group study (n = 72) was conducted in 2 phases to determine the efficacy and safety of amlodipine in cats with naturally occurring hypertension. the placebo group was crossed-over to amlodipine after day 28. a canine vegf elisa (previously validated for feline urine) was used to measure urine and serum vegf. urine vegf concentration was normalised to urinary creatinine (urinary vegf to creatinine ratio [uvc]). univariable linear regression models, followed by a backwards multivariable linear regression model, were performed to identify independent predictors of svegf and uvc. a linear mixed measures model was used to compare the effect of placebo and amlodipine on uvc (28 days) and to investigate potential changes in uvc with long-term amlodipine treatment (90 days). intra-assay and inter-assay cv of svegf measurements were 0.52-2.79 (n = 5) and 5.67-11.05 (n = 4) respectively. dilutional parallelism indicated a mean recovery of 98.8% ae 8.5% (n = 4). urea and urine protein:creatinine (upc) were independent negative and positive predictors of svegf respectively. plasma creatinine was an independent negative predictor of uvc, upc and sodium were independent positive predictors. no association was found between svegf and uvc. no significant changes in uvc or differences between groups were found with 28 days of amlodipine or placebo treatment. mean uvc at screening was 0.403 and 0.441 lg/g after 90 days of amlodipine treatment (p = 0.061), both within the healthy cat reference range (0.171 to 0.682 lg/g). the lack of correlation between urinary and serum vegf suggests that uvc reflects renal vegf production, and is possibly a biomarker of renal stress. uvc does not significantly change with amlodipine treatment suggesting that amlodipine may not cause renal stress when used in cats with hypertension and concurrent ckd. disclosures: this study was sponsored by orion inc. and ceva animal health and used residual samples collected from animals involved in a clinical trial run by orion inc. jonathan elliott provides consultancy advice to the following companies: bayer animal health, ceva animal health, orion inc., elanco animal health, zoetis ltd, boehringer ingelheim, vetoquinol ltd., waltham centre for pet nutrition, idexx ltd. the group is in receipt of research funding from the following companies: novartis animal health, royal canin ltd, zoetis, ceva animal health / orion inc. jonathan elliott serves on the following advisory boards: international renal interest society, european emesis council. diffuse large b-cell lymphoma (dlbcl) is the most frequent subtype of non-hodgkin lymphomas in dogs. in humans, 3 common morphological variants have been recognized by the world health organization (who) classification: centroblastic, immunoblastic and anaplastic. the who classification was recently adapted to canine lymphomas. however, no study clearly correlated prognosis to each morphological variant of canine dlbcl. the objective of this retrospective study was to correlate morphological variants of dlbcl to prognosis, in dogs treated with a standardized chemotherapy protocol. medical records from dogs with a cytological diagnosis of dlbcl between 1999 and 2014 were retrospectively reviewed by a single boarded clinical pathologist. the centroblastic (dlbcl-cp) and immunoblastic morphotypes (dlbcl-ib) were defined as previously described. anaplastic variant is very rare in dogs, and no case meeting all inclusion criteria were diagnosed during the study period. a fourth borderlines morphological variant was identified and distinguished in this study for clinical considerations (immunoblasts rich centroblastics (dlbcl-irc)). it was characterized by the presence of a higher number of immunoblasts compared to dlbcl-cp. complete initial and follow-up clinical information and application of a standardized chemotherapy protocol were part of the main inclusion criteria. statistical analysis was performed using kaplan-meier analysis. fourty-nine dogs were included. thirty-four (69.4%) were dlbcl-cp, 12 (24.5%) were dlbcl-ib and 3 (6.1%) were dlbcl-irc. median first remission duration for dlbcl-cp, dlbcl-ib, dlbcl-irc were respectively 365 and 156.5 and 91 days (p < 0.0001). median overall survival time for dlbcl-cp, dlbcl-ib, dlbcl-irc were respectively 482, 259 and 344.5 days (p = 0.06). a significant shorter time to obtain complete remission (p = 0.006) and a significant longer duration of first remission (p < 0.00001) in dogs with dlbcl-cp in comparison to dlbcl-ib were observed when dlbcl-irc were included in the dlbcl-ib group. interestingly for 2 cases, dlbcl-irc variant was observed in peripheral lymph nodes whereas dlbcl-ib variant was observed in the spleen. moreover, 1/4 recurrent dlbcl-cp and 2/3 of dlbcl-irc displayed progression towards dlbcl-ib variant. in conclusion, this study showed, for the first time, significant prognostic differences between the morphological variants of canine dlbcl, suggesting the prognostic impact of immunoblastic features as it is discussed in humans. disclosures: the residency program of david sayag is supported in part by zoetis. canine lymphoma is a heterogenous group of diseases and evidence exists to describe different behaviours between b-cell and tcell phenotypes of disease. this study aims to describe the response to treatment and survival of canine b-cell multicentric lymphoma (cbcml) cases treated at the royal veterinary college. signalment, clinical findings, staging, treatment, response and survival times were recorded retrospectively. sixty-three cases of cbcml were identified. forty-nine percent presented as stage 5, 35% stage 4, and 16% stage 3. sixty-two percent were substage b and 38% were substage a. forty-four percent received "chop" induction protocols, 30% "cop," 8% "coap," and the rest received various induction protocols. ninety-five percent of dogs responded to induction treatment. median first remission duration (frd) was 108.5 days. thirty-seven dogs (59%) received rescue protocols with a response rate of 60%. median overall survival time (os) was 209 days. follow-up was 1309 days. this study showed that cop protocols followed by doxorubicin rescue therapy gave no significant difference in os compared with both chop induction alone and chop followed by a rescue protocol (p = 0.179). os was significantly increased by increased frd (p = 0.001), absence of an aberrant immunophenotype (p = 0.025), complete response to therapy (p = 0.000), and use of rescue protocol (p = 0.000). frd was significantly increased by use of a chop induction protocol compared with a cop protocol (p = 0.025), and complete response to therapy (p = 0.000). age, bodyweight, sex/neuter status, stage, substage, and cell size had no effect on frd or os. seven (11%) of the dogs had a prolonged os in excess of 2 years, and 4 of these dogs remain alive. dogs in the prolonged os group were more likely to be anaemic on presentation (pcv<37%, p = 0.041), experienced a greater frd (p = 0.012) and were more likely to be treated with a rescue protocol (p = 0.036) than other dogs. no other significant differences in signalment, clinical presentation, stage, substage, or treatments received were found between this group of dogs and others. in this group of dogs, chop induction therapy gave no survival benefit over the cheaper, less intense cop protocol, providing doxorubicin rescue therapy was later employed. the proportion of dogs receiving chop induction versus cop did not significantly differ between dogs with prolonged survival and those without. the use of rescue protocol, complete response to treatment, aberrant immunophenotype and first remission duration were shown to have prognostic relevance. disclosures: no disclosures to report. lymphoma is the most common malignant haemopoietic tumour in the dog. gene expression profiling (gep) of canine lymphoma has highlighted the important signalling pathways including b-cell activation, b-cell receptor and nf-kb signalling. next-generation sequencing offers benefits over microarray technology for gep in identification of novel transcripts and sequence variants. the aim of this study was to examine gene expression and variant calling in canine lymphoma using rna-seq. lymph node samples were collected from 23 canine multicentric lymphoma patients as part of their clinical investigations. diagnosis was confirmed cytologically or histologically and cell lineage established by pcr for antigen receptor rearrangements (parr) and flow cytometry. cdna was prepared from extracted rna and sequencing performed on an illumina nextseq 500 sequencer generating 150 bp paired-end reads. samples were from 18 b-cell tumours (10 stage v, 5 stage iv, 2 stage iii and 1 stage ii) and 5 t cell tumours (2 stage iii, 1 each stage ii, iv, v). 38 million reads (mean) per sample were obtained with 82% mapping to the canine genome. b-and t-cell samples clustered separately on principal component analysis indicating distinct gene expression patterns. 232 genes were upregulated (log2fc >2, q value <0.05) in b-cell lymphomas, many involved in bcr signalling, primary immunodeficiency and haematopoietic cell lineage pathways, innate immune and inflammatory responses. 640 genes were upregulated (log2fc >2, q value <0.05) in t cell lymphomas, most affecting tcr signalling, but also natural killer mediated cytotoxicity, jak-stat signalling, haemopoietic cell lineage and cancer pathways. compared to the reference genome, 4.8 million sequence variants were detected across the 23 samples; 74% not previously described. using the sift (sorting intolerant from tolerant) algorithm, 17% were predicted to be deleterious for protein function. functional analysis of the affected genes indicated many were involved in bcr signalling and cancer-related pathways. some such as bcl10 and map3k14 were affected in almost all cases, although proportionally more frequent in b cell lymphoma. others such as traf3 were exclusive to b cell cases. genes affecting a large number of cases such as bcl10 tended to have a common variant present in 3 or more cases whereas other genes had variants unique to each single case. although it remains to be confirmed if the detected variants represent true mutations rather than polymorphisms, rna-seq of canine lymphoma samples has generated interesting pilot data that need to be expanded with more samples to validate the results. disclosures: manikhandan mudaliar works for glasgow polyomics which is a commercial company within glasgow university and carries out genomic and polyomic assays. the next gen sequencing was done at glasgow polyomics, partly using an ecvim grant. feline large granular lymphocyte (lgl) lymphoma is uncommonly described in the literature and it is caused mainly by t-cell lymphocyte. to date a standard protocol has not yet been established and long term prognosis is poor. a recent study (krick et al. 2008 ) described a median survival time of 57 days (range: 0-267) in cats with lgl lymphoma receiving mainly a cop-based protocol and in few cases adjuvant surgery or orthovoltage radiation therapy. surprisingly, in that study the longest survival time was achieved from a cat in the non treated group (median survival time 2 days; range: 0-288) that received only prednisone and single agent cyclophosphamide. considering these data, and the advantages in treating with more than one alchylating agents t-cell lymphoma in dogs (brodsky et al 2009), the aim of this study was to assess if the sequential use of different alchylating agents was of any benefit in cats with lgl lymphoma. to all owners of cats with a cytological or hystopatological diagnosis of lgl lymphoma that presented to the san marco veterinary clinic from july 2008 till december 2014 were offered a treatment with sequential alchylating agents and prednisone (saa&p) protocol or palliative care with only prednisone. the saa&p protocol consisted of prednisone at 2 mg/kg q24 h and chlorambucil at 2 mg/cat (for cats >4 kg) to q72 h (for cats <4 kg). when despite treatment progressive disease or stable disease plus clinical signs referable to the lgl lymphoma were present chlorambucil was substituted with cyclophosphamide at 25 mg/cat q10 days. finally, when cats stop responding to cyclophosphamide, this drug was substituted with lomustine at 50-60 mg/m 2 q3-5 week. during the study period 28 cats were diagnosed with a lgl lymphoma. on owner request 6 cats were euthanased at the time of diagnosis, 2 cats were sent home with no treatment and lost to follow-up, 10 cats received prednisone alone, and 10 cats received the saa&p protocol. median survival time for cats treated with prednisone alone was 7 days (range: 1-229 days, 95% ci 0-22 days) and for cats treated with the saa&p protocol was 137 days (range: 25-532 days, 95% ci 39-235). survival kaplan-meier curves of the 2 treatment group were significantly different (log rank test = 8.01; p = 0.0042). survival time in cats with lgl lymphoma treated with the saa&p protocol is significantly longer than in cats receiving only prednisone and seems to be longer than historical reported data of cats receiving cop-based protocol. disclosures: no disclosures to report. mast cell tumors are often accompanied by eosinophilic inflammation as they are known to produce eosinophil chemotactic factors. however, little is known about frequency or eventual prognostic significance of blood eosinophilia in mct bearing dogs. thus, the aim of this study was to determine frequency of absolute and relative peripheral blood eosinophilia as well as eosinopenia and to evaluate potential influence on progression free interval (pfi), overall survival time (ost) and tumor specific survival (tss). dogs with mast cell tumors diagnosed between 2008 and 2014 were included into this retrospective study. data were collected from medical records and follow up phone conversations with patient owners or referring veterinarians. medical records were reviewed to rule out underlying clinical conditions other than mct that could cause eosinophilia. tumor diagnosis was made either by fine needle aspirate and/or tumor biopsy. a patient was allocated to the eosinophilic group, when the eosinophil concentration was >0.8*10 3 /ll or the relative percentage was > 4%, respectively. when the eosinophil concentration was <0.1*10 3 /ll patients were categorized as eosinopenic. groups were compared by the pearson chi-square test. the pfi, ost and tss curves were generated by the kaplan-meier product limit method. a log rank test was used to compare the curves. one-hundred dogs were included into this study. absolute eosinophilia was detected in 8/ 100 patients and in 37/100 a relative eosinophilia was present. median concentration of eosinophils was 0.3*10 3 /ll (range, 0-3093) and median relative percentage was 3.0% (range, 0-25%). eosinopenia was found in 24% of all dogs. a positive association between relative eosinophilia and low grade tumors was detected with both patnaik (p = 0.001) and kiupel (p = 0.027) grading system. a positive linear correlation was further noticed between absolute eosinophilia and ost (p = 0.022). positive correlation was confirmed between relative eosinophilia and pfs (p = 0.001), ost (p = 0.001), and tss (p = 0.001). accordingly a negative linear fit was found between eosinopenia and pfi (p = 0.002), ost (p = 0.004) and tss (p = 0.001). data indicate that peripheral blood eosinophilia might serve as an easily available additional prognostic tool for mast cell tumor bearing dogs. disclosures: no disclosures to report. limited literature is available about prognostic factors for canine renal carcinomas. in humans, histologic differentiation and tumor type are strongly associated with outcome. in dogs only one publication so far has reported this association. cox-2 expression is documented in several canine neoplasias with prognostic value in canine mammary carcinomas. in renal carcinomas cox-2 expression has been demonstrated but its significance is not known. the aim of this study was to evaluate clinical and histopathological features of canine renal carcinomas, including cox-2 expression, and to correlate them with outcome. our hypothesis was that advanced disease, higher histological grade and increased cox-2 expression would be associated with shorter survivals. this retrospective multi-institutional study within 20 veterinary institutions, included histologically confirmed cases of canine renal carcinoma undergoing nephrectomy between 1998-2015, with available follow up. histologic features and cox-2 immunostaining scoring were reviewed by 2 independent pathologists where available. signalment, clinical presentation, stage, adjuvant therapy and survival times were recorded and statistical analysis performed. sixty-two cases were included. male to female ratio was 1:1, median age was 8.5 years. cross-breed dogs (n = 10) and labrador retrievers (n = 9) were over-represented. on presentation 6 dogs had metastasis. overall median survival time (mst) was 426 days (18-1945 days) . dogs without metastasis lived longer (mst 420 versus 141 days, p = 0.01). twenty-seven dogs received adjuvant therapy post-nephrectomy, without impact in mst (treatment 420 days, no treatment 532 days, p = 0.5). fifty samples were available for histopathological review, and 30 for cox-2 immunostaining. shorter survival times were seen in solid histological type compared to others (solid 152 days, papillary 532 days, tubular 540 days, p = 0.01). histologic degree of differentiation was associated with mst (well 485 days, moderate 1176 days, undifferentiated 90 days; p = 0.04). vascular invasion was associated with shorter survival (mst 210 days versus 540 days if absent; p = 0.01). marked invasiveness was associated with shorter mst (117 days versus mild = 720 days, moderate 570 days; p = 0.02). patients with low cox-2 expression had longer mst (1176 days) than those with high (203 days, p = 0.01). mitotic index, clear cell type, nuclear morphology, fuhrman nuclear grading, presence of pseudocapsule, necrosis, haemorrhage and type of inflammation were not significantly associated with mst. histopathological findings (degree of differentiation, invasiveness, vascular invasion, solid histologic type), cox-2 expression and metastasis present at diagnosis are strongly associated with survival in canine renal carcinomas and can be used as prognostic factors. disclosures: no disclosures to report. the detection of leishmania infantum-specific antibodies has been extensively exploite